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Nature. Author manuscript; available in PMC 2016 September 17.
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Published in final edited form as:

Nature. 2016 March 17; 531(7594): 335–340. doi:10.1038/nature17188.
Crystal Structures of the M1 and M4 Muscarinic Acetylcholine

Receptors
David M. Thal1,*, Bingfa Sun2,*, Dan Feng2, Vindhya Nawaratne1, Katie Leach1, Christian C.
Felder3a, Mark G. Bures3b, David A. Evans3c, William I. Weis4a,4b, Priti Bachhawat2, Tong
Sun Kobilka2, Patrick M. Sexton1,†, Brian K. Kobilka2,4a,†, and Arthur Christopoulos1,†
1Drug Discovery Biology and Department of Pharmacology, Monash Institute of Pharmaceutical
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Sciences, Monash University, Parkville, 3052, Victoria, Australia

2ConfometRx, Santa Clara, CA, USA
3aNeuroscience, Eli Lilly, Indianapolis, IN, USA
3bComputational Chemistry and Chemoinformatics, Eli Lilly, Indianapolis, IN, USA
3cComputational Chemistry and Chemoinformatics, Eli Lilly, Windlesham, UK
4aDepartment of Molecular and Cellular Physiology, Stanford University School of Medicine,
Stanford, California 94305, USA
4bDepartment of Structural Biology, Stanford University School of Medicine, Stanford, California
94305, USA
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Summary
Muscarinic M1–M5 acetylcholine receptors are G protein-coupled receptors (GPCRs) that regulate
many vital functions of the central and peripheral nervous systems. In particular, the M1 and M4
receptor subtypes have emerged as attractive drug targets for treatments of neurological disorders,
such as Alzheimer's disease and schizophrenia, but the high conservation of the acetylcholine-
binding pocket has spurred current research into targeting allosteric sites on these receptors. Here,
we report the first crystal structures of the M1 and M4 muscarinic receptors bound to the inverse
agonist, tiotropium. Comparison of these structures to each other, as well as the previously
reported M2 and M3 receptor structures, reveals differences in the orthosteric and allosteric
binding sites that contribute to a role in drug selectivity at this important receptor family. We also
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Reprints and permissions information is available at www.nature.com/reprints.

†
Corresponding authors: Correspondence and request for materials should be addressed to P.M.S. (patrick.sexton@monash.edu),
B.K.K (kobilka@stanford.edu) or A.C. (arthur.christopoulos@monash.edu).
*These authors contributed equally to this work.
Author Contributions. D.M.T performed cloning, protein expression, purification, crystallization, data collection, structure
refinement, and radioligand binding assays on the M4 receptor. D.F. purified and crystallized the M1 receptor. B.S. performed data
collection and structure refinement on the M1 receptor. K.L., V.N. and D.M.T. performed mutagenesis and radioligand binding studies
that examined the effects of amino acid substitutions on ligand pharmacology. C.C.F, M.G.B. and D.E. provided the pirenzepine IFD
and active-state M4 homology model. P.B. generated the active-state model of the M1 receptor. T.S.K. provided supervision on the M1
muscarinic receptor production and purification. W.I.W. supervised structure refinement. B.K.K., P.M.S., and A.C. provided overall
project supervision. D.M.T and A.C. wrote the manuscript.
Author Information. Atomic coordinates and structure factors will be deposited in the Protein Data Bank (http://pdb.rcsb.org) as
entries 5CXV and 5DSG for the M1 and M4 receptors, respectively. C.C.F, M.G.B. and D.E. are employees of Eli Lilly. Readers are
welcome to comment on the online version of the paper.
Thal et al. Page 2
report identification of a cluster of residues that form a network linking the orthosteric and
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allosteric sites of the M4 receptor, which provides new insight into how allosteric modulation may
be transmitted between the two spatially distinct domains.
The M1 – M5 muscarinic acetylcholine receptors constitute an important family of Class A

GPCRs activated by the neurotransmitter, acetylcholine1. Both the M1 and M4 receptors
have been associated with learning, memory, and cognition2,3 and have emerged as attractive
targets for the treatment of various central nervous system disorders, including Alzheimer's
disease, schizophrenia and drug addiction4-6. However, the orthosteric acetylcholine-binding
site is highly conserved, and the clinical translation of compounds targeting these receptor
subtypes has remained largely unsuccessful due to adverse side effects from off-target
activity at peripheral M2 and M3 receptor subtypes7-9. Encouragingly, muscarinic receptors
possess spatially distinct allosteric binding sites that offer greater potential for selective
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receptor targeting10-12, and the M1 and M4 receptors are prime examples where highly
selective positive allosteric modulators (PAMs) with central nervous system activity and
preclinical efficacy have been identified4,13-17.
To date, however, the structural basis of drug action at these receptor types has been largely
restricted to mutational analyses18-21, with the only reported muscarinic receptor crystal
structures being of the M2 and M3 subtypes22,23. Thus, to better understand the molecular
basis for orthosteric and allosteric drug interactions with the M1 and M4 receptors, we
sought to obtain high-resolution x-ray crystal structures of both subtypes. To gain additional
insight into potential mechanisms of allosteric modulation, we complemented our findings
with active-state homology modelling to rationalize the effects of targeted mutations on the
interaction between a well-characterized PAM and acetylcholine at the M4 receptor.
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Crystallization of the M1 and M4 receptors

To determine the structures of the M1 and M4 muscarinic receptors, we utilized protein
engineering and lipidic cubic phase methodology24,25. Both receptors were crystallized in
the presence of the high affinity and clinically utilized inverse agonist, tiotropium (Spiriva®),
to stabilize the inactive-state. Intracellular loop 3 (ICL3) of the M1 receptor was replaced
with a T4 lysozyme fusion protein, and in the case of the M4 receptor a minimal T4
lysozyme (mT4L)26 fusion was utilized to aid crystallization (Extended Data Fig. 1). It was
also necessary to remove the first 21 residues of the N-terminus from the M4 receptor to
improve diffraction. The M1 receptor was also crystallized with the N2Q and N12Q
mutations to remove glycosylation sites, and, unintentionally, an N110Q3.37 mutation.
Importantly, the binding affinities of [3H]QNB (M1 receptor), [3H]NMS (M4 receptor),
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acetylcholine or tiotropium were not significantly different at either fusion construct when
compared to the wild-type receptor, suggesting that the alterations did not perturb the
orthosteric site; the M1 N110Q3.37 mutation also had no significant effect on receptor
functionality in the absence of T4 lysozyme (Supplementary Table 1). The M1 and M4
structures were subsequently determined to a resolution of 2.7 Å and 2.6 Å, respectively
(Extended Data Table 1).

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Comparison of muscarinic receptor structures

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Overall, the structures of the M1 and M4 receptor are similar to the previously solved
inactive M2 and M3 receptors22,23, with similar positioning of the seven-transmembrane
(TM1–7) bundle and root mean squared deviations of 0.6 – 0.9 Å (Fig. 1a). Subtle
differences between the receptors are observed on the extracellular and intracellular sides
(Fig. 1b,c) corresponding to regions that are least conserved across the muscarinic subtypes
(Extended Data Fig. 2). For example, the M2 receptor differs from the other receptors in the
tilt and position of TM1 and TM7 (Fig. 1a,b). Notably, the M1 receptor was co-crystallized
with a FLAG peptide co-bound on the intracellular side, which makes extensive contacts
with TM6 and ICL3 (Extended Data Fig. 3a,b), and likely contributes to observed
differences in TM5, TM6, and a variable linkage between TM7–helix8 (Extended Data Fig.
3c). The M1-N110Q3.37 mutation has little effect on the M1 structure other than creating a
slight bulge in TM4 due to the loss of a hydrogen bond with S4.53 (Extended Data Fig. 3d).
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More interestingly, the M4 receptor was crystallized with an intact ionic lock (Extended
Data Fig. 3e) a feature uncommonly seen in other GPCRs and not present in the other
muscarinic structures. It is important to note that the observed differences in the intra- and
extracellular sides of the receptor occur in regions that are solvent accessible or are involved
in crystal packing interactions, which could contribute to the observed perturbations between
subtypes; however, none of the crystal packing interactions grossly affect the structure or the
core of the receptor.
Like the inactive M3 receptor, the M1 and M4 receptors were crystallized in complex with
the inverse agonist, tiotropium, and this binding site is buried deep within the
transmembrane core (Fig. 1d). The binding pose of tiotropium and surrounding residues
between these three structures is nearly identical (Fig. 1d-f), which is not surprising given
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the near absolute conservation of residues lining the orthosteric site in the muscarinic family
(Extended Data Fig. 2). However, this high degree of sequence conservation does not
preclude the possibility of differences in tertiary structure with respect to the orthosteric site.
Indeed, one surprising difference is a change in the rotamer of D1123.32 of the M4 receptor
(Fig. 1 f,g); a residue that is conserved throughout the biogenic amine GPCRs and serves as
the counter ion for positively charged neurotransmitters27. This rotameric change points
D1123.32 away from tiotropium and is accompanied by slight movements of Y4397.39 and
Y4437.43, allowing them to form a network of hydrogen bond interactions between D1123.32
and S852.57, W1083.28, Y4397.39 and Y4437.43, which is distinct from the M1, M2 and M3
muscarinic receptor structures (Fig. 1g).
Further comparison of the M1, M3 and M4 tiotropium bound structures with the M2
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receptor, which was crystallized with the structurally similar inverse agonist, QNB, also
reveal considerable differences around residues D3.32, Y7.39 and Y7.43. These three residues
surround the amine group, which is slightly more bulky for QNB than tiotropium (Fig. 1e-
g). Indeed, previous mutagenesis studies28 on the M1 receptor revealed ligand specific
changes in binding affinities of NMS and QNB upon mutation of Y7.39 and Y7.43 to alanine.
For the ligand NMS, which has a structurally similar tropane ring to tiotropium, a 25- and
48-fold loss of binding affinity was observed for the Y7.39 and Y7.43 mutations, respectively,
whereas little effect was observed for QNB. This suggests a potential role for these two

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residues in stabilizing different inactive-state conformations with QNB potentially making

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compensatory interactions unavailable to NMS. The fact that the orthosteric site of the M4
receptor is in some ways closer to the M1 than the M2 subtypes may also allow some
rationalization of the relative subtype selectivity for canonical orthosteric antagonists such as
pirenzepine, which has long been known to have a rank order potency of
M1>M4>M3>M229. We performed induced fit docking (IFD) experiments of the antagonist
into the inactive-state structures of the M1–M4 receptors. The overall poses for pirenzepine
were very similar, with slight variability in the positioning of the methylpiperazine moiety
(Extended Data Fig. 4a). However, there were still distinct differences in the orientation of
residues D3.32 and Y7.39 between the M1, M3 and M4 subtypes versus the M2 receptor, with
D3.32 oriented towards and Y7.39 away from the methylpiperazine group in the M2 IFD
(Extended Data Fig. 4b). These differences should be interpreted with caution, as it is
possible they reflect a restricted sampling in the IFD protocol, and may not be reflective of a
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genuine M2 specific preference. Nevertheless, these results suggest that the differences in
positions of D3.32 and Y7.39, which surround the methylpiperazine group, could contribute to
the marked difference in potency for pirenzepine between the M1 and M2 subtypes29.
Allosteric binding and cooperativity

A comparison of all four solved muscarinic receptor structures illustrates the strikingly high
degree of conservation of the residues comprising the orthosteric site (Fig. 2, green), thus
providing a structural basis for the difficulty in achieving subtype selectivity when targeting
this region. In contrast, muscarinic receptors possess a large extracellular vestibule that
contains residues contributing to an allosteric site. As shown in Fig. 2 (blue), comparison of
these residues reveals a striking divergence between subtypes, due to differences in amino
acid composition (Extended Data Fig. 2) and likely additional tertiary structure changes that
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arise as a consequence of the dynamic nature of the extracellular loop regions. Also shown
in Fig. 2 (yellow) are residues that have been previously suggested to form the “roof” of the
orthosteric site and “floor” of the allosteric site20,30. These “shared” residues show an
intermediate degree of tertiary structure divergence between subtypes when compared to the
orthosteric and allosteric site residues, and are conserved among all five subtypes with the
exception of the M2 receptor where L in ECL2 is replaced by F.
Comparison of the electrostatic surface potential of each receptor (Fig. 3) also reveals
distinct differences in both the shape and charge distribution of the allosteric site and can
explain why some of the best-studied muscarinic receptor allosteric modulators are cationic
compounds31. For example gallamine32, a prototypical negative allosteric modulator of
muscarinic receptors, has a binding potency order of M2 > M1,M4 > M3,M533. The acidic
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EDGE sequence (Fig. 3b) of the M2 receptor has been shown to be important for gallamine
affinity and cooperativity, and indeed, replacement of M1 residues LAGQ with the EDGE
(Fig. 3b) significantly improved gallamine affinity at the M1 receptor33.
Interestingly, inspection of our M4 receptor data also revealed that the precipitant,
polyethylene glycol 300 (PEG 300), is able to occupy the allosteric binding site of the
inactive-state receptor (Extended Data Fig. 5), a finding consistent with the recent structure
of the M3-mT4L receptor26. Surrounding the PEG 300 molecule are residues that form the

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allosteric site from the top regions of TM2, TM3 and TMs 5–7 (Extended Data Fig. 5b).
Furthermore, PEG 300 sits immediately above the aromatic cage composed of Y1133.33,
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Y4166.51, Y4397.39 and Y4437.43 (Extended Data Fig. 5a). These residues have been
implicated in regulating the dissociation of antagonists from the orthosteric binding site34,
and we confirmed the ability of PEG 300 to act as an allosteric modulator in its own right
through its ability to retard the dissociation of [3H]NMS in a concentration-dependent
manner with a calculated apparent affinity of approximately 10 mM for the [3H]NMS-
occupied M4 receptor (Extended Data Fig. 5c,d).
Our finding above illustrates an inherent difficulty in obtaining inactive-state structures with
prototypical negative allosteric modulators bound in the open muscarinic extracellular
vestibule, as PEG 300 is a required precipitant and is present at concentrations of over 1.0
M. However, a recent breakthrough was the solution of the active-state structure of the
related M2 muscarinic receptor bound to a high efficacy agonist, iperoxo, in the absence or
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presence of the PAM, LY2119620, which preferentially bound in a more tightly closed
vestibule that arises in the active-state35. Because the M4 receptor is most closely related to
the M2 subtype, and M4 receptor PAMs are highly pursued as novel therapeutic agents4,36,
we undertook a combined mutagenesis and molecular modelling study to complement our
structural work and gain additional insights into mechanisms governing positive allosteric
modulation at this muscarinic receptor subtype.
We investigated the interaction between the well-characterized PAM, LY203329813-15,20,21,

and the cognate agonist, acetylcholine. Based on the recent structure of the active M2
receptor bound to the LY2033298 congener, LY211962035,37,38, it is likely that such PAMs
bind to an essentially pre-formed closed state of the extracellular vestibule. As such, residues
whose mutation might alter the cooperativity between acetylcholine and LY2033298 fall into
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three general categories: (i) those that make tighter contacts with the ligands in the closed
state than the open state, (ii) those that are immobilized by the binding of either ligand, such
that the entropic cost is paid by the first binding event, (iii) non ligand-contact residues that
alter the free energy of activation of the receptor and thus the open to closed transition. We
chose to focus on residues within and between the extracellular vestibule and orthosteric
sites, which are likely to reflect the first two categories; mutagenesis of non-contact residues
that govern the free energy of receptor transitions are beyond the scope of the current work.
Because prior mutagenesis studies suggested a role for aromatic residues in receptor
interaction with LY2033298, we generated alanine mutations of selected aromatic residues
near the top of the receptor and applied an allosteric ternary complex model to the data
(Methods) to determine the effect of each mutation on the affinity of acetylcholine (KA) or
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LY2033298 (KB) for the free receptor and the magnitude of positive cooperativity (α)
between the two ligands. We also chose to investigate selected (non-aromatic) residues that
line the proximal and distal ends of ECL2, given the important role this region plays in the
binding of modulators to the extracellular vestibule18,39-41. The results of these experiments
are summarized in Supplementary Tables 2-4 and include prior mutagenesis results from our
laboratory for the same set of ligands. To rationalize our findings, we used the recent active
state M2 receptor structure as a template to generate a homology model of the M4 receptor

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bound to acetylcholine and LY2033298, and compared this to our inactive state crystal
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structure (Fig. 4, Extended Data Fig. 6).
The most dramatic effect on the affinity of the PAM was noted upon mutation of W4357.35
at the top of TM7, with a complete loss in LY2033298 binding, similar to our previous
observations21 upon alanine substitution of F186ECL2 (Fig. 5, Extended Data Fig. 7, and
Supplementary Tables 3-4). Alanine mutations of residues Y1133.33, Y4166.51 and Y4397.39,
which form the roof of the orthosteric site led to significant decreases in cooperativity. A
slight increase in modulator affinity and significant decrease in cooperativity was also noted
with mutation of Y892.61, together with our prior identification of residues W1083.28 and
L1093.29 as likely contributors to the PAM binding pocket20. Comparison of our inactive
state structure to the active state model now provides a mechanistic rationale for our
findings, specifically a contraction of the extracellular vestibule that results predominantly in
an inward movement of N4236.58, F186ECL2 and W4357.35 allowing π-stacking interactions
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to occur with the modulator in the active state (Fig. 4b). With regards to the acetylcholine-
binding pocket, there is a contraction of the pocket mediated by an inward movement of the
top of TM6 to accommodate the large difference in size between acetylcholine and
tiotropium resulting in significant movement of residues Y4166.51, N4176.52, W4136.48 and
Y4397.39 (Fig. 4c). Additionally, D1123.32 is reoriented to interact with the choline head-
group of acetylcholine, and is no longer stabilized by the same hydrogen bond network that
is seen in the inactive state (Fig. 1g).
Importantly, mapping of the amino acid residues that significantly affect the cooperativity
between acetylcholine and LY2033298 upon mutation also identified, for the first time, a
network that appears to link the allosteric and orthosteric sites, involving the interface
between TMs 2, 3, 6 and 7, and extending along the top of ECL2 (Fig. 5; orange coloured
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residues); this network is consistent with views of allosteric modulation that propose a
preferred energetic link between orthosteric and allosteric sites42 but, to our knowledge, has
never been directly mapped before in a GPCR. Interestingly, a comparison of the side chain
locations between the inactive M4 structure and active M4 model for residues in the
allosteric network reveals that the majority of residues at the TM2/3/7 interface that
contribute to cooperativity are not predicted to undergo appreciable movement between
states, whereas comparison of residues further away from the interface (F186ECL2, Y4166.51,
N4236.58, and W4357.35) are predicted to move significantly between the two states
(Extended Data Fig. 8). The TM2/3/7 interface, which forms part of the hydrophobic core of
the receptor, may act as a hinge mediating conformational rearrangements in the
extracellular vestibule between the inactive (open extracellular vestibule) and active (closed
extracellular vestibule) states of the receptor. Disruption of this hinge by mutagenesis alters
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the packing interactions within the interface and might change the energetic barrier between
the open and closed conformations of the receptor leading to either an increase or decrease
in PAM cooperativity. Thus, binding of a PAM to the allosteric site might stabilize the
conformation of the allosteric network residues that are otherwise found in a more dynamic
state. Presumably, structures of the inactive state and active M4 model described here
represent the lowest energy conformations, as they were obtained using crystallography, or
are based on the x-ray structures of the active M2 receptor35 (Extended Data Fig. 8).

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Another noteworthy feature of LY2033298 is that it is selective towards the M4 receptor

versus the M1 receptor when tested against acetylcholine15. This difference in selectivity
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could arise either through differential binding affinities of LY2033298 or through a

difference in the cooperativity between LY2033298 and acetylcholine between the two
subtypes (Extended Data Fig. 9).
Conclusions
Muscarinic receptors remain important drug targets, and designing molecules to selectively
target the orthosteric binding site has proved challenging, as highlighted by the lack of
prominent differences between the receptor subtypes. Alongside the previously determined
M2 and M3 structures, the M1 and M4 structures presented here now offer a near complete
view of the inactive state of this important subfamily of GPCRs. Excitingly, comparison of
these structures clearly reveals a divergence in residues lining the allosteric site, highlighting
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the importance of this region for designing selective drugs. Moreover, our enriched
structure-function analysis of the M4 receptor indicates that it is possible to combine crystal
structure and mutagenesis data to uncover new insights into GPCR allosteric modulation,
and our results point to the TM2/3/7 interface as a network for further studies on the
mechanistic basis of allostery at Class A GPCRs. Together with the recent solution of the
inactive M2 and M3 receptors, as well as the active and PAM-bound M2 receptor, our study
has contributed to an emerging picture of mechanisms of allostery at a therapeutically
important receptor family that may facilitate the design of novel agents targeting a variety of
CNS disorders while avoiding peripheral off-target effects.
Methods
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M1 and M4 receptor expression and purification

The human M4 muscarinic receptor gene (cdna.org) was cloned into a modified pFastBac1
vector to give a receptor containing an amino-terminal FLAG epitope tag and a carboxyl–
terminal 8 × histidine tag. Residues 226–389 of ICL3 were removed and replaced by a
minimal Cys-free T4 lysozyme fusion protein26. The human M1 muscarinic receptor gene
was also cloned into the modified pFastBac1 vector, and residues 219-354 of ICL3 were
removed and replaced by a Cys-free T4 lysozyme fusion protein. Both fusion proteins were
expressed using the Bac-to-Bac Baculovirus Expression System (Invitrogen) in Sf9 cells.
Cells were infected at a density of 4.0–5.0 × 106 cells mL-1, treated with 10 μM atropine,
and harvested at 60 hours. Receptor was solubilized and purified in the presence of
tiotropium as previously described for the M322 receptor using Ni-NTA chromatography,
FLAG affinity chromatography, and size exclusion chromatography. The N-terminus of the
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M4 receptor was removed by cleavage with HRV 3C protease at a concentration of 2%

(w/w) during concentration of the receptor prior to size exclusion chromatography (∼2hr at
4 °C). Post size exclusion chromatography, purified receptor was concentrated to 85 AU
(∼50 mg mL-1) and flash frozen in small aliquots using liquid nitrogen.

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Pharmacology of crystallization constructs

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Sf9 cells expressing wild type M4 or M4-mT4L receptor, as described above, were pelleted
and washed with PBS three times for 1 h each to remove any bound atropine. Cells were
resuspended in binding buffer (10 mM HEPES pH 7.5, 100 mM NaCl, and 10 mM MgCl2)
and flash frozen with liquid nitrogen. Saturation binding assays were carried out using
approximately 20,000 cells per well with 9 different concentrations of [3H]NMS in a total
volume of 0.5 mL for 3 h at 37 °C. Competition binding assays with acetylcholine and
tiotropium were carried out in the presence of a fixed concentration of [3H]NMS over 10
different concentrations of ligand for 3 h at 37 °C. Non-specific binding was measured in the
presence of 10 μM atropine, and reactions were harvested by rapid filtration through GF/B
filters. Data were analysed using Prism 6.0d. Similar methods were applied for binding
assays using wild type M1 and M1-T4L, except that [3H]QNB was used as the radioligand.
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Crystallization
Purified M1-T4L•tiotropium and M4-mT4L•tiotropium were crystallized using lipid cubic
phase technology. Each receptor was reconstituted by mixing the protein solution into 10:1
(w/w) monoolein:cholesterol (Sigma) in 1:1.5 parts w/w protein:lipid ratio using the two-
syringe method24. For the M1 receptor, samples of 50 nL (20–40 nL for M4) were spotted
onto 96-well glass plates and overlaid with 800 nL (600 nL for M4) of precipitant solution
for each well using a Gryphon LCP (Art Robbins Instruments). Glass plates were then
sealed using a glass cover film and incubated at 20 °C. Initial crystals for the M1 receptor
formed after 24 hours in conditions containing 33% PEG 300, 100 mM sodium acetate, and
100 mM Bis-Tris Propane (pH 8.0). For the M4 receptor, initial crystals formed after 24
hours in conditions containing 25–40% PEG 300, 50–100 mM EDTA (pH 8.0), and 100 mM
MES (pH 5.5–6.5). M1 and M4 crystals were harvested using mesh grid loops (MiTeGen)
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and stored in liquid nitrogen before use.
Data collection, processing, and structure determination

X-ray diffraction data were collected at the Advanced Photon Source at Argonne National
Laboratories at GM/CA beamline 23ID-D. Crystals were located by initial rastering using an
80 μm by 30 μm beam with five-fold attenuation and 1 s exposure. Regions that contained
strong diffraction were then sub-rastered using a 10 μm collimated beam with five-fold
attenuation. Data were then collected with the 10 μm beam using no attenuation with 1–2
second exposures and 1 degree oscillations. To prevent radiation damage, data were
collected in wedges of 3–10 degrees before moving onto either a different site on the same
crystal or a new crystal. Diffraction data were processed using HKL2000 (M1 receptor) or
XDS46 (M4 receptor) and statistics are summarized in Extended Data Table 1. Both
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structures were solved by molecular replacement using Phaser47. For the M1 receptor, the
inactive M3 structure22 (PDB: 4DAJ) was split into its receptor and T4L components and
used as corresponding search models. The refinement was carried out using Refmac548 with
manual building in coot49. For the M4 receptor, the inactive M2 structure23 (PDB: 3UON)
and the inactive M3-mT4L26 (PDB: 4U15) were used as search models for the receptor and
mT4L fusion domains, respectively. The resulting model was completed by iterative
refinement in Phenix50 and manual building with Coot49. MolProbity51 was used for

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structure validation, and figures were prepared using PyMol52. Final refinement statistics are
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reported in Extended Data Table 1.
Induced fit docking of pirenzepine

The inactive state structures of M1, M2, M3 (PDB 4U15, chain B) and M4 (chain A)
receptors were processed by the protein preparation wizard of the Schrodinger 2014-2
suite53, after deleting the lysozyme insertion region. Missing sidechains were added by
Prime and hydrogens refined by minimization with the OPLS2.1 force field. Binding grids
were defined using the default settings in Glide, centering the grid on the crystallized
orthosteric ligand in each case. The PEG ligand in the extracellular vestibule of M3 and M4
receptors was deleted before grid generation. The ligand, pirenzepine, was treated with
ligprep software to generate initial protonated 3D structures. Compound structures were
docked using the induced fit docking protocol with default settings, which involves the use
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of the OPLS_2005 force field to refine residues around poses docked by Glide SP, followed
by redocking into the generated receptor conformations, also with Glide SP. The poses with
the lowest induced fit score were selected. This scoring function takes into account an
estimate of the protein conformational penalty along with a protein-ligand interaction
docking score.
Molecular modelling of active M4 receptor

A homology model of a human active-state M4 receptor was constructed using the Prime
program implemented in Maestro version 2014.1 from Schrodinger. The crystal structure of
the M2 receptor with an orthosteric and allosteric agonist bound (PDB: 4MQT) was used as
a template to build the M4 model. The M2-M4 sequence alignment generated by Prime
needed no adjustment due to the overall significant sequence homology between the two
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isoforms. The initial M4 receptor model was built with the allosteric ligand (LY2119620)
present in the M2 crystal structure bound in the M4 allosteric site and with iperoxo bound in
the orthosteric site (as also present in the M2 structure). The binding mode of LY2119620 in
M4 was used as a guide to manually dock LY2033298 into the M4 allosteric binding site. In
addition, iperoxo from the M4 model was manually modified into acetylcholine (ACh). The
M4-ACh-LY2033298 complex was then subjected to 500 steps of energy minimization
(MacroModel implemented in Maestro 2014.1 from Schrodinger53) in order to optimize key
interactions in the binding sites. The resulting model of ACh and LY2033298 bound to M4
was used in subsequent modelling studies described in this paper.
Molecular modelling of active M1 receptor

The active state of the M1 receptor was modelled based on the active state structure of M2
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bound to iperoxo (PDB: 4MQT), using the automated protein structure homology modelling
web server Swiss-Model54,55. The nanobody structure was removed and the resulting
coordinates were used as a template to model the M1 primary sequence without intracellular
loop 3 residues (resi: 213-240). The model was built using Promod-II, minimized by
steepest descent energy minimization using a GROMOS96 force field and the quality was
assessed by the QMEAN scoring function. ACh and LY2033298 were docked in the M1
homology model using Swiss-Dock56, using steric and chemical considerations such as
shape, charge complimentary, and keeping the protein structure constant. The top scoring

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clusters were evaluated manually based on chemical and steric considerations to pick the
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favourable pose. Due to static docking, the top four ACh poses did not affect the docking
results for LY2033298. For ACh, the selected pose is in the trans conformation similar to the
M4•ACh•LY2033298 model. Finally, the structures with the ligand were energy minimized
using Chimera with standard Steepest Descent and Conjugate Gradient steps.
Receptor mutagenesis and generation of cell lines

DNA encoding the human M4 mAChR with a triple HA20 or cmyc21 tag at its amino
terminus was subjected to QuikChange site-directed mutagenesis (Stratagene, La Jolla, CA)
to generate M4 mAChR sequences with the desired amino acid substitutions. DNA
constructs in pEF5/frt/V5 (Invitrogen) were stably expressed in FlpIn™ CHO cells, which
were maintained in high-glucose Dulbecco's modified Eagle's medium containing 10% FBS,
16mM HEPES, and 400 μg/ml hygromycin B.
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Radioligand binding assays

Cell membranes were prepared as described previously14,57. [3H]QNB affinity (KA) at the
M4 WT receptor and mutants was determined by saturation binding assays, performed by
incubating varying concentrations of [3H]QNB with 10–100 μg of membranes at 37 °C for 1
h, in a final volume of 0.5-1 ml binding buffer (20 mM HEPES, 100 mM NaCl, and 10mM
MgCl2 at pH 7.4).
Radioligand inhibition binding assays were performed by co-incubating 10–100 μg of

membranes with a KA concentration of [3H]QNB (determined in saturation assays,
Supplementary Table 2) and varying concentrations of the non-radiolabeled test compound
in 0.5–1 ml binding buffer in the presence of the guanine nucleotide, GppNHp (100 μM),
which was used to promote receptor-G protein uncoupling. These experiments determined
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the concentration of ACh that inhibited 20% [3H]QNB binding, defined as the IC20
concentration of ACh, which was used in subsequent interaction studies between [3H]QNB,
ACh, and LY2033298. These experiments were performed by co-incubating 10–100 μg of
membranes, an IC20 concentration of ACh, and a KA concentration of [3H]QNB with
increasing concentrations of LY2033298 in binding buffer containing GppNHp (100 μM).
The reaction was left to reach equilibrium for 3 h at 37 °C. For all experiments, nonspecific
binding was defined in the presence of 10 μM atropine, total binding was determined in the
absence of the test ligand, and vehicle effects were determined with 0.1% DMSO. The
assays were terminated by vacuum filtration through GF-B glass fibre filters, which were
washed three times with ice-cold 0.9% NaCl. [3H]QNB radioactivity was measured using a
Packard 1600 TR liquid scintillation beta counter. Due to a lack [3H]QNB binding, affinity
data for W164A4.57 were determined from functional pERK1/2 experiments performed as
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previously described20,21.
Data Analysis
Data were analysed using Prism (GraphPad, San Diego, CA). For radioligand saturation
binding, nonspecific and total binding data were analysed as described previously58.
Inhibition binding curves between [3H]QNB and ACh were fitted to a one-site binding

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model58. Interaction experiments between [3H]QNB, ACh and LY2033298 were fitted to the
following allosteric ternary complex model20,21,59:
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where Y is the specific radioligand binding, Bmax is the total number of receptors, [A], [B],
and [I] are the concentrations of radioligand, allosteric modulator, and unlabeled orthosteric
ligand, respectively, KA, KB, and KI are the equilibrium dissociation constants of the
radioligand, allosteric modulator, and unlabelled orthosteric ligand, respectively, and α′ and
α are the cooperativity factors between allosteric modulator and the radioligand or unlabeled
orthosteric ligand, respectively. The value of α′ was taken as 1 when the binding of
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[3H]QNB changed by less than 10% at 10-5 M LY2033298 relative to zero LY2033298, and
was fixed as such for all analyses. Otherwise, the value of α′ was determined using a global
fit to the allosteric ternary complex model. Statistical differences between pharmacological
parameters at WT versus mutant M4 receptors were determined by oneway ANOVA with
Dunnett's post hoc test, where p<0.01 was considered statistically significant.
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Extended Data
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Extended Data Figure 1. Crystallization construct design, purification and crystallization

a-b, Crystallization constructs used for the (a) M1 receptor and (b) M4 receptor. All
constructs contain an N-terminal FLAG epitope (coloured yellow), C-terminal histidine tag
(coloured purple), and a T4L lysozyme fusion protein (coloured red). For the M4 receptor,
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initial constructs diffracted out to 4 Å, however, the diffraction data appeared to suffer from
a lattice translocation disorder and were unsolvable. The final crystallization construct
contained a shortened N-terminus with a HRV 3C cleavage site shown in dark green and a
minimal T4 lysozyme fusion (mT4L)26, shown in red. c, Snake-plot diagram of the best
diffracting M4 mAChR construct coloured according to (a). Residues coloured blue are
single point mutations from this study, and residues coloured orange are previously studied
mutations20,21. d, Size exclusion chromatography trace of purified monodispersed M4-

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mT4L bound to tiotropium. e, Crystals of M4-mT4L obtained in lipidic cubic phase and
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observed under circularly polarized light.

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Extended Data Figure 2. Sequence conservation across the muscarinic receptor subfamily
a-c, The sequence alignment of the human M1 – M5 receptors (d) was determined on the
ConSurf server to calculate amino acid conservation scores60,61. Conservation scores for
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each residue were mapped62,63 onto the M4 structure and coloured as a gradient from blue
(highly conserved) to red (least conserved) with views from the (b) extracellular and (c)
intracellular sides. The radius of the cartoon increases as the residues at each position
become more poorly conserved. Tiotropium and PEG 300 from the M4 structure are shown
as spheres and coloured with carbon in white, oxygen in red, nitrogen in blue, and sulphur in
yellow. d, Amino acid sequences of the human M1–M5 receptors were aligned using the
ClustalW2 server64. Alpha helical regions are shown as blue boxes as determined by the

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consensus of the M1–M4 structures. The most conserved residue in each TM (X.50) is in
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bold lettering. Regions of the N-terminus, C-terminus and ICL3 regions are removed for
space and clarity. Insertion points of the T4 lysozyme fusion proteins between TM5 and
TM6 are underlined with bold lettering. Residues from the orthosteric binding-site are
highlighted in red and allosteric binding-site residues in blue. Residues that contribute to
both sites are coloured in yellow.
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Extended Data Figure 3. Distinct structural features for the M1 and M4 receptors
The receptors shown are aligned and coloured as in Figure 1. a,b, The M1 receptor was
crystallized with the FLAG peptide (DYKDDDD; coloured cyan sticks) co-bound on the
cytoplasmic surface. Residues of the M1 receptor within 4 Å of the FLAG peptide are shown
as magenta coloured sticks with views from the (a) membrane and (b) cytoplasmic side. c,
The linkage between TM7 and helix 8 of the M1 receptor undergoes a bend starting with a
change in rotamer of residue Y7.53, which may be a result of perturbations in TM6 due to the
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FLAG peptide. d, The M1-N110Q3.37 mutation causes a slight bulge in TM4 due to the loss
of a hydrogen bond with S4.53. e, Chain B of the M4 receptor has an intact ionic lock with
R3.50 forming hydrogen bonds with T6.34 and E6.30.

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Extended Data Figure 4. Induced fit docking of pirenzepine into the M1–M4 structures
The receptors shown are aligned and coloured as in Figure 1. a, Superposition of the poses
of pirenzepine from the IFD experiments. b, Comparison of the pirenzepine poses for the
M1 and M4 receptor with residues that contribute to the orthosteric site of the M1–M4
receptors (several residues omitted for clarity).
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Extended Data Figure 5. PEG 300 occupies the allosteric binding site of the inactive M4 receptor
a, The cross section of the solvent accessible surface area of the M4 receptor is coloured
blue. Tiotropium and PEG 300 are shown as spheres with respective carbons coloured white
and peach. The aromatic cage of covering tiotropium is highlighted in orange b, View from
the extracellular side with residues that contact PEG 300 shown as spheres. c, Dissociation
kinetics of [3H]NMS in the presence of PEG 300. [3H]NMS was incubated with M4-mT4L
membranes at 37 °C for 3 h, followed by addition of 10 μM atropine ± PEG 300 at the
indicated concentrations and time points. Representative data from three experiments,
performed in duplicate, fit to a one phase exponential decay are shown. d, PEG 300 has an
apparent binding affinity for the NMS-occupied receptor of approximately 10 mM (LogIC50
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= -1.95±0.02).

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Extended Data Figure 6. Ligand interaction diagrams for the M4 receptor

a,b The molecular interactions between the (a) orthosteric and (b) allosteric binding sites are
shown by the program MOE65 for the inactive (M4•tiotropium structure) and active states
(M4• acetylcholine •LY2033298 model). Residues with a bold outline were selected in this
study or others20,21 as single point mutations.
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Extended Data Figure 7. Identification of key residues that govern LY2033298 affinity and
binding cooperativity with acetylcholine (ACh) at the M4 receptor
Competition between a fixed concentration of [3H]QNB and increasing concentrations of
ACh (black circles), LY2033298 (blue triangles) or LY2033298 in the presence of an IC20
concentration of ACh (red squares) are shown. The curves drawn through the points
represent the best global fit of an extended ternary complex model. For data sets where the
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binding of [3H]QNB changed by less than 10% at 10-5 M LY2033298 relative to zero
LY2033298, the value of α′ was fixed to 1 (connecting line shown). Data points represent
the mean ± s.e.m. of at least three experiments performed in triplicate.

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Extended Data Figure 8. Comparison of cooperativity network residues between the inactive and
active-states
a,b, Chemical structures of (a) the M4 ligands used in this study and (b) the M2 ligands
from the active-state crystal structures (PDB: 4MQT and 4MQS). c-f, Mapping of the
allosteric network onto the (c,d) inactive M4 structure (blue residues), M4 active-state model
(orange residues) and (e,f) the inactive (yellow residues) and active-state M2 structures
(magenta and green residues) with views from the (c,e) membrane or (d,f) extracellular
surface. Ligands are coloured according to element: carbon, cyan; oxygen, red; nitrogen,
blue; sulphur, yellow; chlorine, green.
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Extended Data Figure 9. LY2033298 binding to active-state M1 and M4 models

Comparison of active-state M1 (green) and M4 (orange) models bound to LY2033298 and
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acetylcholine, with acetylcholine and LY2033298 shown as sticks and coloured according to
element: carbon, white; oxygen, red; nitrogen, blue; sulphur, yellow; chlorine, green. Several
residues surrounding LY2033298 are shown as sticks and coloured according to receptor.
M4-N4236.58 is predicted to undergo significant movement between the inactive and active
states to form a hydrogen bond with the methoxy group of LY2033298. In the M1 receptor
this residue is a serine (S3886.58) and is unable to form a similar hydrogen bond. However,
mutation of N4236.58 to alanine at the M4 receptor results in no loss of LY2033298 affinity,
but does result in a 6-fold loss in cooperativity between acetylcholine and LY2033298
(Supplementary Table 3). This is suggestive of selectivity being derived through
cooperativity as a possible mechanism between the M1 and M4 receptors. Additional
determinants for M1 and M4 selectivity could also arise through differences in residues on
TMs 2 and 7, which contribute to (I932.65) or sit proximal to (D4327.32 and S4367.36) the
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allosteric network.

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Extended Data Table 1

Data collection and refinement statistics
Data collection* M1-T4L•tiotropium M4-mT4L•tiotropium

Beamline GM/CA GM/CA
23-ID-D 23–ID-D
Number of crystals 8 64
Space group P 21 21 21 P 1 21 1
Cell dimensions
a, b, c (Å) 58.0, 72.2, 175.7 48.5, 172.0, 60.7
α, β, γ (°) 90, 90, 90 90, 94.4, 90
Resolution (Å) 30.00–2.70 (2.80–2.70) 28.99–2.60 (2.69–2.60)
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Rmerge (%) 15.5 (83.6) 21.3 (94.7)

<I/σ> 8.7 (1.9) 8.2 (1.5)
CC1/2 (%) N.D.† 99.0 (54.5)
Completeness 97.1 (98.2) 99.7 (97.9)
Multiplicity 4.3 (4.2) 13.1 (4.9)
Refinement
Resolution (Å) 30.00–2.70 28.53–2.60
No. of reflections working / test set 19,223 / 1,011 30,299 / 2,146
Rwork/Rfree (%) 22.8 / 27.5 22.7 / 24.0
No. of atoms (Chain A / Chain B)
Protein 3439 3058 / 3115
Ligands 210 176 / 167
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Average B-factors (Chain A / Chain B; Å2) 73.6 74.0 / 70.5

Receptor 62.5 63.9 / 64.6
T4 lysozyme 64.4 99.5 / 85.3
Tiotropium 66.5 47.4 / 47.9
Allosteric site PEG 300 – 74.9 / 81.6
Waters 58.1 53.7 / 46.7
Other ligands 74.8 80.8 / 78.4
RMS deviation from ideality
Bond length (Å) 0.009 0.002
Bond angles (°) 1.39 0.61
Ramachandran statistics‡
Favored regions (%) 96.2 99.2
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Allowed regions (%) 3.6 0.8

Outliers (%) 0.2 0.0
*
Highest shell statistics in parenthesis
†
N.D., Not determined, because the structure was solved before CC1/2 values were introduced71.
‡
As calculated by Molprobity

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Supplementary Material
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Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
This work was funded by Program Grant APP1055134 of the National Health and Medical Research Council
(NHMRC) of Australia (A.C., P.M.S.). Portions of this work were supported by a Lilly Research Award Program
grant. W.I.W. and B.K.K. were supported by the Mathers Foundation. A.C. is a Senior Principal, and P.M.S. a
Principal, Research Fellow of the NHMRC. GM/CA @ APS has been funded in whole or in part with Federal funds
from the National Cancer Institute (Y1-CO-1020) and the National Institute of General Medical Science (Y1-
GM-1104). Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Basic Energy
Sciences, Office of Science, under contract No. W-31-109-ENG-38.
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Figure 1. Structural comparison of the M1–M4 receptors

a, The overall view of the muscarinic structures is shown as cartoons aligned to the M3
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receptor, with the M1 coloured in green, M2 in yellow (PDB: 3UON), M3 (PDB: 4U15,
chain A) in orange and M4 (chain A) in blue. Root mean squared deviations for the
alignment (excluding T4L fusions) of M1, M2 and M4 versus the M3 receptor are 0.86 Å,
0.81 Å, and 0.62 Å, respectively. The ligand, tiotropium, for the M4 receptor is shown as
sticks and coloured according to element: carbon, light blue; oxygen, red; nitrogen, dark
blue; sulphur, yellow. b,c Comparison of the views from (b) the extracellular side and (c) the
intracellular side d, M1 and M4 residues involved in tiotropium binding are shown as sticks
(several residues are omitted for clarity). The black dashed line indicates a bidentate
hydrogen bond between N6.52 and tiotropium. e, Superposition of tiotropium from the M1,
M3 and M4 structures and QNB from the M2 structure. The arrow indicates the main
structural difference between tiotropium and QNB. f, Comparison of the orthosteric binding
site of the M1–M4 receptors with orthosteric site residues shown as sticks. g, The rotameric
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change of D1123.32 is stabilized by a network of hydrogen bonds.

Thal et al. Page 27
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Figure 2. Comparison of orthosteric and allosteric binding site residues across the M1–M4
receptors
a,b The M1 – M4 receptors are aligned to the M3 muscarinic receptor (PDB: 4U15, chain
A) and are shown as grey coloured cartoons with views from the (a) membrane and (b)
extracellular side. Residues in ECL2 are numbered relative to the position of the disulphide-
bonded cysteine. Carbon atoms are coloured by site with the orthosteric residues in green,
dark blue for allosteric conserved residues, light blue for allosteric nonconserved residues,
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and yellow for residues that contribute to both sites. Oxygen atoms are coloured red,
nitrogen blue, and sulphur yellow. Non-conserved residues are labelled according to either
the most common residue or by the residue at the M4 receptor. Residues K6.62 (M1), D6.63
(M1) and S6.63 (M3) are shown as alanine due to a lack of electron density on the side
chains.
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Thal et al. Page 28
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Figure 3. Electrostatic and surface properties of the different muscarinic receptor structures
a, Electrostatic potentials (+ 5 kT/e in blue and − 5 kT/e in red) mapped on the surfaces of
the M1 – M4 receptor structures calculated at pH 7.0 using the programs PDB2PQR43,44
and APBS45. b, Residues in ECL2 that make up the EDGE sequence at the M2 receptor and
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the corresponding regions at the other subtypes are shown as sticks. Negatively charged
residues in the sequence alignment are coloured red.
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Thal et al. Page 29
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Figure 4. Model of an active M4 receptor bound to acetylcholine and LY2033298

a, Comparison of the M4•tiotropium receptor structure (blue) versus an active-state model
(orange) bound to acetylcholine and LY2033298 based on the active
M2•iperoxo•LY2119620 structure (PDB: 4MQT) as viewed from the membrane. The active
M4 model was aligned to the M4•tiotropium structure (chain A, excluding T4L) with a root
mean squared deviation of 0.9 Å. b,c Cross-sectional views of the (b) allosteric site and (c)
orthosteric site as viewed from the extracellular side with a 90° rotation relative to the
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membrane from (a). Residues surrounding each site are shown as sticks (several removed for
clarity). Acetylcholine and LY2033298 are shown as (a) spheres or (b,c) sticks and coloured
according to element: carbon, cyan; oxygen, red; nitrogen, blue; sulphur, yellow; chlorine,
green. Acetylcholine is in the trans conformation and aligns in a similar pose to iperoxo (c,
transparent yellow sticks).

Thal et al. Page 30
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Figure 5. A cooperativity network at the M4 receptor

a, Changes in either LY2033298 binding affinity (ΔpKB, coloured black) or cooperativity
(ΔLog α, coloured orange) relative to wild-type M4 are shown for each mutation. Mutations
that differ significantly from wild type are indicated by asterisks. Cooperativity and binding
values for F186+1 and W4357.35 were not determined due to a lack of LY2033298 binding
(see Supplementary Tables 2-4). b,c Residues from (a) were mapped onto the M4 active-
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state model and coloured as orange sticks with translucent spheres with views from (b) the
membrane and (c) the extracellular side. LY2033298 and acetylcholine are shown as sticks
and coloured the same as in Figure 4.

Astrocytic β2-adrenergic receptors mediate
hippocampal long-term memory consolidation
Virginia Gaoa,b, Akinobu Suzukib,1, Pierre J. Magistrettic,d, Sylvain Lengacherd,e, Gabriella Polloninia,
Michael Q. Steinmana, and Cristina M. Alberinia,2
a
Center for Neural Science, New York University, New York, NY 10003; bFriedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY
10029; cDivision of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology, Thuwal 23955-6900,
Kingdom of Saudi Arabia; dBrain Mind Institute, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland; and
e
Department of Psychiatry, Centre for Psychiatric Neuroscience, Lausanne University Hospital, 1008 Lausanne-Prilly, Switzerland
Edited by James L. McGaugh, University of California, Irvine, CA, and approved June 7, 2016 (received for review March 28, 2016)
Emotionally relevant experiences form strong and long-lasting neurons and therefore have been studied largely in neuronal/synaptic
memories by critically engaging the stress hormone/neurotransmitter models (reviewed in ref. 17). However, in addition to being
noradrenaline, which mediates and modulates the consolidation of expressed in pre- and postsynaptic compartments of neurons,
these memories. Noradrenaline acts through adrenergic receptors βARs are also found in other cell types, particularly in astrocytes
(ARs), of which β2-adrenergic receptors (βARs) are of particular impor- (18–21). More specifically, it has been suggested that β2ARs in
tance. The differential anatomical and cellular distribution of βAR sub- the nervous system are expressed predominantly in glia (18, 21–
types in the brain suggests that they play distinct roles in memory 23), whereas β1ARs are found primarily in neurons, at synaptic
processing, although much about their specific contributions and mech- junctions (18, 22, 23). This suggestion raises the question of
anisms of action remains to be understood. Here we show that astro- which βAR subtype (β1AR or β2AR) and which βAR-expressing
cytic rather than neuronal β2ARs in the hippocampus play a key role in cells mediate hippocampal memory consolidation.
the consolidation of a fear-based contextual memory. These hippocam- NA activates glycogenolysis and subsequent lactate release
pal β2ARs, but not β1ARs, are coupled to the training-dependent release (24), a process known to occur mainly in astrocytes and not in
of lactate from astrocytes, which is necessary for long-term memory neurons; in fact, glycogen is stored almost exclusively in astro-
formation and for underlying molecular changes. This key metabolic cytes (25, 26). Glycogenolysis and its activation by NA are critical
role of astrocytic β2ARs may represent a novel target mechanism for for memory consolidation in chicks (27). Furthermore, in the rat
stress-related psychopathologies and neurodegeneration. hippocampus, glycogenolysis, which results in lactate increase
and astrocyte–neuron lactate transport, mediates memory con-
astrocyte | memory | hippocampus | β-adrenergic receptor | lactate solidation (28, 29), in vivo hippocampal long-term potentiation,
and the induction of learning-dependent molecular changes, in-
cluding activity-regulated cytoskeletal protein (Arc) and phos-
E motional arousal and the stress hormone noradrenaline (NA)
mediate and modulate memory consolidation, the process re-
quired to stabilize long-term memory (1). NA contributes to this
phorylation of cAMP response element-binding protein at serine
133 (pCREB) and cofilin (p-cofilin) (29). Using the hippocampus-
effect through the activation of α- and β-adrenergic receptors dependent task inhibitory avoidance (IA), here we asked which
(βARs) in brain regions such as the amygdala and the hippocampus, astrocytic and/or neuronal β1ARs and/or β2ARs in the hippocam-
which are critical for encoding and consolidating memories (2). Of pus mediate memory consolidation.
particular interest is the action of NA through βARs because
blockers of these receptors, such as propranolol, have been reported Significance
to interfere with memory consolidation and strengthening (3–5)
and have been suggested as potential therapeutics to treat anxiety Experiences are remembered long-term when these memories are
disorders, including panic disorder (6) and posttraumatic stress formed in a state of arousal and heightened emotion. The arousal-
disorder (7). All three subtypes of βARs—β1, β2, and β3—are induced release of noradrenaline is critical for modulating
present in the central nervous system with distinct distributions (8): consolidation, the process that establishes long-term memory. Al-
In the rat hippocampus, a region required for the consolidation of though the effects of pharmacological manipulation of adrenergic
explicit/episodic memories, β1ARs predominate, and their pattern signaling on memory stability are already being investigated in
of distribution is different from that of β2ARs and β3ARs (9–11), the clinical setting, how adrenergic receptors mediate long-term
suggesting possible distinct roles for these receptor subtypes. memory consolidation remains unclear. This study reports a
Pharmacological investigations using agonists (e.g., NA itself) and previously unidentified mechanism with important translational
antagonists (e.g., propranolol) that bind to both β1ARs and β2ARs implications: The noradrenergic receptors that in the hippocam-
have suggested βARs have a similar role in memory encoding, pus mediate memory consolidation are β2-adrenergic receptors
modulation, and retrieval in humans and rodents (3, 5, 12). Studies (β2ARs) expressed in astrocytes. These receptors are necessary for
on animal models lacking β1ARs or treated with selective β1AR the learning-evoked release of lactate from astrocytes, which
agonists or antagonists revealed the critical role of this adrenergic then is required to support the neuronal molecular changes es-
sential for long-term memory formation.
receptor subtype in both synaptic plasticity and memory formation,
with particular emphasis on retrieval (12–14). Genetic deletion of Author contributions: V.G., A.S., and C.M.A. designed research; V.G., A.S., S.L., G.P., and
β2ARs results in impaired memory modulation by stress or cortico- M.Q.S. performed research; P.J.M. and S.L. contributed new reagents/analytic tools; V.G.,
sterone and impaired hippocampal plasticity, in agreement with a A.S., G.P., and C.M.A. analyzed data; and V.G., P.J.M., and C.M.A. wrote the paper.
Downloaded at Indonesia: PNAS Sponsored on November 10, 2020
pharmacologically established role of β2ARs in amygdala-dependent The authors declare no conflict of interest.
memory modulation and its effect on hippocampal function and in This article is a PNAS Direct Submission.
prefrontal cortex (2, 15, 16). However, little is known about the 1
Present address: Department of Biochemistry, Graduate School of Medicine and Phar-
role of hippocampal β2ARs in hippocampus-dependent long- maceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan.
term memory formation and processing. 2
To whom correspondence should be addressed. Email: ca60@nyu.edu.
In general, the functional contributions of βARs to memory This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
processes have been thought to result mainly from their effect on 1073/pnas.1605063113/-/DCSupplemental.
8526–8531 | PNAS | July 26, 2016 | vol. 113 | no. 30 www.pnas.org/cgi/doi/10.1073/pnas.1605063113

Results not at 7 d after training (P < 0.0001; two-way repeated-measures
βARs Mediate Memory Consolidation via Astrocytic Lactate. A bilateral ANOVA followed by Bonferroni’s post hoc tests) (Fig. 1B). To-
injection of the β1+β2AR antagonist propranolol into the rat dorsal gether, these results indicate that the critical role of β1ARs and/or
hippocampus (dHC) 15 min before training had no effect on short- β2ARs in memory consolidation is likely dependent on lactate and
term memory tested 1 h after training (Fig. 1A) but significantly hence on astrocytic mechanisms.
impaired long-term memory tested 1 d and 7 d after training (Fig. Propranolol injected bilaterally into the dHC 15 min before
1B), suggesting that hippocampal β1ARs and/or β2ARs mediate IA training also blocked the learning-dependent increase of molecular
memory consolidation. Given that NA promotes glycogenolysis and changes known to underlie synaptic plasticity and memory formation;
lactate release from astrocytes (30), which are necessary for memory these included the immediate early gene Arc 30 min after training
consolidation (29), we tested whether the effect of propranolol (Fig. 1C), and phosphorylation of CREB (Fig. 1D) and of calcium
on memory consolidation is linked to astrocytic lactate release. We calmodulin kinase II α (CaMKIIα) (Fig. 1E) 20 h after training, time
points at which these molecular changes have been previously
found that coadministration of L-lactate with propranolol fully and
established as learning dependent (29, 31). Lactate coadministration
persistently rescued memory impairment. In contrast, an equicaloric
rescued all these molecular impairments (P < 0.01; one-way
concentration of D-glucose had no effect. A threefold higher ANOVA followed by Newman–Keuls post hoc test). The levels
concentration of glucose rescued memory impairment at 1 d but of total CREB and CaMKIIα remained unchanged (P > 0.05)
(Fig. S1 and Table S3). Hence, βARs in the hippocampus engage
astrocytic mechanisms to support memory formation.
A IA Training
Test 1 B IA Training
Test 1 Test 2 β2ARs, but Not β1ARs, Mediate the Learning-Dependent Lactate
15 min 1h 15 min 1d 6d Increase and Long-Term Memory Formation. Next we sought to
Inject dHC Inject dHC
** *** dissociate the roles of the βAR subtypes, β1AR and β2AR, in
*** *** regulating training-induced astrocytic lactate release and mem-
****** ory consolidation. In agreement with our previous finding (29),
Mean latency (s)
Mean latency (s)
*** *
400 400
300 300
we found that training leads to a significant increase in lactate
200
levels in the dHC as measured by microdialysis; this increase
200
lasted for more than 60 min and returned to baseline by ∼90 min.
100 100
7 7 11 13 8 7 9 11 13 8 7 9
However, this lactate increase was completely abolished by a
0
Test 1
0
Test 1 Test 2 systemic (i.p.) injection of propranolol or of the β2AR-selective
Trained - Vehicle Trained - Propranolol + D-Glucose antagonist ICI 118,551, but not the β1AR-selective antagonist
Trained - Propranolol Trained - Propranolol + 3x D-Glucose betaxolol, given 30 min before training (P < 0.0001; two-way
Trained - Propranolol + L-Lactate ANOVA followed by Bonferroni post hoc tests) (Fig. 2A). Com-
pared with vehicle-injected control rats, propranolol- and ICI
IA Training IA Training 118,551-injected rats, but not betaxolol-injected rats, had signifi-
Euthanasia Euthanasia
cantly impaired long-term memory when tested 1 d after training
15 min 30 m 15 min 20 h
(P < 0.0001; one-way ANOVA followed by Bonferroni post hoc
Inject dHC Inject dHC
tests) (Fig. 2A). The efficacy of betaxolol was confirmed in parallel
C kDa D kDa E kDa
by reproducing the previously established effect of this compound
50 pCREB
50
pCaMKIIα 50 on retrieval of contextual fear conditioning (CFC) (Fig. S2 and
Arc
37
37 Table S4) (12). In addition, as with systemic injections, bilateral
50 50 50 injection of ICI 118,551 into the dHC 15 min before training
actin actin actin 37 impaired long-term memory when tested 1 d and 7 d after training,
37 37
whereas injection of betaxolol had no effect (P < 0.001; two-way

250 * * *
300 250 repeated-measures ANOVA followed by Bonferroni post hoc tests)
% of Naive-Vehicle
% of Naive-Vehicle
% of Naive-Vehicle
* * * * * *
200 200 (Fig. 2B). Following the 7-d test, rats received a reminder footshock
200 (RS)—the unconditioned stimulus in a different context—and
150 150
100 100 testing 1 d later showed that the memory deficit persisted, sug-
100
50 50 gesting that the amnesia was likely caused by impaired consolidation
8 8 6 9 7 9 8 5 9 5 9 10 6 10 6 rather than by inhibition of memory expression (P < 0.01; Student’s
0 0 0
t test) (Fig. 2B). Hence, both the IA-induced increase in hippo-
campal lactate and long-term memory consolidation require β2ARs
Naïve - Vehicle Trained - Propranolol but not β1ARs. We therefore focused our subsequent investigations
Trained - Vehicle
Trained - L-Lactate
Trained - Propranolol + L-Lactate
on β2ARs.
Fig. 1. L-lactate rescues the impairment of long-term IA memory and un- Astrocytic, Not Neuronal, Hippocampal β2ARs Mediate Long-Term
derlying molecular mechanisms produced by propranolol. Memory retention is Memory Formation. The expression and distribution of β2ARs on
expressed as mean latency ± SEM (in seconds). (A) Short-term memory tested different cell types is not clear; although some studies suggest that
β2ARs are expressed mainly in glia (18, 21–23), others report that
NEUROSCIENCE
1 h after training following a bilateral dHC injection of propranolol given

15 min before training (n = 7). (B) Long-term memory tested 1 d after training they are found on both astrocytes and neurons (20). To determine
(test 1) and 6 d later (test 2) following a bilateral dHC injection of vehicle or the selective functional contribution of astrocytic and/or neuronal
propranolol, in the presence or absence of L-lactate, or of an equicaloric β2AR in long-term memory formation, we used cell-specific virus-
concentration of D-glucose, or of a three times higher concentration (3×) of
mediated knockdown of the receptors. The astrocyte-specific pro-
D-glucose, given 15 min before training (n = 7–13). (C–E) Densitometric
Western blot analysis and representative images for Arc (C), pCREB (D), and
moter glial fibrillary acidic protein (short gfaABC1D) was packaged
into adeno-associated virus 9 (AAV9), and the neuron-specific
pCaMKIIα (E) in dHC extracts from untrained (naive) rats injected with vehicle
and rats injected 15 min before training with vehicle, L-lactate, propranolol, or human synapsin (hSyn) promoter was packaged in AAVDJ;
propranolol + L-lactate and killed 30 min (for Arc) or 20 h (for pCREB and both were tagged with eGFP and injected into the dHC. Two
pCaMKIIα) after training. Data are expressed as mean protein percentage ± weeks after injection, histochemical staining revealed that the
SEM of mean values (100%) in vehicle-injected naive rats. Each sample protein expressed fluorescence of eGFP was distributed throughout the
densitometric value was normalized to that of relative actin stained on the dHC (Fig. 3A). The AAV9-short gfaABC1D-eGFP expression
same blot (n = 5–10). *P < 0.05; **P < 0.01; ***P < 0.001. Numeric values and was selectively astrocytic, as revealed by its colocalization with
detailed statistical analyses are reported in Tables S1 and S2. the endogenous astrocytic marker GFAP but not with the
Gao et al. PNAS | July 26, 2016 | vol. 113 | no. 30 | 8527
A Inject i.p. IA Training
to assess the expression of β2AR or in the presence of the β2AR
Test inhibitor ICI 118,551 to assess the expression of β1AR. Using this
30 min
Microdialysis 1d
method, we quantified the distribution of β1AR and β2AR binding
20 min 90 min and confirmed the previously described differential distributions of
*** these receptors in the hippocampus (Fig. 4A) (10). We observed
*** ***
*
350
that β2AR binding occurs in the lacunosum moleculare and the
L-Lactate concentration
200 *** 300

*** ** ** stratum oriens subregions (Fig. 4B), which are enriched in astro-
in % of baseline
Mean latency (s)

* ** 250 cytes rather than neurons (Fig. 4C), whereas β1AR binding was
150 200 Naive diffused throughout the hippocampus (Fig. 4B).
150 Vehicle Compared with relative Scr controls, the infection of both gfa-Sh
100 100 Propranolol and hSyn-Sh resulted in a significant decrease in β2AR binding
50 Betaxolol without affecting the binding levels of β1AR (P < 0.001 Student’s
0
7 7 7 6 ICI 118,551 t test) (Fig. 5 A and B), providing evidence for the specific
Test
10
20
30
40
50
60
70
80
90
0
0
-1
Minutes following training
B IA Training RS
A Bregma: -2.1 mm -3.0 mm -4.4 mm
Test 1 Test 2 Test 3
15 min 1d 6d 1d 1d
Inject dHC
** ** **
Mean latency (s)
400 1mm
300 B CA1
C CA
1
200 Vehicle eGFP eGFP CA3

DG
Betaxolol GFAP NeuN DG

100 CA
3
ICI 118,551 1mm 1mm

14 7 6 14 7 6 8 6
0
Test 1 Test 2 Test 3 CA1 CA3 DG CA1 CA3 DG
Fig. 2. Propranolol and ICI 118,551, but not betaxolol, block both training-
eGFP
eGFP
evoked L-lactate increase in the dHC and long-term IA memory formation.
(A, Left) The relative concentration of L-lactate in the dHC was measured by
microdialysis in freely moving rats injected i.p. with vehicle, propranolol,
betaxolol, or ICI 118,551 30 min before training. Untrained (naive) rats in-
GFAP
NeuN
jected with vehicle were used as controls. Baseline data were collected for
20 min before training, and data collection continued for 90 min after training.
Data are expressed as the percent of baseline L-lactate concentration ± SEM
(mean of the first two samples set at 100%; n = 4–7). (Right) Long-term
merge
merge
memory retention of the rats tested in microdialysis expressed as mean la-
tency (in seconds) ± SEM (n = 6–7). (B) Long-term memory tested 1 d (test 1) 100 μm 100 μm
and 7 d (test 2) after training and following an RS (test 3) in rats that re-
ceived a bilateral dHC injection of vehicle, ICI 118,551, or betaxolol 15 min
before training. Data are expressed as mean latency (in seconds) ± SEM (n =
D Bregma: -2.4 mm -3.5 mm -4.2 mm
6–14). **P < 0.01; ***P < 0.001. Numeric values and detailed statistical
analyses are reported in Tables S5, S6, and S7.
1mm
neuronal marker NeuN (Fig. 3 B and C) or with the microglial E CA1

F CA1
marker ionized calcium-binding adaptor molecule 1 (Iba1) (Fig. S3). eGFP DG eGFP
Conversely, AAVDJ-hSyn-eGFP expression was found selectively in NeuN CA3
GFAP DG
CA3
neurons and not astrocytes (Fig. 3 D–F). 1mm 1mm
To knock down β2ARs, we used two methods: shRNA miR CA1 CA3 DG CA1 CA3 DG
(Sh) or antisense (AS) sequences against β2ARs. shRNA were
eGFP
eGFP
designed by Vector Biolabs (SI Materials and Methods). The Sh
and AS sequences were engineered into the astrocyte-specific
virus, resulting in AAV9-short gfaABC1D-eGFP-β2AR-Sh and
AAV9-short gfaABC1D-β2AR-AS (henceforth referred to as
NeuN
GFAP
“gfa-Sh” and “gfa-AS,” respectively) or into the neuron-specific

virus, resulting in AAVDJ-hSyn-eGFP-β2AR-Sh and AAVDJ-
hSyn-β2AR-AS (henceforth referred to as “hSyn-Sh” and “hSyn-AS,”
merge
respectively). Relative scrambled (Scr) or sense (S) sequences were

merge
used as controls.
First, we measured the ability of the Sh and AS viruses to affect 100 μm 100 μm
the selective expression of the β2AR protein. Using β1AR/β2AR-

Fig. 3. AAV under the short gfaABC1D or the hSyn promoter selectively
knockout mouse brains (32) we used Western blot analyses and/or
targets astrocytes or neurons, respectively. dHC infection of AAV9-short
immunohistochemistry to conduct specificity testing of several
gfaABC1D-eGFP (A–C) or AAVDJ-hSyn-eGFP (D–F) 2 wk after bilateral in-

available antibodies indicated to bind β2ARs specifically, but we jection (A, D). Shown are representative images of rostral-to-caudal brain
failed to identify any specific reactivity (Table S8). Thus, to sections [bregma −2.4 to −4.4 mm (45)]. All images of whole dHC are com-
quantify β2AR and β1AR protein levels, we used receptor auto- posite tile scans. In AAV9-short gfaABC1D-eGFP–infected cells, eGFP ex-
radiography binding assays. As previously established (10), this pression colocalizes with immunostaining of GFAP (B) but not NeuN (C) in
method is based on the quantification of iodo-(-)-cyanopindolol subregions CA1 and CA3 and the dentate gyrus (DG). In AAVDJ-hSyn-eGFP–
([125I] CYP) binding in the presence of serotonergic inhibitors (SB infected cells, eGFP expression colocalizes with NeuN (E) but not GFAP (F) in
224289 and WAY 100,635) and of the β1AR inhibitor CGP-20712A subregions CA1 and CA3 and the dentate gyrus.
8528 | www.pnas.org/cgi/doi/10.1073/pnas.1605063113 Gao et al.

A Bregma -3.6mm negative Similar results were obtained using gfa-AS and hSyn-AS. Four
weeks after infection, both gfa-AS and hSyn-AS significantly
reduced β2AR binding compared with S controls (P < 0.05; Stu-
dent’s t test), whereas β1AR binding remained unchanged (Fig. 6 A
and B). Gfa-AS had no effect on short-term memory (Fig. 6C) but
2AR 1AR significantly impaired long-term memory tested at 1 d and 7 d after
training (Fig. 6D). The memory impairment was rescued by a bi-
lateral injection of lactate into the dHC 15 min before training (P <
0.0001; two-way repeated measures ANOVA followed by Bonfer-
roni post hoc tests) (Fig. 6D). In contrast, hSyn-AS had no effect
on long-term memory (P > 0.05; one-way ANOVA) (Fig. 6E).
B Together, these data show that β2ARs expressed by hippo-
campal astrocytes rather than neurons are the critical effectors of
the adrenergic-mediated effect on long-term memory formation.
Or
CA1
Or
Pyr
CA1 Specifically, astrocytic β2ARs play a critical role in memory
Pyr
Rad
Rad
LMol consolidation by activating lactate release that supports long-
LMol CA2 MoDG
CA2 MoDG DG term memory formation and its underlying molecular changes.
DG
CA3 CA3 Discussion

Using two distinct, virus-mediated, cell-specific knockdown ap-
proaches, we showed that hippocampal β2ARs, but not β1ARs,
C anti-GFAP anti-NeuN and specifically β2ARs expressed on astrocytes but not on neu-
rons, are required for IA memory consolidation. The critical
Or Or
action of the hippocampal β2ARs is coupled to the training-
CA1 CA1
Pyr
Rad
Pyr
Rad evoked lactate release.
CA2 LMol
MoDG
LMol
MoDG These results together with the previously established roles for
β1ARs in synaptic plasticity and CFC memory retrieval of both rats
CA2
CA3 DG DG
CA3 and mice (12, 14) show that β1ARs and β2ARs contribute differen-
tially to hippocampal memory function and processes. We speculate
1mm 1mm that in the hippocampus β1ARs may activate neuronal responses
during retrieval and that β2ARs activate astrocytic metabolic coupling
Fig. 4. β2AR and β1AR autoradiography. (A) Anatomical schematic repre- to neurons to mediate memory consolidation. This differential role
sentation of coronal brain sections at bregma −3.6 mm (45) relative to
of β1ARs and β2ARs in memory processes seems to be part of a
representative examples of receptor autoradiography: negative control and
[125I] CYP binding to β2AR or β1AR. (B) dHC autoradiography for β2AR or β1AR
broader picture of distinct, and in some cases opposite, roles of the
binding. (C) Composite tile scan images of immunostaining with anti-GFAP
β1ARs and β2ARs in memory processes such as consolidation and
(astrocytes) or anti-NeuN (neurons) antibodies of a dHC section shown at the retrieval which involve different brain regions in different memory
same bregma coordinates. DG, dentate gyrus; LMol, stratum lacunosum systems (13, 33). Our findings of a cell-specific role of β2ARs in
moleculare; MoDG, stratum moleculare of the dentate gyrus; Or, stratum hippocampal astrocytes may contribute to explaining controversial
oriens; Pyr, stratum pyramidale; Rad, stratum radiatum. data on systemic or genetic ablation vs. intraregional inhibition of
receptor subtypes (12, 15). Understanding these differential roles and
contributions of glial vs. neuronal βARs, perhaps in different neu-
knockdown of β2AR in astrocytes or neurons, respectively. Notably, ronal populations (excitatory vs. inhibitory neurons) as well as in
the knockdown of the astrocyte-selective virus was more robust different brain regions, is key to unraveling the mechanisms of
(about 50%) than that of the neuron-selective virus (about 20%), NA-mediated responses. This understanding also should help the
as is consistent with a more abundant β2AR binding codistribution clinical use of βAR antagonists or agonists, and the understanding of
with astrocytes than with neurons and with previous literature the relative behavioral outcomes.
suggesting higher expression of β2AR in glia (18, 21–23). Similar to the results described in chicks (34), our data showed
Next we determined the effect of virus-mediated knockdown that astrocytic β2ARs in the hippocampus play a critical role in
of β2ARs on memory retention. gfa-Sh bilaterally injected into memory consolidation. Although the abundance of βAR subtypes
the dHC 2 wk before training had no effect on short-term does not correlate in chick and mammalian brains (34), it seems
memory tested 1 h after training (Fig. 5C) but impaired long-term that β2ARs expressed by astrocytes have conserved roles in memory
memory 1 d and 7 d later (P < 0.0001; two-way repeated-measures formation by controlling glycogenolysis (27). In addition, lactate
ANOVA followed by Bonferroni post hoc tests) and following an production from aerobic glycolysis coupled to β2AR stimulation
RS (P < 0.01; one-way ANOVA followed by Bonferroni post hoc does not seem to occur exclusively in the brain; in fact, it has also
tests) (Fig. 5D). Lactate injected bilaterally into the dHC 15 min been described as occurring in muscle during shock states associated
with a reduced or maintained blood flow (35).
before training fully and persistently rescued the memory impair-
Our results are consistent with several previous findings concerning
NEUROSCIENCE
ment (Fig. 5D). In contrast, 2 wk after hippocampal injection, functional regulation, mechanisms of action, and anatomical target-
hSyn-Sh elicited no change in long-term memory retention coming of NA in the mammalian brain, where NA, produced by the locus
pared with Scr controls (P > 0.05; two-way repeated-measures coeruleus (LC), projects diffusely to a variety of brain areas, including
ANOVA) (Fig. 5E). To rule out the contribution of behavioral the neocortex (36) and the hippocampus (37, 38). First, because NA
ceiling retentions and/or suboptimal time of infection to the lack of is released in the extracellular space from junctional varicosities along
an effect of neuronal β2AR knockdown on memory, we also tested its fibers, it can act on extrasynaptic receptors, which are localized on
the effect of a longer, 4-wk infection of hSyn-Sh and of a weaker astrocytes in particular (39). Second, in the hippocampus the highest
memory evoked by milder-intensity training (0.6 mA footshock) noradrenergic innervation is found in the dentate gyrus and in the
given either 2 or 4 wk after infection. Memory tested 1 d and 7 d stratum lacunosum moleculare, where we found an enrichment of
after training showed no differences in any of these conditions (P > β2ARs (Fig. 4B). Third, one cellular consequence of NA release
0.05; two-way repeated-measures ANOVA) (Fig. S4 and Table S9), during LC firing in particular behavioral states or evoked by gluta-
indicating that neuronal β2ARs are not critical for IA memory matergic inputs is glycogenolysis (30). Fourth, NA signaling is nec-
consolidation. essary for long-term potentiation (17), memory consolidation (2), and
A β2AR β1AR B β2AR β1AR (42). If this is the case, direct hippocampal delivery of lactate
Receptor binding *** therefore might be more efficient.
Receptor binding
300 700
***600
300 600
500
Because the dysregulation of β2ARs in astrocytes is associated
500 with multiple sclerosis, Parkinson’s disease, and Alzheimer’s
(dpm)
(dpm)
200 200 400
400
300 300 disease (43), we suggest that the disruption of the metabolic
100 200 100 200
100
role of astrocytic β2ARs in regions supporting cognitive func-
100
0
6 6
0 6 6 0 6 6
0 6 6 tions may contribute to the pathological features of those dis-
gfa-Scr gfa-Sh hSyn-Scr hSyn-Sh eases. Hence, targeting astrocytic β2ARs mechanisms may help
prevent or repair these disorders and/or their precipitation
C D E by stress.
IA Training IA Training RS IA Training
Test 1 Test 1 Test 2 Test 3 Test 1 Test 2
1h 15 min 1 d 6d 1d 1d 1d 6d Materials and Methods
Inject All animal protocols complied with the National Institutes of Health Guide
dHC * *** for the Care and Use of Laboratory Animals (44) and were approved by the
400 * *** * ** Icahn School of Medicine at Mount Sinai Institutional Animal Care and
Mean latency (s)
Mean latency (s)
Mean latency (s)

800 600 Use Committee or by the New York University Animal Welfare Committee.
300
600 Adult male Long–Evans rats were implanted with cannulae and/or injected
400 with AAV bilaterally into the dHC (from bregma: anteroposterior, −4 mm;
200
400 mediolateral, ±2.6 mm; and dorsoventral, −3.5 mm) as described in ref. 31.
100 200 IA training (0.9 mA for 2 s) and testing were performed as described in ref.
200
31. Drug injections (i.p.) of propranolol (Sigma-Aldrich), betaxolol (Tocris
0 6 6
0
9 8 8 7 9 8 8 7 9 8 7 7
0
12 12 12 12
Bioscience), or ICI 118,551 (Sigma) were done at 10 mg/kg. Intra-dHC injec-
Test 1 Test 1 Test 2 Test 3 Test 1 Test 2 tions (1 μL per side) of propranolol (5 μg/μL), betaxolol (2.2 μg/μL), ICI 118,551
gfa-Scr gfa-Scr-Vehicle gfa-Sh-Vehicle hSyn-Scr (5 μg/μL), L-lactate (100 mM), or D-glucose (50 mM or 150 mM, as indicated)
gfa-Sh gfa-Scr-L-Lactate gfa-Sh-L-Lactate hSyn-Sh were performed as described previously (29, 31). For Western blot analysis,
Fig. 5. ShRNA-mediated knockdown of β2AR in astrocytes but not in dHC protein extracts were done as described in ref. 31; primary antibodies
neurons impairs IA long-term memory. This impairment is rescued by were rabbit anti-pCREB (1:2,000; Cell Signaling), rabbit anti-CREB (1:1,000;
L-lactate. (A and B) β2AR and β1AR binding, expressed in disintegrations per
Millipore), rabbit anti-Arc (1:1,000; Synaptic Systems), rabbit anti-pCaMKII
minute (dpm), (n = 6) measured 2 wk after injection of gfa-Scr or gfa-Sh (A) (1:5,000; Cell Signaling), and mouse anti-CaMKII (1:4,000; Millipore). Immu-
or hSyn-Scr or hSyn-Sh (B). (C) Short-term memory tested 1 h after training nohistochemistry was performed using mouse anti-GFAP (1:400; Millipore),
and expressed as mean latency ± SEM (in seconds) in rats injected with
either gfa-Scr or gfa-Sh 2 wk before training (n = 6). (D) Long-term
memory tested 1 d (test 1) and 7 d (test 2) after training and after an RS
(test 3), expressed as mean latency ± SEM (in seconds), in rats injected with A β2AR β1AR B β2AR β1AR
either gfa-Scr or gfa-Sh 2 wk before training (n = 7–9) and with a bilateral 300 700 300 * 600
Receptor binding
Receptor binding
*
dHC injection of vehicle or L-lactate administered 15 min before training. 600 500
200 500 200 400
(E) Long-term memory tested 1 d (test 1) and 7 d (test 2) after training,
(dpm)
(dpm)
400 300
expressed as mean latency ± SEM (in seconds), in rats injected with either 300
hSyn-Scr or hSyn-Sh 2 wk before training (n = 12). *P < 0.05; **P < 0.01; 100 100 200
200
100
***P < 0.001. Numeric values and detailed statistical analyses are reported 8 8
100
8 8 6 6 6 6
0 0 0 0
in Tables S10 and S11.
gfa-S gfa-AS hSyn-S hSyn-AS
C D E
the induction of plasticity and memory genes such as Arc, pCREB, IA Training IA Training IA Training
Test 1 Test 1 Test 2 Test 1 Test 2
and pCaMKIIα in the hippocampus in vivo (Fig. 1).
1h 15 min 1 d 6d 1d 6d
Our findings that the training-dependent lactate increase in the
hippocampus requires β2ARs and not β1ARs and that supplying Inject dHC
lactate rescues the IA memory impairment caused by hippocampal 800

400 800
astrocytic β2AR knockdown strengthens the conclusion that *** **
Mean latency (s)
Mean latency (s)
Mean latency (s)
astrocytically generated lactate is a key mediator of hippocampus- 300 600 ** 600

dependent memory formation under arousing conditions (29). 400
200 400
Our results do not dissect whether this lactate originates solely
from glycogenolysis or also from glutamate-mediated glycolysis, as 100 200 200
proposed by the astrocytic–neuronal lactate shuttle hypothesis 5 6
0 12 13 10 12 12 13 10 12
0
12 11 12 11
0
(40). As suggested by Hertz and Gibbs (41), the level of stress/ Test 1 Test 1 Test 2 Test 1 Test 2
arousal evoked by the task may dictate whether glycogenolysis gfa-S gfa-S-Vehicle gfa-AS-Vehicle hSyn-S
and/or glycolysis are recruited to provide the lactate necessary gfa-AS gfa-S-L-Lactate gfa-AS-L-Lactate hSyn-AS
for memory consolidation. It is possible that the relevance of the Fig. 6. Antisense-mediated knockdown of β2AR in astrocytes but not in
experience, and thus the experience-evoked stress level, deter- neurons impairs IA long-term memory. The impairment is rescued by L-lac-
mines whether astrocytic β2ARs are recruited as essential mech- tate. (A and B) β2AR and β1AR binding expressed in disintegration per
anisms of memory consolidation. minute (n = 6) measured 4 wk after injection of gfa-S or gfa-AS (A) or hSyn-S
Furthermore, similar to previous observations with the glyco- or hSyn-AS (B). (C) Short-term memory tested 1 h after training and
genolysis inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol hydrochloride expressed as mean latency ± SEM (in seconds) of rats injected with either
(DAB) (29), a direct supply of glucose into the hippocampus was gfa-S or gfa-AS 4 wk before training (n = 5 or 6). (D) Long-term memory
tested 1 d (test 1) and 7 d (test 2) after training, expressed as mean latency ±
unable to replicate the effect of lactate, and only at higher

SEM (in seconds) of rats injected with either gfa-S or gfa-AS 4 wk before
concentrations were the memory impairments caused by β2ARs
training (n = 10–13). A bilateral dHC injection of vehicle or L-lactate was ad-
disruption transiently rescued. Although further experiments are ministered 15 min before training. (E) Long-term memory tested 1 d (test 1)
needed to dissect this issue, one possible explanation is that ac- and 7 d (test 2) after training, expressed as mean latency ± SEM (in seconds)
tivity-dependent processes promote glucose entry into astrocytes of rats injected with either hSyn-S or hSyn-AS 4 wk before training (n = 11 or
rather than directly into neurons. Glucose then would be metabo- 12). *P < 0.05; **P < 0.01; ***P < 0.001. Numeric values and detailed sta-
lized into lactate and finally transported from astrocytes to neurons tistical analyses are reported in Tables S12 and S13.
8530 | www.pnas.org/cgi/doi/10.1073/pnas.1605063113 Gao et al.

mouse anti-NeuN (1:200; Millipore), or rabbit anti-Iba1 (1:400; Wako). Micro- ACKNOWLEDGMENTS. We thank Dr. Karl Deisseroth (Stanford University)
dialysis samples, collected every 10 min, were analyzed using the Abcam Lac- for generously providing pAAV-hSyn plasmids; Dr. Mark Kay (Stanford Uni-
tate Fluorescence Assay Kit as described in ref. 29. Receptor autoradiography versity) for generously providing the pRC-DJ plasmid used to produce
was performed as described in ref. 10; [125I] CYP (Perkin-Elmer) containing AAVDJ; Dr. Bernard Schneider (École Polytechnique Fédérale de Lausanne)
for generously providing the pAAV-short gfaABC1D-eGFP plasmid used to
1 μM SB 224289 (Tocris), 1 μM WAY 100635 (Tocris), and 100 nM CGP 20712 produce AAV9; and Dr. Alberto Julio Kaumann (University of Murcia) for
(Tocris) or 50 nM ICI 118,551 (Sigma) was used. Data were analyzed using one- helpful advice concerning βAR radiolabeling. This work was supported by
way or two-way ANOVA or two-way repeated-measures ANOVA followed by NIH Grant R01 MH100822 and the McKnight Memory and Cognitive Disorder
Bonferroni or Newman–Keuls post hoc tests or Student’s t test. Award (to C.M.A.) and NIH Grant F30 MH098570 (to V.G.).
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iso
Journal of Mental Disorders

al D rder Ayano, J Ment Disord Treat 2016, 2:2
nt
s
urna of Me
DOI: 10.4172/2471-271X.1000120
&T
reatmen and Treatment
l
Jo
t
ISSN: 2471-271X
Research Article
Research Article OpenAccess
Open Access
Dopamine: Receptors, Functions, Synthesis, Pathways, Locations and

Mental Disorders: Review of Literatures
Getinet Ayano*
Chief Psychiatry Professional and mhGap Coordinator at Research and Training Department, Amanuel Mental Specialized Hospital, Addis Ababa, PO box: 1971, Addis
Ababa, Ethiopia
Abstract
Dopamine is monoamine neurotransmitter. Dopamine is produced in the dopaminergic neurons in the ventral
tegmental area of the substantia nigra, midbrain and the arcuate nucleus of the hypothalamus. In the periphery,
dopamine is found in the kidney where it functions to produce renal vasodilation, diuresis, and natriuresis. Dopamine
neurons are more widely distributed than those of other monamines and it is found in hypothalamus, olfactory bulb,
the midbrain substantia nigra and ventral tegmental area and in the periaqueductal gray and retina.
There are five subtypes of dopamine receptors, D1, D2, D3, D4, and D5, which are members of the large
G-protein coupled receptor super family. The dopamine receptor subtypes are divided into two major subclasses:
types 1 and 5 are similar in structure and drug sensitivity, and these two receptors are referred to as the "D1like"
group or class of receptors. Dopamine receptor types 2, 3, and 4 are called the "D2like" group. Dopamine plays
central role in pleasurable reward behavior, inhibition of prolactin production (involved in lactation), sleep, mood,
attention, learning, behavior, control of nausea and vomiting and pain processing. In addition it also involved in
controlling movement, emotion and cognition.
Due to extensive localization of dopamine receptor to brain areas and its role in wide range of functions,
dopaminergic dysfunction has been implicated in the pathophysiology of schizophrenia, mood disorders, obsessive
compulsive disorder (OCD), autism spectrum disorders, attention deficit–hyperactivity disorder (ADHD), tourette's
syndrome, substance dependency, Parkinson's disease and other disorders.
Keywords: Dopamine; Receptors; Synthesis; Locations; Mental the D2 receptor, implicating this subtype as an important site of
disorders antipsychotic drug action [3,4].
Dopamine Receptors D1 receptor has high affinity for the antagonist SCH 23390 and
relatively low affinity for butyrophenones such as haloperidol. D1
There are five subtypes of dopamine receptors, D1, D2, D3, D4, and receptor activation stimulates cyclic adenosine monophosphate (cAMP)
D5, which are members of the large G-protein coupled receptor super formation, D2 receptor stimulation produces the opposite effect. In
family [1]. The dopamine receptor subtypes are divided into two major addition to the stimulation of adenylate cyclase, D1 receptors may
subclasses: types 1 and 5 are similar in structure and drug sensitivity, also stimulate phosphoinositide turnover and modulate intracellular
and these two receptors are referred to as the "D1like" group or class calcium levels [1,3].
of receptors. Dopamine receptor types 2, 3, and 4 are also similar in
structure and are, therefore, grouped together as the "D2like" group The D1 receptors are found in high concentration in the
[2]. Dopamine receptors are typically couple to Gs and Gi mediated hypocampus, caudate, putamen, nucleus accumbens, hypothalamus,
transduction systems [3]. substantia nigra pars reticulata, olfactory tubercle and frontal and
temporal cortex [3,5]. D1 receptors have been implicated in the cognitive
The ultimate effect of D1-like activation (D1 and D5) can be functions of dopamine such as the control of working memory and
excitation (via opening of sodium channels) or inhibition (via opening attention. D1 receptors contribute significantly to the CNS effects of
of potassium channels); the ultimate effect of D2-like activation (D2, cocaine, suggesting the involvement of other receptors in addition to
D3, and D4) is usually inhibition of the target neuron [2]. The effect of the D2 receptor, in mediating rewarding effects of drugs of abuse [1,3,5].
dopamine on a target neuron depends on which types of receptors are
present on the membrane of that neuron and on the internal responses D1 and D5 receptors have a higher degree of homology with each
of that neuron to the second messenger cAMP [2]. D1 receptors are the other than with the D2–4 subtypes. D5 receptor has 50% homology
most numerous dopamine receptors in the human nervous system and
D2 receptors are the second most abundant receptors. D3, D4, and D5
receptors are present at significantly lower levels [2]. *Corresponding author: Getinet Ayano, Chief Psychiatry Professional and mhGap
Coordinator at Research and Training Department, Amanuel Mental Specialized
D1 and D5 receptors mostly involved in post synaptic inhibition. Hospital, Addis Ababa, PO box: 1971, Addis Ababa, Ethiopia, Tel: +251-9-27-17-
D2, D3, and D4 receptors are involved in both pre-and postsynaptic 29-68; E-mail. ayanogetinet@yahoo.com
inhibition. D2 receptors regulates mood, emotional stability in the Received July 18, 2016; Accepted July 26, 2016; Published August 02, 2016
limbic system and movement control in the basal ganglia [3,4].
Citation: Ayano G (2016) Dopamine: Receptors, Functions, Synthesis, Pathways,
D1 and D2 receptors were distinguished on the basis of differential Locations and Mental Disorders: Review of Literatures. J Ment Disord Treat 2: 120.
doi:10.4172/2471-271X.1000120
binding affinities of a series of agonists and antagonists, distinct
effectors mechanisms, and distinct distribution patterns within Copyright: © 2016 Ayano G. This is an open-access article distributed under the
terms of the Creative Commons Attribution License, which permits unrestricted
the CNS. It was subsequently found that the therapeutic efficacy use, distribution, and reproduction in any medium, provided the original author and
of antipsychotic drugs correlated strongly with their affinities for source are credited.
J Ment Disord Treat, an open access journal Volume 2 • Issue 2 • 1000120

ISSN: 2471-271X
Citation: Ayano G (2016) Dopamine: Receptors, Functions, Synthesis, Pathways, Locations and Mental Disorders: Review of Literatures. J Ment
Disord Treat 2: 120. doi:10.4172/2471-271X.1000120
Page 2 of 4
with D1. This structural similarity is reflected in the similar affinities effects of antipsychotic drugs are thought to result from the blockade of
of a wide variety of dopaminergic drugs for these two receptors. The these striatal dopamine receptors [10,11].
main distinguishing feature of their binding profiles is that the binding
The Mesolimbic Pathway, where the dopaminergic projections
affinity of dopamine is higher for the D5 receptor than that for the D1
originate in the ventral tegmental area, and then spread to the
receptor. D5 receptor is expression in nucleus of thalamus suggesting
amygdala, pyriform cortex, lateral septal nuclei and the nucleus
that role in pain stimuli [1,3,6].
accumbens. In this pathway dopamine functions in emotion and
D2 receptor is highly expressed in basal ganglia, septum, ventral reward systems. Mesolimbic dopamine mediates pleasure in the brain.
tegmental area and nucleus accumbes. D3 receptors mediate positive It is released during pleasurable situations and stimulates one to seek
regulatory influences of dopamine on production of neurotension. D4 out the pleasurable activity or occupation. This means food, sex, and
receptors homology with D2 and D3 41% and 39% respectively and several drugs of abuse are also stimulants of dopamine release in
they are found in hippocampus and frontal cerebral cortex [6-8]. the brain, particularly in areas such as the nucleus accumbens and
prefrontal cortex. In addition dopamine plays major role in addictions
Although the D1like receptors are mentioned as a primary target
in this pathway. All known drugs of abuse activate the mesolimbic
for antipsychotic drugs, several findings indicate that they are not
clinically relevant. Of the 3 D2 like receptors, only the D2 receptor pathway, and plastic changes in this pathway are thought to underlie
itself is blocked by antipsychotic drugs in direct relation to their clinical drug addiction. Antipsychotic drugs that decrease positive symptoms
antipsychotic potencies [3]. of schizophrenia by blocking dopamine receptors in the mesolimbic
pathway [10,12,13].
The D1 receptors are linked to adenylate cyclase which, when
activated, produces cyclic AMP as a secondary messenger. The D2 The Mesocortical Pathway, In which the dopaminergic fibers also
receptors are not positively linked to adenylate cyclase and may arise from the A10 region (the ventral tegmental area) and project to the
owe their physiological effects to their ability to inhibit this enzyme. frontal cortex and septohippocampal regions. Mesocortical dopmine
The D2 receptors are probably the most important postsynaptic mediates cognitive and emotional behaviour. Levels of dopamine in
receptors mediating behavioural and extrapyramidal activity. Most the brain, especially the prefrontal cortex, help in improved working
therapeutically effective neuroleptics block the D2receptors, while memory and attentions. However, this is a delicate balance and as levels
drugs like bromocriptine, which is a dopamine receptor agonist used in increase or decrease to abnormal levels, memory suffers. Antipsychotic
the treatment of parkinsonism, activate them. The correlation between drugs worsen negative symptoms of schizophrenia by blocking
the antagonist effect of a series of neuroleptics on brain [3,4]. dopamine receptors in the mesocortical pathway [10,12,13].
Agonist stimulation of D1 receptors results in cyclic The Tuberoinfundibular Pathway which originates in the arcuate
adenosinemonophosphate (cyclic AMP) synthesis followed by nucleus ofthe hypothalamus (arcuate and paraventricular nuclei)
phosphorylation of intracellular proteins, including dopamine- and and projects to pituitary gland (the median eminence). Dopamine in
AMP-regulated phosphoprotein (DARPP-32). The receptor binding this pathway inhibit prolactin release. Antipsychotic drugs that block
affinity of a dopamine agonist independent on the degree of association dopamine receptors in the pituitary may thus disinhibit prolactin
of the receptor and the guanine nucleotide binding regulatory protein, release and cause galactorrhea. Dopamine is the main neuroendocrine
which is regulated by guanosinetriphosphate (GTP) and calcium inhibitor of the secretion of prolactin from the anterior pituitary
or magnesium ions. Thus the D1 receptor may exist in a high or low gland. Dopamine produced by neurons in the arcuate nucleus of the
agonist affinity state depending on the balance between GTP (which hypothalamus is released in the hypothalamo-hypophysial blood
favors low affinity) and the divalent cations (which favor high affinity). vessels of the median eminence, which supply the pituitary gland.
The high affinity D1 receptor is classified as a D5 receptor [4,6]. This acts on the lactotrope cells that produce prolactin. These cells can
produce prolactin in absence of dopamine. Dopamine is occasionally
The D3 and D4 receptors appear to be largely restricted to the limbic called prolactin-inhibiting factor (PIF), prolactin-inhibiting hormone
areas of the rat and human brain. These receptors are of particular
(PIH), or prolactostatin [14] (Table 2).
interest as they have a high affinity for such atypical neuroleptics as
clozapine. Such findings suggest that the D3 and D4 receptors in the Dopamine Synthesis
human brain may mediate the antipsychotic actions of many typical
and atypical neuroleptics. The restriction of these receptors to the Dopamine is synthesized from the amino acid tyrosine, which is
limbic regions may lead to the development of neuroleptics which are taken up into the brain via an active transport mechanism. Tyrosine
specifically targeted to these areas [6,7] (Table 1). is produced in the liver from phenylalanine through the action of
phenylalanine hydroxylase. Tyrosine is then transported to dopamine
Dopamine System and Functions containing neurons where a series of reactions convert it to dopamine
[15,16]. Within catecholaminergic neurons, tyrosine hydroxylase
There are four major pathways for the dopaminergic system in the
catalyzes the addition of a hydroxyl group to the meta position of
brain:
tyrosine, yielding L-dopa. This rate-limiting step in catecholamine
The Nigro-Stiatal Pathway, In which fibres originate from the synthesis is subject to inhibition by high levels of catecholamines (end-
substantia nigra (pars compacta) and project rostrally to become widely product inhibition). Because tyrosine hydroxylase is normally saturated
distributed in the basal ganglia (caudate nucleus and the putamen). In with substrate, manipulation of tyrosine levels does not readily impact
this pathway dopamine plays a significant role in movement (the control the rate of catecholamine synthesis. Once formed, L-dopa is rapidly
of motor function and in learning new motor skills). Degeneration of converted to dopamine by dopa decarboxylase, which is located in
the nigrostriatal system causes Parkinson's disease [9-11]. Dopamine the cytoplasm. It is now recognized that this enzyme acts not only on
cell bodies in the pars compacts division of this region send ascending L-dopa but also on all naturally occurring aromatic L-amino acids,
projections to the dorsal striatum (especially to the caudate and including tryptophan, and thus it is more properly termed aromatic
putamen) and thereby modulate motor control. The extrapyramidal amino acid decarboxylase [15,16].

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Page 3 of 4
Receptors Locations Functions

Found in high concentration in mesolimbic, nigrostratal and mesocortical
Voluntary movements, regulate growth and development, regulations of
areas , such as substancia nigra, olfactory bulb, nucleus accumbens,
D1 feeding, affect, attentions, reward, sleep, impulse control, reproductive
cuadate, putamen, striatum, Expressed in low level in cerebellum,
behaviors, working memory, learning, control of rennin in kidney
hippocampus, thalamus, hypothalamus, kidney
Expressed in high levels in as substancia nigra, olfactory bulb, cuadate, Involved in working memory, reward-motivation functions regulate blood
putamen, ventral tagemental area(VTA), nucleus accumbens Found in low pressure, renal functions, gastrointestinal motility, vasodilatations, regulate
D2
level in hypothalamus, septum, kidney, cortex, heart, blood vessels, adrenal locomotion-presynatic receptors inhibit locomotion and post synaptic
glands, gastrointestinal tract, sympathetic ganglia receptors activate locomotion
Expressed only in CNS and it is not found outside the CNS. Found in Involved in endocrine function cognitions, emotions, regulations of
D3
olfactory bulb, nucleus accumbens locomotor functions and modulates endocrine functions
Substancia nigra, hippocampus, amygdala, thalamus, hypothalamus,
kidney, frontal cortex, heart, blood vessels, adrenal glands, gastrointestinal Regulations of renal functions, gastrointestinal motility, vasodilatations,
D4
tract, sympathetic ganglia, globus palidum, Lowest receptor found in CNS blood pressure, modulations of cognitive functions
than all dopamine receptors
Substancia nigra, hypothalamus, hippocampus, dental gyrus, kidney, heart, Involved in pain process, affective functions, endocrine functions of
D5
blood vessels, adrenal glands, gastrointestinal tract, sympathetic ganglia dopamine
Table 1: Summary of dopamine receptors, locations and functions.
Pathway Function
Nigrostriatal Movement and sensory stimuli
Mesolimbic Pleasure and reward seeking behaviors, addiction, emotion, perception
Mesocortical Cognition, memory, attention, emotional behavior, and learning
Tuberoinfundibular Control of the hypothalamic pituitary endocrine system, inhibition of prolactin secretions
Table 2: Summary of dopamine pathways and major functions.
Dopamine and Mental Disorders dopamine release in the striatum. The dopamine input to the striatum
is provided by a very dense network of axon terminals arising from cell
Due to extensive localization of dopamine receptor to brain areas bodies in the midbrain–substantia nigra pars compacta and ventral
and its role in wide range of functions, dopaminergic dysfunction tegmental area. The increased locomotor activity and stereotypy caused
has been implicated in the pathophysiology of schizophrenia, mood by psychostimulants seem especially to involve dopamine release in
disorders, obsessive compulsive disorder (OCD), autism spectrum ventral and dorsal parts of striatum, respectively. The ventral striatum
disorder, attention deficit–hyperactivity disorder (ADHD), tourette's includes the “core” and “shell” of the nucleus accumbens, blockade of
syndrome, substance dependency, Parkinson's disease and other dopamine neurotransmission in this region attenuates most rewarding
disorders. effects of addictive drugs, such as conditioned place preference The
The role of dopamine in schizophrenia dopaminergic projection to ventral striatum has therefore been intensely
investigated for its potential involvement in addictions[26-28].
Dopamine is among the common neurotransmitters involved in
pathogenesis of schizophrenia, largely based on patients’ responses to The role of dopamine in attention-deficit hyperactivity
psychoactive agents [17-20]. The role of dopamine in schizophrenia disorder (ADHD)
is based on the dopamine Hypothesis which evolved from two
Dopamine is among the common neurotransmitters involved in
observations. First, drug group which blocks dopamine function,
pathogenesis of Attention-Deficit Hyperactivity Disorder (ADHD).
known as the phenothiazines, could reduce psychotic symptoms.
Defects in dopamine metabolism have long been implicated in the
Second, amphetamines, which increase dopamine release, can induce
etiology of ADHD. The impulse and behavior problems found in
a paranoid psychosis and exacerbate schizophrenia and that disulfiram
Attention-Deficit Hyperactivity Disorder (ADHD) appear related to
inhibits dopamine hydroxylase and exacerbates schizophrenia [17-19].
low levels of Dopamine in the brain. Stimulants increase catecholamine
The role of dopamine in mood disorders concentrations by promoting their release and blocking their uptake.
Stimulants has been helpful in treating hyperactivity. Other drugs that
The findings on dopamine in mood disorders suggest that have reduced hyperactivity include tricyclic drugs and monoamine
decreased dopamine activity is involved in depression, while increased oxidase inhibitors (MAOIs), which indicate role of dopamine in
dopamine function contributes to mania [21]. The role of dopamine in Attention-Deficit Hyperactivity Disorder (ADHD) [29,30].
mood disorders is based on evidence that drugs that reduce dopamine
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Author manuscript
J Cell Signal. Author manuscript; available in PMC 2016 August 08.
Author Manuscript

J Cell Signal. 2016 June ; 1(2): . doi:10.4172/jcs.1000111.
Angiotensin Receptors: Structure, Function, Signaling and

Clinical Applications
Khuraijam Dhanachandra Singh and Sadashiva S Karnik*
Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, 9500 Euclid
Avenue, Cleveland, USA
Author Manuscript
Abstract
Angiotensinogen – a serpin family protein predominantly produced by the liver is systematically
processed by proteases of the Renin Angiotensin system (RAS) generating hormone peptides.
Specific cell surface receptors for at least three distinct angiotensin peptides produce distinct
cellular signals that regulate system-wide physiological response to RAS. Two well characterized
receptors are angiotensin type 1 receptor (AT1 receptor) and type 2 receptor (AT2 receptor). They
respond to the octapeptide hormone angiotensin II. The oncogene product MAS is a putative
receptor for Ang (1–7). While these are G-protein coupled receptors (GPCRs), the in vivo
angiotensin IV binding sites may be type 2 transmembrane proteins. These four receptors together
regulate cardiovascular, hemodynamic, neurological, renal, and endothelial functions; as well as
cell proliferation, survival, matrix-cell interactions and inflammation. Angiotensin receptors are
important therapeutic targets for several diseases. Thus, researchers and pharmaceutical companies
Author Manuscript
are focusing on drugs targeting AT1 receptor than AT2 receptor, MAS and AngIV binding sites.
AT1 receptor blockers are the cornerstone of current treatment for hypertension, heart failure,
renal failure and many types of vascular diseases including atherosclerosis, aortic aneurism and
Marfan syndrome.
Keywords
Angiotensin; AT1 receptor; AT2 receptor; MAS and AngIV binding site; ARBs; RAS
Introduction
Renin Angiotensin System (RAS) produces hormonal peptides which signal through cell
Author Manuscript
surface receptors classified as angiotensin receptors. Recent International Union of Basic

and Clinical Pharmacology (IUPHAR) review entitled “Angiotensin Receptors: Interpreters
of Pathophysiological angiotensinergic stimuli” covered >7255 research articles published in
the last 15 years and highlighted enormous development in angiotensin receptor research
[1]. The previous review on this topic by de Gasparo et al. [2] is a classic on most cited
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
*
Corresponding author: Sadashiva S Karnik, Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, 9500
Euclid Avenue, Cleveland, USA, Tel: 2164441269; Fax: 2164449263; karniks@ccf.org.
Singh and Karnik Page 2
articles list of Pharmacological Reviews. The current review, also published in

Author Manuscript
Pharmacological Reviews therefore has a high standard to meet in the coming decade.
Literature covered for the IUPHAR review demonstrated that AT1 receptor studies
dominated this research area in the past fifteen years followed by AT2 receptor, MAS and
the so called AT4 receptor. Arguably the conflicting results reported on insulin regulated
amino peptidase as the cognate receptor for angiotensin IV appears to be a major setback. In
contrast, discovery of MAS as a putative Ang (1–7) receptor is a major stimulus of research
activity. Continuation of this trend seems to be reflected in our analysis of research literature
for years 2013–2015 (Figure 1). AT1 receptor research exceeds the steady number of
publication on AT2 receptor, the rising trend for MAS and a clear trending decline for the
AngIV binding site (Figure 1).
The review is organized into major sections covering AT1 receptor, AT2 receptor, AT4
Author Manuscript
binding site, MAS and devoted a section for absence of AT3 receptor in angiotensin receptor
nomenclature system. Within each section, advances in structure-function, pharmacology,
experimental models, genetics, signalling, expression profile and pathophysiological aspects
are discussed with extensive citations provided for >1100 peer reviewed papers. This review
is a must read for students and researchers interested in RAS physiology and pathology as
well as drug developers.
AT1 receptor– Significant advances took place on almost all aspects of research on AT1
receptor, classically thought to be the sole mediator of all effects of RAS. Recent elucidation
of crystal structures of human AT1 receptor bound with the antagonists ZD7155 [3] and
Olmesartan [4] facilitates discussion of future mechanistic studies in specific structural
details. The crystal structure confirms the postulates 7TM α-helical architecture of AT1
Author Manuscript
receptor with three extracellular loops (ECL1-3) and three intracellular loops (ICL1-3). The
C-terminal region is highly disordered. ECL2 of AT1 receptor exhibits a β-hairpin secondary
structure which serves as an epitope for the agonistic autoantibodies in preeclampsia and
malignant hypertension [5,6]. AngII bound AT1 receptor crystal structure is currently
unavailable.
AT1 receptor blockers (ARBs) are selective non-peptide antagonists in clinical use for the
treatment of high blood pressure and are also being examined for various other human
cardiovascular disorders [1]. Eight ARBs –azilsartan, eprosartan, candesartan, irbesartan,
losartan, telmisartan, olmesartan and valsartan– are available for clinical use. Most of the
ARBs excepting telmisartan do not cross the blood brain barrier (BBB) in pre-clinical trials
suggesting their efficacy in brain pathological conditions. From the crystal structure and
molecular docking simulation critical ligand binding residues (Arg167, Tyr35 and Thr84)
Author Manuscript
identified may facilitate further refinement and development of novel ARBs [3,4]. Inverse
agonism of most ARBs is also observed in several studies [7]. Biased agonism of AngII
analogs was described and their potential application in heart failure therapy is evaluated in
clinical trials.
Significant advances made in defining pathophysiology are extensively reviewed.

AT1receptor knockout mice develop polyurea and abrupt vasodepressin signalling observed

in the inner medulla [8]. Genetic association studies found that AT1 receptor A1166C
Author Manuscript
(rs5186) polymorphism is associated with essential hypertension, increased aortic stiff [9]
and myocardial infarction [10]. Naturally occurring missense variantsmay directly (A163T,
T282M and C289W) or indirectly (L48V, L222V and A244S) influence ligand binding and
AT1 receptor signals. AT1 receptor signalling is mediated through G-proteins, G-protein
independent β-arrestin, reactive oxygen species, non-receptor type tyrosine kinases, small G-
proteins, transactivation of receptor tyrosine kinases. Furthermore, interacting scaffold,
mechanical stress, heterodimerization; and signalling through phosphorylation,
desensitization, and internalization may also be involved. Abnormal activation of AT1
receptor leads to a number of pathophysiologies including cardiovascular remodeling and
hypertrophy, vascular inflammation and atherosclerosis, endothelial dysfunction, oxidative
stress, extra cellular matrix deposition, insulin resistance, angiogenesis and cancer,
autoantibodies and malignant hypertension [1].
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AT2 receptor
The AT2 receptor shares approximately 34% amino acid sequence homology with AT1
receptor [1]. Physiological functions of AT2 receptor are not clearly defined till now, but 15
years of research devoted to this protein have further detailed physiological modulations by
AT2 receptor including those promoted by discovery of small molecule agonists and
antagonists. Beneficial effects of AT2 receptor have long been unclear due to its low
expression in adults. Both AngII and AngIII bind to AT2 receptor with affinity in nano
molar range and do not distinguish it from AT1 receptor. Even though the AT2 receptor
recognizes the same physiological ligands, the pharmacophore of AT2 receptor is very
distinct from that of AT1 receptor. Two non-peptide chemical compounds PD123319
(ditrifluoro acetate) and PD123177 (trifluoro acetate salt) defines the pharmacology and
Author Manuscript
functions of this receptor [11–15]. Recent discovery of a selective AT2 receptor non-peptide
agonist, compound 21, may expedite exploration of distinct roles of AT2 receptor in many
physiological and pathophysiological states. AT2 receptor became new therapeutic target for
the treatment of neuropathic pain. A few molecules like PD123319 [16,17] and EMA401
[18] are in clinical trials but treatment is limited due to poor efficacy and unfavourable side
effects.
Expression of AT2 receptors is predominant in distinct brain areas such as the locus
coeruleus and [19] and the amygdaloid nucleus [20]. Though, its expression declines after
birth, it is expressed at low levels in the normal adult cardiovascular system, adrenal gland,
kidney, brain, uterine myometrium and skin [21]. AT2 receptor Knock-out (KO) mouse
shows higher blood pressure than wild type animals without any growth abnormalities.
Developmental apoptosis of mesenchymal cells is not altered in the AT2 receptor KO mice
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but increased risk for renal diseases as well as inhibition of pressure natriuresis, vascular
hypertrophy and exacerbation of heart failure were observed [22–24]. Beneficial AT2
receptor functions from the knock out mouse study could be protective counteracting blood
pressure regulation by the AT1 receptor. Pharmacological modulation of AT2 receptor also
suggests its role in antidiuretic and antinatriuretic functions. Studies on genetic
polymorphism of this gene revealed their association with mental retardation, ventricular
structural changes, metabolic disorders, congenital urinary tract abnormalities etc. [1]. The

intra signal transduction process of AT2 receptor is unique among the GPCRs and is
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different from the AT1 receptor mediated signalling. AT2 receptor signalling involves G-
protein, protein phosphatases [Dual specificity protein phosphatase 1(MKp-1), Protein
phosphatase 2A (PP2A), Src homology phosphatase-1 (SHP-1)] and scaffolding protein,
nitric oxide/cGMP ion channel protein and ion channel protein and constitutive activity
(ligand independent activity of AT2 receptor) [1]. The pathological and physiological roles
of AT2 receptor include regulation of vascular response, cardiac growth response and
fibrosis response in other tissues. The development of agonists and antagonists against AT2
receptor for therapeutic use is crucial and in early stage, hence extensive studies are
warranted.
AT3 receptor
Although existence of AT3 receptor subtype displaying unique pharmacology was reported
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[25,26] no literature is available confirming the existence of a distinct gene for this receptor
in humans to date.
AT4 receptor
High affinity membrane binding sites for the [125I] AngIV peptide was termed as AT4
receptor in 1995 [27]. They are concentrated predominantly in brain and to different extents
in heart, kidney, adrenals and blood vessels. This receptor does not bind the analogues of
AngII, [Sar1]AngII, [Sar1,Ile8]AngII, Sar1,Ala8]AngII, Ang(1–7), AngII and the non-
peptide inhibitors of AT1 and AT2 receptors losartan, PD123177 and CGD42112A [1].
Histo-autoradiographic mapping studies of AngIV binding site determined the higher
concentration of its binding in brain which was then linked to regulation of cognitive sensory
and motor functions. Albiston et al (2001) identified [125I] AngIV peptide binding protein
Author Manuscript
as insulin regulated amino peptidase (IRAP, EC 3.4.11.3 also called LNEP for Leucyl-N-
exopeptidase) [28] which is a type 2 TM protein of the gluzincin amino peptidase family
[29,30].
Several independent observations in recent publications have cast some doubt regarding the
identity of IRAP as the only AT4 receptor. For instance, peptide antagonists of AngIV
binding sites and small molecule inhibitors of IRAP activity produced divergent
physiological effects [31,32]. Moreover, IRAP knockout mice were not altered in their
cognitive behavioural response to AngIV. Several other type II membrane proteins have been
reported as potential AT4 receptor candidates [33–35]. Therefore, understanding etiology
and treatment of memory dysfunctions associated with dementia and degenerative diseases
through AT4 receptor is significantly delayed.
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MAS
MAS is a candidate receptor for endogenously produced RAS peptide hormone Ang (1–7)
[36]. It remained orphan until the neuropeptide FF was shown to activate G-protein
signalling through this receptor. The action of Ang (1–7) through MAS is proposed to be
production of arachidonic acid and activation of nitric oxide synthase which may not involve
cAMP, IP3 and calcium signalling. MAS exhibits highest expression in brain and testis.
Becker et al. (2007) has observed that MAS expression in brain regions is important for

cardiovascular regulation [37]. Altered heart rate and decreased blood pressure was observed
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in KO mice and it was thought to be due to imbalance in the nitric oxide (NO) and reactive
oxygen species (ROS) [1]. In vivo studies show possible protective role of MAS through
Ang (1–7) mediated activation making it an enticing drug target. The pathophysiology of
MAS may be related to heart, kidney, vasculature, brain and reproductive organs. Above
conclusions are made from in vivo physiological and mouse KO studies. Although
independent research groups supported some of the findings, extensive pharmacological
studies are required to consolidate the conclusion that MAS is Ang (1–7) receptor as well as
elucidate its ligand activation mechanism.
Overall remark
The enormous development in angiotensin receptor research has been addressed on the
structure, pharmacological, signalling, physiological and pathophysiological state. Study on
Author Manuscript
AT1 receptor dominates in the field of angiotensin receptor research including the recent
solving of its crystal structure which opened new avenues for structure based drug discovery
and development. In the near future, we anticipate establishment of structures of other
angiotensin receptors. However, research on other angiotensin receptors is in nascent state
and extensive study is warranted.
Acknowledgments
We thank Russell Desnoyer for comments and suggestions on this manuscript and the National Institutes of Health
Grants R01 HL57470 and R01 HL115964 (to S. S. K.) for support.
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Figure 1.
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Received: 27 August 2018 Revised: 21 February 2019 Accepted: 18 April 2019
DOI: 10.1111/bph.14696
Themed Section: New Uses for 21st Century BJP
REVIEW ARTICLE
Histamine, histamine receptors, and neuropathic pain relief
Ilona Obara1,2 | Vsevolod Telezhkin3 | Ibrahim Alrashdi2 | Paul L. Chazot4
1
School of Pharmacy, Newcastle University,
Newcastle upon Tyne, UK Histamine, acting via distinct histamine H1, H2, H3, and H4 receptors, regulates vari-
2
Institute of Neuroscience, Newcastle ous physiological and pathological processes, including pain. In the last two decades,
University, Newcastle upon Tyne, UK
3
there has been a particular increase in evidence to support the involvement of H3
School of Dental Sciences, Newcastle
University, Newcastle upon Tyne, UK receptor and H4 receptor in the modulation of neuropathic pain, which remains
4
Department of Biosciences, Durham challenging in terms of management. However, recent data show contrasting effects
University, Durham, UK
on neuropathic pain due to multiple factors that determine the pharmacological
Correspondence responses of histamine receptors and their underlying signal transduction properties
Ilona Obara, School of Pharmacy, The Faculty
of Medical Sciences, Newcastle University,
(e.g., localization on either the presynaptic or postsynaptic neuronal membranes). This
King George VI Building, Newcastle upon Tyne review summarizes the most recent findings on the role of histamine and the effects
NE1 7RU, UK.
Email: ilona.obara@ncl.ac.uk
mediated by the four histamine receptors in response to the various stimuli associ-
ated with and promoting neuropathic pain. We particularly focus on mechanisms
underlying histamine‐mediated analgesia, as we aim to clarify the analgesic potential
of histamine receptor ligands in neuropathic pain.
LINKED ARTICLES: This article is part of a themed section on New Uses for 21st
Century. To view the other articles in this section visit http://onlinelibrary.wiley.
com/doi/10.1111/bph.v177.3/issuetoc
1 | NEUROPATHIC PAIN AND ITS disorders (channelopathies) replaced the term “dysfunction.” Second,
TREATMENT to avoid misdiagnosis of neuropathic pain as another type of pain orig-
inating from the nervous system, such as spasticity and rigidity of the
Neuropathic pain was first defined by the International Association for muscles and bone (e.g., musculoskeletal pain), the term “nervous sys-
the Study of Pain as “pain initiated or caused by a primary lesion tem” was replaced by the term “somatosensory system” (Finnerup
or dysfunction in the nervous system” (Merskey & Bogduk, 1994). et al., 2016). This revised definition of neuropathic pain describes the
Fourteen years later, Treede et al. (2008) revised this definition and nature of this condition more precisely and is, therefore, now widely
redefined it as “pain arising as a direct consequence of a lesion or dis- accepted and approved by the Neuropathic Pain Special Interest Group
ease affecting the somatosensory system.” In the revised definition of of the International Association for the Study of Pain. Neuropathic pain
neuropathic pain, two terms have received particular attention. First, can be divided into two subtypes, peripheral or central, based on the
the term “disease” which refers to all types of abnormal conditions anatomical location of the lesion or the disease, within the peripheral
including inflammation, autoimmune syndromes, and ion channel nervous system (PNS) or central nervous system (CNS), respectively.
Abbreviations: CaM, calmodulin; DRG, dorsal root ganglion; GSK3β, glycogen synthase kinase 3β; IP3, inositol triphosphate; KO, knockout; LC, locus coeruleus; PIP2, phosphatidylinositol 4,5‐
bisphosphate; PNS, peripheral nervous system; SP, substance P
--------------------------------------------------------------------------------------------------------------------------------
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided
the original work is properly cited.
© 2019 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.
580 wileyonlinelibrary.com/journal/bph Br J Pharmacol. 2020;177:580–599.

OBARA ET AL. 581
BJP
It is estimated that the worldwide prevalence rate of neuropathic pain 1988), partial sciatic ligation (Seltzer, Dubner, & Shir, 1990), spinal nerve
in the general population lies between 7% and 10%; however, this figure ligation (Kim & Chung, 1992), and spared nerve injury (Decosterd &
differs for different countries (van Hecke, Austin, Khan, Smith, & Woolf, 2000) models. These models aim to simulate some of the clinical
Torrance, 2014). The highest prevalence rates for neuropathic pain were features of neuropathic pain, in the preclinical setting (e.g., allodynia and
recorded in Canada (17.9%) and in the United States (9.8–12.4%; hyperalgesia), because a large proportion of peripheral neuropathic pain
VanDenKerkhof et al., 2016; Yawn et al., 2009), while a relatively low models, which are currently used in research, share alterations in hind‐
prevalence rate was noted in Austria and Netherlands (3.3% and 1%, limb cutaneous sensory thresholds following partial injury of a peripheral
respectively; Bouhassira, Lanteri‐Minet, Attal, Laurent, & Touboul, (usually sciatic) nerve. This is associated with the development of neuro-
2008; Gustorff et al., 2008; Harifi et al., 2013). In the United Kingdom, pathic pain symptoms, such as hyperalgesia and allodynia (Ma et al., 2003;
France, and Brazil, it is reported that 7–10% of chronic pain sufferers Wall et al., 1979). There are also limitations associated with the use of ani-
have been affected by neuropathic pain (Fayaz, Croft, Langford, mal models of neuropathic pain. These limitations are linked to challenges
Donaldson, & Jones, 2016). As a consequence of extended life expec- associated with (a) reliable and objective measures of behavioural
tancy, it is predicted that the worldwide prevalence rate of neuropathic responses to noxious stimuli, since animals cannot self‐report and the
pain is likely to increase further, because this type of chronic pain occurs experimenter can be biased, (b) appropriateness of the outcome mea-
with many common age‐related diseases. Neuropathic pain is triggered sures, for example, sleep disturbance to reflect spontaneous pain, and
by a lesion within the somatosensory system, trauma, or to toxic effects (c) complexity of mechanisms underlying the development of neuropathic
of certain medications (Colloca et al., 2017; Yu et al., 2013). Pathological pain and their relevance to humans (Colleoni & Sacerdote, 2010).
conditions that are responsible for the development of neuropathic pain Despite advances in the understanding of the underlying causes and
through injury include metabolic diseases (e.g., diabetic neuropathy), mechanisms leading to the development and maintenance of neuropathic
infection (e.g., postherpetic neuralgia), vascular disease (e.g., stroke), pain, 40% of Europeans who suffer from chronic pain did not achieve sat-
trauma (e.g., orofacial neuropathy), and cancer (Campbell & Meyer, isfactory pain control (Breivik, Collett, Ventafridda, Cohen, & Gallacher,
2006). Neuropathic pain is a complex condition that can either be con- 2006), and to date, no medication has shown long‐term efficacy and toler-
stant or periodic and presents with a range of different symptoms. These ability for neuropathic pain conditions. A significant contributing factor to
symptoms can increase throughout the day with clinically relevant these limited therapeutic strategies is that neuropathic pain has different
morning–evening differences and can be affected by gender and under- aetiology and pathophysiology to any other type of chronic pain, making
lying aetiology (Gilron, Bailey, & Vandenkerkhof, 2013). Sufferers of neu- the management of this type of chronic pain particularly difficult and chal-
ropathic pain have ongoing, spontaneous pain that has a significant lenging (Finnerup et al., 2015). Consequently, there is a pressing need for
negative impact on quality of life and daily functioning, including physical, the identification of new therapeutic strategies to improve management
emotional, and social well‐being (Jensen, Chodroff, & Dworkin, 2007). of neuropathic pain that will directly improve the outcome for pain sufferers.
The mechanisms underlying neuropathic pain are complex and multidi- The histamine system has been a target for multiple therapeutic inter-
mensional. Numerous pathophysiological and biochemical changes cause ventions. Recently, growing evidence has supported the use of selective
morphological and functional adaptations in the nervous system, including ligands of histamine H3 receptor and H4 receptor for the treatment of
an increase in excitatory neurotransmitters and neuropeptides, for exam- neuropathic pain (Bhowmik, Khanam, & Vohora, 2012; Chaumette et al.,
ple, histamine, bradykinin, 5‐HT, and glutamate, which leads to hyperexcit- 2018; Popiolek‐Barczyk et al., 2018; Sanna, Mello, Masini, & Galeotti,
ability (Baron, Binder, & Wasner, 2010). Likewise, injured peripheral nerve 2018). Approval in the European Union for the use of pitolisant (Wakix™),
fibres give rise to an intense and prolonged input of ectopic activity to the an antagonist/inverse agonist of H3 receptors for the treatment of narco-
CNS and may induce secondary changes to the excitability of the spinal lepsy (Kollb‐Sielecka et al., 2017), presents an opportunity to explore its
cord dorsal horn neurons (Colloca et al., 2017; Ossipov & Porreca, 2005). clinical use for other conditions. Consequently, it seems an appropriate
These morphological changes and functional adaptations lead to abnormal time to reconsider the histamine system as a therapeutic target for the
sensory signs in patients with neuropathic pain presented as, for example, management of neuropathic pain. This review aims to summarize the most
allodynia (pain due to a stimulus that does not normally activate the noci- recent findings on the role of histamine and its effects, mediated by differ-
ceptive system), hyperalgesia (an increased response to a stimulus that is ent subtypes of histamine receptors, on neuropathic pain, with particular
normally painful), or sensory loss (hypoesthesia). Clinically, neuropathic regard to the mechanisms underlying histamine‐mediated analgesia.
pain manifests as evoked pain and presents in many ways such as burning,
tingling, prickling, shooting, electric shock‐like, jabbing, squeezing, spasm, 2 | HISTAMINE, HISTAMINE RECEPTORS,
or cold (Rice, Finnerup, Kemp, Currie, & Baron, 2018). AND PAIN
Animal models of neuropathic pain are essential in understanding the
plethora of mechanisms that may drive neuropathic pain, allowing the
2.1 | Histamine and pain
field to identify potential therapeutic targets for the effective manage-
ment of this condition. Extensive research in the pain field has developed Histamine (2‐(4‐imidazolyl)‐ethylamine), one of the most extensively
and characterized a wide variety of animal models of neuropathic pain. studied amino acid‐derived neurotransmitters in the CNS and PNS, is
The four most commonly used models, also adopted in some studies involved in various physiological and pathological processes, including
discussed in this review, are chronic constriction injury (Bennett & Xie, sleep‐waking cycle, homeostasis, synaptic plasticity, and learning
582 OBARA ET AL.
BJP
(Panula et al., 2015; Parsons & Ganellin, 2006; Pini, Obara, Battell, 2.2 | Histamine receptors and pain
Chazot, & Rosa, 2016). Histamine is synthesized from the amino acid
L‐histidine through oxidative decarboxylation via catalysis with the his- Excitatory histamine receptor signalling in nociceptive pathways is
tidine decarboxylase enzyme (Bodmer, Imark, & Kneubuhl, 1999; Haas, associated with increased pain symptoms (Gangadharan & Kuner,
Sergeeva, & Selbach, 2008) and is arguably the most pleiotropic mole- 2013; Mobarakeh et al., 2000), whereas inhibition of histamine
cule in the human and animal body, being present in many cell types receptor signalling predominantly causes neuroprotective and
(Lindskog, 2017). Histamine is released by neuronal and non‐neuronal antinociceptive effects (Bhowmik et al., 2012; Chazot & Care, 2005;
sources and is responsible for many physiological processes, including Popiolek‐Barczyk et al., 2018). Different subtypes of histamine recep-
the contraction of smooth muscles of the lungs, uterus, and intestine, tors are expressed in both presynaptic and postsynaptic neuronal mem-
secretion of gastric acid in the stomach, and vasodilation, and modula- branes (Brown, Stevens, & Haas, 2001; Parsons & Ganellin, 2006;
tion of heart rate and contractility (Parsons & Ganellin, 2006). Hista- Zhang et al., 2013). Presynaptic histamine receptors function as
mine also functions as a neurotransmitter, within the nervous system, autoreceptors or heteroreceptors providing either positive or negative
regulating a variety of body functions, such as temperature control, feedback regulation of neurotransmitter release from the axon
memory, wakefulness, and pain sensation (Panula & Nuutinen, 2013). terminals into the synaptic cleft (Nieto‐Alamilla, Marquez‐Gomez,
Histamine is a key mediator in the processing of nociceptive infor- Garcia‐Galvez, Morales‐Figueroa, & Arias‐Montano, 2016). It could be
mation, acting in an antinociceptive manner in the CNS while, con- concluded that the resultant excitatory or inhibitory physiological
versely, in a nociceptive manner in the PNS (Khalilzadeh, Azarpey, effect of histamine receptors depends on the action of the neurotrans-
Hazrati, & Vafaei Saiah, 2018). In the PNS, histamine is released in mitter and the subsequent downstream cascade.
response to tissue injury/damage, and, through the sensitization of Specificity of localization of histamine receptors in different parts of
polymodal nociceptors resulting in increased firing rates, it contributes the nervous system, on either presynaptic or postsynaptic membranes,
to the generation of pain hypersensitivity. In neuropathic pain, hista- is determined by their physiological relevance (Parsons & Ganellin,
mine released in the periphery by mast cells has been shown to play 2006). Among the four subtypes of histamine receptors, H1 and H2
an important role in the development of hypersensitivity following receptors are predominantly identified postsynaptically (Brown et al.,
nerve injury. This pathological process is associated with recruitment 2001; Connelly et al., 2009; Zhang et al., 2013), with the location of
of macrophages and neutrophils, and as histamine is a powerful the H4 receptor requiring further investigation (Connelly et al., 2009).
chemoattractant of mast cells, it regulates this recruitment (Smith, Cross‐desensitization and agonist‐induced heterodimerization of H1
Haskelberg, Tracey, & Moalem‐Taylor, 2007; Zuo, Perkins, Tracey, & and H2 receptors (Alonso et al., 2013) may suggest a possible partner-
Geczy, 2003). Interestingly, it was also observed that peripherally act- ship between histamine receptors. Expression of H3 receptors was ini-
ing histamine could interact with mechanisms underlying pruritus (itch) tially reported as exclusively presynaptic in the rat cerebral cortex
and pain. Findings suggest that low concentrations of histamine, acting (Arrang, Garbarg, & Schwartz, 1983; Clark & Hill, 1996), while postsyn-
on sensory neurons, produce pruritus with a high concentration leading aptic expression of H3 receptors could not be completely excluded
to pain (Baron, Schwarz, Kleinert, Schattschneider, & Wasner, 2001; (Nieto‐Alamilla et al., 2016). H3 receptors are predominantly expressed
Hough & Rice, 2011; LaMotte, Simone, Baumann, Shain, & Alreja, in neurons and, together with H4 receptors, have higher affinity (nM
1987; Parsons & Ganellin, 2006; Pini et al., 2016; Simone, Alreja, & range) for histamine than H1 and H2 receptors (μM range; Parsons &
LaMotte, 1991). There is also evidence to show that histamine‐induced Ganellin, 2006). Expression of H3 and H4 receptors on the opposite
itch can convert into pain associated with neuropathic hyperalgesia sides of the synaptic cleft may contribute to their effects in neuropathic
(Baron et al., 2001). Indeed, multiple itch pathways were identified indi- pain, although the neuronal topology of the H4 receptor still remains
cating the presence of distinct itch‐generating types of neuron, one controversial. The use of selective ligands for histamine receptors has
responsible for transmitting itch sensation and the other, ultimately, led to a better understanding of the physiological and pathophysiolog-
for transmitting pain (Usoskin et al., 2015). In contrast to histamine ical roles of these receptors. The next section summarizes the effects
activity in PNS, multiple behavioural studies have shown that histamine produced by histamine receptor ligands on neuropathic pain.
injected directly into the various brain areas (e.g., somatosensory cortex Besides their presynaptic or postsynaptic localization, the physio-
or hippocampus) attenuated pain (Erfanparast, Tamaddonfard, Farshid, logical effects of histamine receptors are, to a great extent, determined
& Khalilzadeh, 2010; Tamaddonfard & Hamzeh‐Gooshchi, 2014). by the type of guanine nucleotide‐binding proteins (G‐proteins) to
Histamine exerts its effects via four distinct GPCR subtypes: H1, which they are coupled (Leung & Wong, 2017). The difference in
H2, H3, and H4 receptors. These receptors differ in their pharmacology underlying signalling pathways may directly determine the effect on
and signal transduction properties (Panula et al., 2015; Parsons & pain perception produced by selective ligands, even when they act at
Ganellin, 2006; Simons & Simons, 2011). Thus, histamine has differen- the same histamine receptor, as described below.
tial effects on neuropathic pain dependent upon the histamine recep-
tor subtype it is bound to. As mentioned previously, this review aims 2.2.1 | H1 receptor
to summarize histamine‐mediated effects on neuropathic pain. There-
fore, the next sections of this review focus on mechanisms underlying H1 receptors are excitatory receptors, which couple with Gq‐type pro-
histamine‐mediated analgesia. teins, leading to downstream activation of PLC and hydrolysis of
OBARA ET AL. 583
BJP
FIGURE 1 Diagram illustrating histamine

receptor signalling—Gq pathway. Histamine
binds to histamine receptors (H1, H2, or H3
receptor subtypes) that are coupled with the
Gq‐type protein. Gq α subunit activates PLC
which hydrolyses phosphatidylinositol 4,5‐
bisphosphate (PIP2), subsequently producing
DAG, that remains in the inner leaflet of the
plasma membrane activating PKC, and water
soluble inositol triphosphate (IP3), which binds
to inositol triphosphate sensitive receptor
(IP3R) and stimulates Ca2+ release from
endoplasmic reticulum. This intracellular Ca2+
forms a complex with calmodulin (CaM) Ca2
+
CaM and induces PKC‐dependent
phosphorylation. This suppresses the activity
of potassium voltage‐gated channels type 7
(Kv7 channels), which depolarizes the neurons,
and leads to the augmentation of neuronal
excitability, which manifests as increased pain
symptoms
phosphatidylinositol 4,5‐bisphosphate (PIP2) to produce DAG and ino- (Mojtahedin, 2016). Gs α subunit stimulates AC with consequent
sitol triphosphate (IP3). DAG subsequently activates PKC at the mem- augmented production of cAMP and consequent activation of PKA
brane, while IP3 diffuses and binds to IP3 receptors on the endoplasmic (Brown et al., 2001). Elevated cAMP concentration up‐regulates
reticulum to mobilize stored calcium (Ca2+). These changes cause PIP2 PKA, which as reported for hippocampal neurons, could activate ligand
depletion and increased intracellular concentration of Ca2+. This gated α‐AMPA receptors with resultant depolarization and increase of
2+
increased concentration of Ca activates PKC‐dependent phosphory- neuronal firing (Park et al., 2016). Also, as it was reported for after‐
lation and forms a complex with calmodulin (Ca2+CaM), both of which hyperpolarization in enteric neurons, PKA inhibits small conductance
suppress potassium voltage‐gated type 7 (Kv7) channels (Figure 1), Ca2+‐activated potassium (KCa) channels with resultant augmented
leading to depolarization and increased nociception (Brown & neuronal excitability (Figure 2; Vogalis, Harvey, & Furness, 2003).
Passmore, 2009; Chen, Li, Hiett, & Obukhov, 2016).
2.2.3 | H3 receptor
2.2.2 | H2 receptor
Presynaptic H3 receptors are coupled with Gi (AC inhibitory) proteins
H2 receptors are postsynaptic, predominantly associated with AC via (Nieto‐Alamilla et al., 2016; Schlicker & Kathmann, 2017). Gi α
coupling to Gs and, in a similar fashion to H1 and H3 receptors, Gq pro- subunit‐mediated AC inhibition results in a decreased intracellular
teins, both pathways initiating excitatory downstream signalling. Thus, concentration of cAMP and subsequent down‐regulation of PKA
H2 receptor inhibition yielded efficient antinociceptive effects (Nieto‐Alamilla et al., 2016). In sympathetic and sensory neurons, it

receptor signalling—Gs pathway. Histamine
binds to the H2 receptor subtype that is
coupled with the Gs‐type protein. Gs α subunit
activates AC, with subsequent production of
cAMP, which then stimulates PKA. PKA‐
dependent phosphorylation activates ligand
gated α‐AMPA receptors, which open and
facilitate influx of Na+ and, less commonly,
Ca2+. PKA also suppresses K+ efflux through
small conductance Ca2+‐activated potassium
channels (SK channels). Both the activation of
AMPA receptors and inhibition of SK channels
depolarize the neurons, with consequent
augmentation of their excitability and
increased pain signalling
584 OBARA ET AL.
BJP
was reported that H3 receptor activation stimulated dissociation of dependent phosphorylation (Dzamko, Zhou, Huang, & Halliday,
Gi β and γ subunits from Gi α subunit, which then inhibited 2014). PKB, via phosphorylation, inactivates GSK3β, which decreases
voltage‐gated Ca2+ influx through N‐, P‐, and Q‐type Ca2+ channels neuronal excitability (Paul et al., 2016), inhibits neuronal inflammation
(Zamponi & Currie, 2013), and stimulated G protein‐coupled (Maixner & Weng, 2013), and, subsequently, relieves pain. PKB‐
inwardly rectifying potassium (Kir) channels (Luscher & Slesinger, dependent phosphorylation that activates the MAPK/ERK cascade
2010). Both mechanisms could hyperpolarize presynaptic neurons, was also reported to be a highly efficient neuroprotective mechanism
reduce neuronal excitability, and produce pain relief (Figure 3). for chronic inflammatory and neuropathic pain (Cruz & Cruz, 2007;
Besides the Gi pathway, postsynaptic activation of H3 receptors Figure 3).
was reported to stimulate PLC in a subpopulation of striatal neurons,
with subsequent activation of the IP3 pathway followed by increased 2.2.4 | H4 receptor
intracellular concentrations of Ca2+ (Rivera‐Ramírez et al., 2016). Thus,
it is analogous to the mechanisms described for H1 /H2 receptors The role of H4 receptors in the nervous system is poorly understood
coupled to Gq proteins (Figure 1). Similarly, the H1 and H2 receptor (Schneider & Seifert, 2016). H4 receptors are known to be coupled
Gq cascade PLC signalling pathways modulate neuronal excitability to Gi proteins, and their downstream pathways are postulated to be
with resultant potential facilitation of pain sensitivity. Furthermore, similar to those described for H3 receptors (Figure 3). Compared to
H3 receptor activation was established to inhibit glycogen synthase the other three types of histamine receptors, the H4 receptor is not
kinase 3β (GSK3β) and MAPK/ERK pathways (Bongers et al., 2007; expressed abundantly in the CNS and PNS. By quantitative single‐cell
Morita, Aida, & Miyamoto, 1983). These effects are translated via Gi Ca2+ imaging, it was demonstrated that histamine induces a Ca2+
β and γ subunits (Lai et al., 2016), which up‐regulate the PI3K pathway increase in a subset of sensory neurons (3–10%) via activation of the
with subsequent production of phosphatidylinositol 3,4,5‐ H1 and H4 receptors as well as inhibition of the H3 receptor. It is
triphosphate (PIP3) from PIP2, which results in the recruitment of assumed that the decreased threshold in response to H3 receptor
PKB (Akt). PKB is initially activated by phosphoinositide‐dependent antagonism, which accounts for the analgesic effect of H3 receptor
kinase 1 (PDK1) and mammalian target of rapamycin complex 2‐ antagonists, activates H1 and H4 receptors on sensory neurons, which

receptor signalling—Gi pathway. Histamine
binds to the histamine receptors (presynaptic
and postsynaptic H3 or H4 receptor subtypes)
that are coupled with Gi‐type protein. The Gi
α subunit inhibits AC with subsequent
suppression of cAMP production and
inhibition of PKA activity. Also, Gi β and γ
subunits can inhibit Ca2+ influx through
voltage‐gated N‐, P‐, and Q‐type Ca2+
channels and stimulate G protein‐coupled
inwardly rectifying potassium (GIRK) channels,
with resultant K+ efflux. Both effects on N‐
type Ca2+ and GIRK channels result in the
development of hyperpolarization,
attenuation of neuronal excitability, and
resultant pain relief. Besides Gi α subunit
effects, H3 receptor activation could produce
analgesic effects through Gi β and γ subunits,
which up‐regulate the PI3K pathway, with the
subsequent production of
phosphatidylinositol 2,4,5‐trisphosphate
(PIP3) from phosphatidylinositol 4,5‐
bisphosphate (PIP2). PIP3 recruits PKB (Akt),
which phosphorylates and inactivates
glycogen synthase kinase 3β (GSK3β). In
parallel, PKB (Akt)‐dependent
phosphorylation additionally activates the
MAPK/ERK cascade. The action on both
GSK3β and MAPK/ERK decreases neuronal
excitability, inhibits mechanisms of neuronal
inflammation, and, therefore, produces pain
relief
OBARA ET AL. 585
BJP
in turn results in the excitation of histamine‐sensitive afferents and, detected prior to nerve injury, suggesting that the sensory modalities
therefore, may result in a modulation of pain sensitivity (Rossbach evoked by histamine acting via H1 receptors in normal and neuro-
et al., 2011). pathic pain states may result in different effects. This demonstration
of the potential up‐regulation of H1 receptor number in injured affer-
ent nerves further supports the involvement of H1 receptors in the
3 HISTAMINE RECEPTOR LIGANDS AND
| regulation of neuropathic pain hypersensitivity, presumably expressed
NEUROPATHIC PAIN on unmyelinated C‐fibres (Kashiba et al., 2001). The earliest electro-
physiological study to probe the histaminergic system in neuropathic
pain transmission reported that daily injections of astemizole, a
3.1 | H1 and H2 receptor ligands and neuropathic
pain CNS‐sparing H1 receptor antagonist, acting via peripheral histamine‐
sensitive C‐fibres, blocked ectopic spontaneous discharges from the
The best‐known roles for the H1 receptor are regulation of vasodila- neuroma and suppressed autotomy following sciatic and saphenous
tion and bronchoconstriction on multiple cell types, including endo- neurectomy (Seltzer et al., 1991; Yu et al., 2013). More recently, i.c.
thelial and smooth muscle cells, while the H2 receptor is primarily v. injection of histamine blocked mechanical and thermal hypersensi-
involved in the modulation of gastric secretion on parietal cells tivity associated with neuropathic pain (Sanna et al., 2015; Wei
(Barocelli & Ballabeni, 2003; Simons, 2003). There is also evidence et al., 2016). However, these pain‐modulatory effects of histamine
for their expression in the nervous system, where they regulate some can vary depending on the dose of histamine administered i.c.v., due
neuronal functions (Haas et al., 2008; Kashiba, Fukui, & Senba, 2001; to an action on H2 receptors and the involvement of adrenoceptors
Murakami et al., 1999). (Wei et al., 2014; Wei et al., 2016).
Both H1 and H2 receptors have been implicated in the role of his- To further support the role of H1 and H2 receptors in the regula-
tamine in nociception and chronic pain (Table 1). Interestingly, with tion of pain, separate studies using knockout (KO) mice lacking H1
the discovery of H1 and H2 receptor ligands in the 1950s, controlled and H2 receptors demonstrated that these mice displayed significantly
clinical studies using these H1 and H2 receptor antagonists reported lower responses to nociceptive stimuli when compared to their wild‐
mild analgesic activity and their potential as analgesic adjuvants, par- type controls (Mobarakeh et al., 2002; Mobarakeh, Takahashi,
ticularly in conditions where pain was induced by histamine. Most of Sakurada, Kuramasu, & Yanai, 2006). Interestingly, the antinociceptive
the clinical studies focused on diphenhydramine (first generation H1 phenotype of H2 receptor KO mice was relatively less prominent
receptor antagonist) and showed its analgesic potential in the treat- when compared to H1 receptor KO mice, suggesting a potentially dis-
ment of dysmenorrhea, atypical head and face pain, trigeminal neural- tinct role for these receptors in the modulation of pain. Indeed, behav-
gia, and thalamic syndrome (Rumore & Schlichting, 1986). In addition, ioural studies using a model of neuropathic pain, induced by the partial
diphenhydramine, when combined with opioids, showed its potential ligation of the sciatic nerve, showed that the CNS‐permeable H1
as an analgesic adjuvant in refractory cancer pain (Santiago‐Palma, receptor antagonist mepyramine, but not the H2 receptor CNS‐
Fischberg, Kornick, Khjainova, & Gonzales, 2001). In addition to clini- sparing antagonist cimetidine, blocked the effects of histidine on neu-
cal evidence for the analgesic potential of H1 and H2 receptor antago- ropathic pain hypersensitivity and spinal microglia activity (Yu et al.,
nists, preclinical studies identified the expression of H1 and H2 2016). In addition, Jaggi et al. (2017) suggested that the H1 receptor
receptors in nociceptive pathways and, therefore, further supported plays a more important role in a vincristine‐induced model of neuro-
the roles of H1 and H2 receptors in the regulation of pain. There are pathic pain, when compared to H2 receptors. However, Khalilzadeh
limited anatomical data available for H2 receptors, despite the report et al. (2018) observed different behavioural effects upon tibial nerve
of H2 receptor mRNA expression in human spinal cord (Murakami transection‐induced neuropathic pain with respect to the extent of
et al., 1999). The potential involvement of H1 receptors in the modu- brain penetration of the ligands, in a study focused on centrally
lation of neuropathic pain has been investigated more extensively. In active and centrally sparing H1 and H2 receptor antagonists.
studies using in situ hybridization techniques in the guinea pig, the Specifically, both chlorpheniramine, a centrally and peripherally
H1 receptor mRNA was shown to be expressed in about 15–20% of active H1 receptor antagonist, and fexofenadine, an H1 receptor,
the central trigeminal and lumbar dorsal root ganglion (DRG) neurons. centrally sparing antagonist, were found to profoundly decrease
These sensory neurons are fundamental to nociceptive processes, the mechanical hypersensitivity associated with the development of
potentially responding to histamine by acting on H1 receptors. These neuropathic pain. In contrast, while ranitidine, a widely used cen-
neurons are exclusively small in size and coexpress isolectin B4, but trally permeable H2 receptor antagonist, also improved mechanical
not substance P (SP) or CGRP, suggesting characteristics of unmyelin- hypersensitivity, famotidine, a centrally sparing H2 receptor antago-
ated C‐fibres involved in acute nociception. Interestingly, the potential nist, was ineffective. These results indicate that both blood brain
role of H1 receptors in the regulation of neuropathic pain sensitivity barrier penetrating and poorly penetrating histamine H1 receptor
can be explained by the marked (up to fourfold) increase in H1 recep- antagonists can block neuropathic pain hypersensitivity, but only
tor expression in the mainly small‐sized DRG neurons, 1–5 days after a the blood brain barrier penetrating histamine H2 receptor antagonist
crush injury of the sciatic nerve. Moreover, this study showed new can generate an analgesic effect in neuropathic pain. In line with
characteristics of peptidergic (SP/CGRP) sensory neurons not this observation, histamine‐induced mechanical hypersensitivity
586
TABLE 1 A summary of the effects produced by histamine receptor ligands in animal models of neuropathic pain
Drug Model Strain Test Effect Reference

BJP
H1 antagonists
Astemizole Neurectomy Sabra rats Score i.p. suppressed autotomy Seltzer, Paran, Eisen, & Ginzburg, 1991
Chlorpheniramine TNT Wistar rats Acetone i.p. reduced allodynia and prevented cold plate Khalilzadeh et al., 2018
von Frey avoidance behaviour
Double plate
Open field
PLSN Wistar rats Hargreaves i.p. suppressed and alleviated hyperalgesia Zuo et al., 2003
Randall‐Selito
Fexofenadine TNT Wistar rats Acetone p.o. reduced allodynia and prevented cold plate Khalilzadeh et al., 2018
Double plate
Open field
Loratadine Peripheral axotomy Sprague–Dawley Score of autotomy i.p. did not block analgesic effects of histidine on Yu et al., 2013
rats pain behaviour but alone suppressed autotomy
Mepyramine PLSN Sprague–Dawley von Frey Intrathecally, i.c.v. blocked analgesic effects of Yu et al., 2016
(pyrilamine) rats IR laser histidine on pain behaviour
SNL Hannover‐Wistar von Frey Intrathecally did not attenuate the Wei, Viisanen, You, & Pertovaara, 2016
rats Radiant heat antihypersensitivity effect of histamine
SNL Hannover‐Wistar von Frey test Into LC did not attenuate the antihypersensitivity Wei, Jin, Viisanen, You, & Pertovaara,
rats Radiant heat effect of histamine and alone failed to influence 2014
pain
PLSN Rats von Frey i.c.v was ineffective Huang, Adachi, Nagaro, Liu, & Arai, 2007
Promethazine Vincristine induced Wistar albino rats Pinprick i.p. reduced hyperalgesia and allodynia Jaggi, Kaur, Bali, & Singh, 2017
Acetone
Hot plate
H2R antagonists
Cimetidine PLSN Sprague–Dawley von Frey Intrathecally, i.c.v. did not block histidine's analgesic Yu et al., 2016
rats IR laser effects on pain behaviour
PLSN Wistar rats Hargreaves i.p. suppressed and alleviated hyperalgesia Zuo et al., 2003
Randall‐Selito
Famotidine TNT Wistar rats Acetone p.o. was ineffective in all tests Khalilzadeh et al., 2018
von Frey
Double plate
Open field
PLSN Sprague–Dawley von Frey i.p. reduced allodynia and hyperalgesia Yue et al., 2014
rats Diode laser
OBARA
(Continues)
ET AL.
OBARA
TABLE 1 (Continued)
ET AL.

Ranitidine TNT Wistar rats Acetone i.p. reduced allodynia and prevented cold plate Khalilzadeh et al., 2018
Double plate
Open field
Vincristine induced Wistar rats Pinprick i.p. reduced hyperalgesia and allodynia Jaggi et al., 2017
Acetone
Hot plate
PLSN Rats von Frey i.c.v. increased hypersensitivity Huang et al., 2007
Zolantidine SNL Hannover‐Wistar von Frey i.t. attenuated the antihypersensitivity effect of Wei et al., 2016
rats Radiant heat histamine
SNL Hannover‐Wistar von Frey Into LC attenuated histamine's analgesic effect but Wei et al., 2014
rats Radiant heat alone failed to influence pain
H3R antagonists and H3R antagonists/inverse agonist
A‐960656 SNL Hannover‐Wistar von Frey Into LC reduced hypersensitivity Wei et al., 2014
rats Radiant heat
SNL Hannover‐Wistar von Frey i.t. reduced hypersensitivity Wei et al., 2016
rats Radiant heat
SNL Sprague–Dawley von Frey p.o. reduced hypersensitivity Cowart et al., 2012
rats
Ciproxifan SNI BL6 mice Hargreaves i.p. was ineffective Zhang et al., 2012
Dynamic plantar
aesthesiometer
E‐162 CCI Swiss CD1 mice von Frey i.p. reduced hypersensitivity Popiolek‐Barczyk et al., 2018
Cold plate
Tail‐flick
GSK189254 CCI Random‐hooded rats von Frey p.o. reduced allodynia and hyperalgesia Medhurst et al., 2008
Randall‐Selito
SNL Sprague–Dawley von Frey i.p. reduced allodynia Hsieh et al., 2010
rats
BJP
GSK334429 CCI Random‐hooded rats von Frey p.o. reduced allodynia and hyperalgesia Medhurst et al., 2008
Randall‐Selito
Pitolisant SNI BL6 mice Hargreaves i.p. was ineffective, high doses increased thermal Zhang et al., 2012
Dynamic plantar but not mechanical hypersensitivity
aesthesiometer
S38093 CCI and oxaliplatin Sprague–Dawley Randall‐Selito p.o. reduced hypersensitivity Chaumette et al., 2018
induced rats Tail‐immersion (10°C)
(Continues)
587
588
BJP
TABLE 1 (Continued)

ST‐889 SNI BL6 mice Hargreaves i.p. was ineffective Zhang et al., 2012
Dynamic plantar
aesthesiometer
Thioperamide PLSN Rats von Frey i.c.v. increased but i.p. reduced hypersensitivity Huang et al., 2007
PLSN Sprague–Dawley Randall‐Selito i.p. increased hypersensitivity Smith et al., 2007
rats
H4R agonists
ST‐1006 SNI CD1 mice Hargreaves i.c.v. reduced allodynia and hyperalgesia Sanna et al., 2015
Dynamic plantar
aesthesiometer
VUF‐8430 SNI CD1 mice Hargreaves i.c.v. reduced allodynia and hyperalgesia Sanna et al., 2015
Dynamic plantar
aesthesiometer
SNI CD1 mice Hargreaves i.t.reduced allodynia and hyperalgesia Sanna, Lucarini, et al., 2017
Dynamic plantar
aesthesiometer
PLSN Sprague–Dawley Randall‐Selito i.p. reduced hypersensitivity Smith et al., 2007
rats
H4R antagonists
JNJ7777120 PLSN Sprague–Dawley Randall‐Selito i.p. increased hypersensitivity Smith et al., 2007
rats
SNL Sprague–Dawley von Frey i.p. reduced allodynia Hsieh et al., 2010
rats
JNJ10191584 SNI CD1 mice Hargreaves p.o. blocked the analgesic effect of i.c.v. ST‐1006 Sanna et al., 2015
Dynamic plantar and VUF‐8430 and was ineffective alone
aesthesiometer
SNI CD1 mice Hargreaves p.o. blocked the analgesic effect of i.t. VUF‐8430 Sanna et al., 2015
Dynamic plantar and was ineffective alone
aesthesiometer
TR‐7 CCI Swiss CD1 mice von Frey i.p. reduced hypersensitivity Popiolek‐Barczyk et al., 2018
Cold plate
Tail‐flick
Abbreviations: CCI, chronic constriction nerve injury; LC, locus coeruleus; PLSN, partial ligation of sciatic nerve; SNI, spared nerve injury; SNL, spinal nerve ligation; TNT, tibial nerve transection.
OBARA
ET AL.
OBARA ET AL. 589
BJP
was prevented by spinal pretreatment with zolantidine, a brain receptors are confusing as these drugs have different effects on the
penetrating H2 receptor antagonist, as well as localized peripheral nociceptive threshold depending on the pain model used, the nocicep-
administration of cimetidine (H2 receptor antagonist) and chlorphen- tive stimulus selected, together with the affinity and selectivity for the
iramine (H1 receptor antagonist) into the plantar side of the hindpaw histamine receptors, and the dose and routes of administration (Huang
(Zuo et al., 2003). et al., 2007; Smith et al., 2007). Several studies have reported inhibi-
Taken together, these results indicate that the brain histamine, tory effects on pain following activation of H3 receptors using agonists
acting particularly via central H1 and H2 receptors, may be involved (Cannon et al., 2003; Hasanein, 2011). The involvement of H3 recep-
in the modulation of neuropathic pain. These studies consistently sup- tors in neuropathic pain has been implicated using a range of H3
port the idea that CNS‐permeable H1 and H2 receptor antagonists receptor antagonists/inverse agonists (Table 1). The antagonism at
may potentially be used as analgesics for patients with neuropathic H3 receptors results in reduced mechanical and cold hypersensitivity
pain. The involvement of central H2 receptors in the regulation of neu- associated with neuropathic pain (chronic constriction injury model
ropathic pain hypersensitivity was also demonstrated in studies where or spinal nerve ligation model) as shown in studies using E‐162 or
histamine (presumably postsynaptically‐ induced) facilitated mechani- GSK189254, selective H3 receptor antagonists, where its strong anal-
cal hypersensitivity mediated by NMDA receptors as well as, in a gesic effect was observed after a single systemic (i.p.) dose (Hsieh
dose‐dependent manner, Nav1.8 channel expression in primary et al., 2010; Popiolek‐Barczyk et al., 2018). In addition, repeated, orally
afferent neurons in the sciatic nerve and L4/L5 DRG (Wei et al., delivered doses of GSK189254, GSK334429, S38093, and A‐960656,
2016; Yue et al., 2014). While sodium channels are responsible for selective H3 receptor antagonists/inverse agonists, significantly
the development and maintenance of neuropathic pain (Ossipov & reduced paw withdrawal threshold to mechanical stimuli or elicited
Porreca, 2005; Yue et al., 2014), the above studies highlight the an analgesic effect in the vocalization test of neuropathic pain (chronic
importance of histamine acting via H2 receptors in the regulation of constriction injury model or spinal nerve ligation model), showing
mechanisms associated with neuropathic pain states. The influence comparable efficacy to pregabalin or gabapentin, which are both used
of the H2 receptor on non‐neuronal cells (mast cells) is discussed later clinically as first‐line treatments (Chaumette et al., 2018; Cowart et al.,
in this review. 2012; Medhurst et al., 2008). The analgesic efficacy of S38093 was
also confirmed in other models of neuropathic pain with different
aetiologies, such as diabetic and chemotherapeutic agent‐induced
3.2 | H3 receptor ligands and neuropathic pain
neuropathy, where the drug again showed analgesic potency similar
H3 receptors are mostly presynaptic, expressed as autoreceptors on to pregabalin and gabapentin (Chaumette et al., 2018). Analgesia
histaminergic neurons involved in the negative feedback control induced by the blockade of the H3 receptor is possibly the result of
of histamine levels (Arrang et al., 1983; Hough & Rice, 2011), while the regulation of histamine levels in the CNS, as depolarization
H3 heteroreceptors on postsynaptic nonhistaminergic neurons also activates histamine synthesis in nerve endings, a process that is con-
regulate negatively the release of neurotransmitters, such as ACh, trolled by H3 autoreceptors (Arrang et al., 1983; Hough & Rice,
dopamine, 5‐HT, and noradrenaline (Blandina, Munari, Giannoni, 2011). Indeed, Wei et al. (2016) proposed that blocking the
Mariottini, & Passani, 2010; Gemkow et al., 2009; Giannoni et al., autoinhibitory H3 receptor on histaminergic terminals in the pontine
2010). Since the cloning of H3 receptors (Lovenberg et al., 1999), locus coeruleus (LC), which receives efferent projections from the his-
there has been an increased interest within the pharmaceutical indus- taminergic tuberomammillary nucleus, facilitated endogenous release
try in developing ligands for this receptor to target several diseases, of histamine leading to neuropathic hypersensitivity inhibition through
including neuropathic pain. This interest was strongly fuelled by the the regulation of descending noradrenergic pathways. In addition,
report of H3 receptor expression in nociceptive pathways, suggesting there is accumulating evidence to support the idea that the analgesic
its functional involvement in the regulation of nociceptive transmis- effects of H3 receptor antagonists/inverse agonists in neuropathic
sion (Cannon et al., 2007). Indeed, the histamine H3 receptor consists pain can be partially mediated by α2 adrenoceptor desensitization
of several functional isoforms expressed in both the CNS and PNS, (induced by H3 receptor antagonists/inverse agonists) in the LC
particularly along the ascending nociceptive pathway and descending and spinal cord. This suggests an inhibitory role for the central
pain‐control pathway, that are critical for the processing of nocicep- heteroreceptor noradrenergic transmission in the efficacy of H3 recep-
tive information. Within the CNS, this receptor has been found in var- tor antagonists/inverse agonists. In agreement with this idea, systemic
ious brain areas, such as thalamus, hypothalamus, prefrontal cortex, administration of α2 adrenoceptor agonists or nerve injury‐induced
and periaqueductal grey area (Drutel et al., 2001), and in the spinal activation of these α2 adrenoceptors decreases the firing activity of
cord (Cannon et al., 2007; Heron, Rouleau, Cochois, Pillot, & Schwartz, LC noradrenergic cells, resulting in the dampening of noradrenaline
2001; Medhurst et al., 2008). In the periphery, the expression of H3 release in the terminal area (e.g., prefrontal cortex or spinal cord) and
receptors has been identified in DRG, superior cervical ganglia, and promoting neuropathic hypersensitivity by attenuating descending
dermal tissues (Cannon et al., 2007; Medhurst et al., 2008). inhibition (Chaumette et al., 2018; Wei et al., 2010; Wei et al.,
However, while the localization of H3 receptors strongly suggests 2014). In contrast, treatment with H3 receptor antagonists/inverse
its functional involvement in the regulation of nociceptive transmis- agonists restores LC and decreases α2 adrenoceptor activity, respec-
sion, pharmacological studies using agonists and antagonists of H3 tively, potentially leading to relief in neuropathic pain hypersensitivity
590 OBARA ET AL.
BJP
(Chaumette et al., 2018; Wei et al., 2010). To further support this antagonists, blocked the secondary mechanical allodynia in the
proposed mechanism, it was shown that bilateral lesion of the LC, capsaicin‐induced model of pain. Secondary mechanical hypersensitiv-
transection of the spinal cord, or direct injection of a α2 agonist ity is known to be exclusively signalled by A‐fibres and amplified by
(fadolmidine) into the LC reversed the antihyperalgesic effect pro- sensitized dorsal horn neurons (Magerl, Fuchs, Meyer, & Treede,
duced by H3 receptor antagonists, A‐960656 or GSK189254 2001; Treede & Magerl, 2000). Thus, presumably reducing the sensi-
(Chaumette et al., 2018; McGaraughty, Chu, Cowart, & Brioni, tivity of H3 receptor‐positive A‐fibres with selective H3 receptor
2012; Wei et al., 2014). In line with this, electrophysiological studies antagonists resulted in a diminished input to the dorsal horn and the
performed in anaesthetized animals indicated that, after systemic subsequent amplification of the A‐fibres response, confirming the
administration, GSK189254 dose‐dependently decreased both potential role for H3 receptors in the modulation of central sensitiza-
evoked and spontaneous firing of wide dynamic range neurons in tion processes. In contrast, thermal (heat) hypersensitivity is generally
neuropathic, but not sham‐operated rats (McGaraughty et al., regarded as a sign of the peripheral sensitization of C‐fibres, which do
2012). However, analgesia induced by the blockade of the H3 recep- not express H3 receptors (Cannon et al., 2007; Gold & Gebhart, 2010).
tor can also be mediated via H3 heteroreceptors that regulate other The only study that reported a significant increase in thermal (radiant
neurotransmitters' release; the blockade of the H3 receptor is known heat in Hargreaves test), but not mechanical, threshold in the spared
to increase the release of ACh, dopamine, 5‐HT, noradrenaline, and nerve injury model of neuropathic pain used the selective H3 receptor
SP in the CNS (Blandina et al., 2010; Gemkow et al., 2009; Giannoni inverse agonist pitolisant (Wakix™). The drug produced this unex-
et al., 2010). pected effect at a dose 5× higher than its clinically relevant dose,
Interestingly, the majority of the behavioural observations pub- and pharmacological analysis of this effect suggested at least partial
lished indicate that H3 receptor antagonists/inverse agonists do not involvement of transient receptor potential cation channel subfamily
produce any antinociceptive effects in naïve rodents, suggesting a pos- V member 1 (TRPV1), without any contribution of H3 receptors
sibility that H3 receptors are not involved or tonically activated in (Zhang et al., 2012). Interestingly, the H3 receptor antagonist/inverse
nociception (at least in relation to acute mechanical nociception), but agonist E‐162, at a dose that produced a significant reduction in
are critical for pathological pain states, particularly for mechanical mechanical hypersensitivity, also attenuated the response to cold in
hypersensitivity (Chaumette et al., 2018; McGaraughty et al., 2012). neuropathic pain (Popiolek‐Barczyk et al., 2018). The signalling of cool
Also, H3 receptor KO mice showed unaltered response to mechanical temperatures that become aversive in neuropathic pain is known to be
pinch (Cannon et al., 2003), and multiple studies suggest modality mediated via the TRPM8 receptor, a member of the TRP channel fam-
(mechanical vs. heat) and intensity (preferential responses to low‐ ily (Knowlton et al., 2013). It was reported that the number of TRPM8‐
intensity tail pinch stimulation; Cannon et al., 2003) with specific positive Aδ‐fibres (but not C‐fibres) increases after nerve injury (Ji,
antinociceptive effects mediated by H3 receptors. H3 receptor Zhou, Kochukov, Westlund, & Carlton, 2007); thus, it is possible that
antagonists/inverse agonists at a dose that produced a significant H3 receptor‐positive A‐fibres are probably sensitive to cooling and
reduction of mechanical hypersensitivity in neuropathic pain did not may contribute to cold hypersensitivity in neuropathic pain.
attenuate heat hypersensitivity indicating that the antihyperalgesic The most significant inconsistencies in behavioural outcomes
effect was due to selective depression of spinal sensory rather than in neuropathic pain can be found in studies on the role of a first‐
motor neurons (Wei et al., 2014; Wei et al., 2016). To support this, generation imidazole‐based molecule, thioperamide (H3 receptor
in situ hybridization studies revealed H3 receptor mRNA transcripts antagonist, H3 /H4 receptor inverse agonist), in the regulation of
in the sensory neurons of the dorsal horn and DRG (Heron et al., mechanical hypersensitivity in neuropathic pain. On the one hand,
2001). Moreover, receptor autoradiography studies, using [3H] blocking H3 receptors (and H4 receptors) by thioperamide resulted in
GSK189254, showed specific H3 receptor binding sites in the a significant enhancement of mechanical hyperalgesia in a rat model
dorsal horn of the spinal cord and DRG, confirming these as sites of neuropathic pain induced by partial ligation of the sciatic nerve.
of action of H3 receptor antagonists within structures receiving Specifically, i.c.v. (Huang et al., 2007) or s.c. injection (Smith et al.,
histaminergic innervation and are critical for processing of pain infor- 2007) of thioperamide directly into the operated hindpaw resulted in
mation (Medhurst et al., 2008). The modality‐ and intensity‐specific a significantly reduced mechanical withdrawal threshold as compared
antinociceptive effects of H3 receptor activation/inhibition may also to controls. On the other hand, systemic (i.p.) injection of thioperamide
suggest involvement of a specific population of sensory fibres that significantly increased mechanical withdrawal threshold indicating an
regulate mechanical hypersensitivity. In line with this, immunohisto- analgesic effect (Huang et al., 2007). The reason for this discrepancy
chemical studies identified localization of H3 receptors (confirmed by may lie in the drug's dual affinity for both H3 and H4 receptors (e.g.,
H3 receptor KO mice) on medium‐size cell bodies in DRG and on the effect of thioperamide on neurotransmitter release in the anterior
small‐calibre periarterial, peptidergic Aδ fibres that ramified in dorsal hypothalamic area of rats is nonreversible by an H3 receptor agonist,
horn laminae I, II, and V and coexpress immunoreactivity for acid‐ suggesting the involvement of H4 receptors; Yamamoto, Mochizuki,
sensing ion channel 3 and 200‐kD neurofilament protein. This strongly Okakura‐Mochizuki, Uno, & Yamatodani, 1997) and on the behav-
supports the involvement of H3 receptors in the regulation of mechan- ioural effects resulting from the route (localized vs. systemic) and
ical sensitivity (Cannon et al., 2007). In addition, Medhurst et al. (2007) dose of thioperamide administration. In addition, the involvement of
showed that GSK207040 and GSK334429, selective H3 receptor other histaminergic mechanisms of action in the behavioural effects
OBARA ET AL. 591
BJP
produced by thioperamide is suggested by the observation that Interestingly, H4 receptor deficiency does not support a role for H4
thioperamide increases the density of intact mast cells in the injured receptors in the physiological maintenance of pain threshold, as H4
nerve (Smith et al., 2007). While nerve injury causes a decrease in receptor‐KO mice did not show any change in thermal or mechanical
mast cell numbers as a consequence of degranulation (Zuo et al., nociceptive thresholds, suggesting that the H4 receptor is specifically
2003), thioperamide's action leads to an opposite effect that may rep- involved in the regulation of hypersensitivity associated with patho-
resent a prevention of mast cell degranulation and stabilization or logical chronic pain induced by nerve injury (Sanna, Ghelardini, et al.,
redistribution of mast cells in the injured nerve that theoretically 2017). This observation in H4 receptor‐KO neuropathic mice is partic-
would result in the inhibition of hyperalgesia, rather than its enhance- ularly important as H4 receptor mRNA expression in humans and
ment. This effect could be linked to the observation that histamine rodents supports their involvement in the regulation of neuronal func-
(acting through H1 and H3 receptors) inhibits the release of the pro‐ tion, including regulation of neuropathic pain. The controversy around
inflammatory cytokine TNF‐α from alveolar macrophages (Sirois, the generation of consistently specific H4 receptor antibodies high-
Ménard, Moses, & Bissonnette, 2000), and antagonism of H3 recep- lights the need for cautious interpretation of some of the immunohis-
tors on macrophages resulted in an increase in TNF‐α and, subsequent, tochemical outcomes (Beermann, Seifert, & Neumann, 2012; Gutzmer
enhancement of mechanical hyperalgesia (Smith et al., 2007). et al., 2012; Schneider & Seifert, 2016). In line with the observation
Taken together, the interpretation of the thioperamide data is from H4 receptor KO mice, blockade of H4 receptors by the specific
complicated further since the drug has high affinity, not only for H3 H4 receptor antagonist JNJ7777120, injected s.c. directly into the
and H4 receptors but also for 5‐HT3 receptors (Leurs et al., 1995). operated hindpaw, resulted in a significant increase in mechanical
Studies with more selective H3 receptor antagonists/inverse agonists hyperalgesia compared to controls (Smith et al., 2007). Subsequently,
suggest that these ligands may be beneficial for the improvement of activation of H4 receptors by localized administration of potent and
mechanical and cold hypersensitivity associated with neuropathic selective agonists, ST‐1006 (i.c.v.) and VUF8430 (i.c.v., intrathecally,
pain, particularly given their ability to modulate histamine levels, as and s.c. directly into the operated hindpaw), resulted in a significantly
well as several neurotransmitters, including ACh, histamine, noradren- reduced mechanical and thermal withdrawal threshold in mice sub-
aline, dopamine, and SP. However, due to the wide presynaptic and jected to neuropathic pain induced by spared nerve injury or partial
postsynaptic distribution of H3 receptors throughout the CNS and nerve ligation of the sciatic nerve (Sanna et al., 2015; Sanna, Lucarini,
PNS, more research is certainly needed to clarify the involvement of et al., 2017; Smith et al., 2007). The analgesia produced by VUF8430
peripheral, spinal, and brain H3 receptors in various neuropathic pain has been shown to be associated with a reduction in neuroinflamma-
states, thus determining their full potential in neuropathic pain. tion and oxidative stress mediated by neuronal H4 receptors in the
spinal cord and sciatic nerve (Sanna, Lucarini, et al., 2017), and
3.3 | H4 receptor ligands and neuropathic pain the involvement of H4 receptors in the behavioural effects produced
by ST‐1006 and VUF8430 was confirmed with JNJ10191584, H4
The H4 receptor, which has low homology with other histamine recep- receptor antagonist also known as VUF6002, which fully prevented
tors, can be primarily found in bone marrow, intestinal tissue, spleen, the analgesic effects produced by these H4 receptor agonists (Sanna
thymus, and also in various immune cells, such as T cells, mast cells, et al., 2015).
neutrophils, and eosinophils, showing modulatory effects on these Interestingly, similar to the H3 receptor, pharmacological studies
cells, including activation, migration, and production of cytokines using agonists and antagonists of H4 receptors demonstrate that these
and chemokines, suggesting its principal role in the regulation of drugs can have different effects on the nociceptive threshold depend-
immune/inflammatory mechanisms (Takeshita, Sakai, Bacon, & ing on the routes of administration and target cells (Popiolek‐Barczyk
Gantner, 2003; Zhu et al., 2001). Interestingly, recent reports also indi- et al., 2018; Sanna et al., 2015). In contrast to the studies above that
cate the presence of H4 receptors on peripheral sensory nerves, in the used H3 receptor agonists/antagonists after localized application, the
DRG, with more intense staining of small‐ and medium‐diameter cells, antagonism of H4 receptors produced by systemic administration
and in the spinal cord, especially laminae I and II (Sanna, Lucarini, et al., resulted in the alleviation of mechanical and cold hypersensitivity
2017; Strakhova et al., 2009). This neuronal localization supports H4 associated with neuropathic pain (chronic constriction injury model).
receptors involvement in the regulation of neuronal function related Studies using TR‐7, a selective H4 receptor antagonist, elicited a strong
to the modulation of nociceptive transmission (Sanna, Ghelardini, analgesic effect after a single systemic (i.p.) dose, which was as effec-
Thurmond, Masini, & Galeotti, 2017; Sanna, Lucarini, et al., 2017). tive as morphine, a gold standard in pain treatment (Popiolek‐Barczyk
The involvement of H4 receptors in both acute (Galeotti, Sanna, & et al., 2018). In addition, JNJ7777120 reduced mechanical hypersensi-
Ghelardini, 2013) and persistent inflammatory pain (Hsieh et al., 2010) tivity after a systemic (i.p.) administration in neuropathic pain (chronic
is relatively well documented, and recently, the role of H4 receptors in constriction injury model and spinal nerve ligation model; Hsieh et al.,
the modulation of neuropathic pain was identified in H4 receptor‐KO 2010). Given that H4 receptors are expressed on the immune cells, in
mice through the observation that these animals, when subjected to addition to the well‐documented involvement of H4 receptors in the
neuropathic pain, induced by spared nerve injury of sciatic nerve, regulation of immune/inflammatory mechanisms (Takeshita et al.,
showed enhanced hypersensitivity to mechanical and thermal stimuli 2003; Zhu et al., 2001), it is possible that the antinociceptive action
compared to wild‐type controls (Sanna, Ghelardini, et al., 2017). of H4 receptor antagonists, particularly after systemic administration,
592 OBARA ET AL.
BJP
may result from a reduction in ongoing inflammatory processes at the Mast cells are professional cellular suppliers of histamine and
site of nerve injury, since the analgesic effect produced by contribute to the histamine‐based effects in neuropathic pain. For
JNJ7777120 was weaker (secondary) than its anti‐inflammatory effect example, mast cell depletion prevented mechanical allodynia in a
(Hsieh et al., 2010). An underlying mechanism may be associated with mouse model of postoperative pain (Kaur, Singh, & Jaggi, 2017).
stabilization of mast cells that are known to regulate the recruitment Recently, it was shown that the administration of azelastine hydro-
of neutrophils and macrophages and, subsequently, to modulate the chloride, a second‐generation H1 receptor antagonist and mast cell
development of hyperalgesia in neuropathic pain (Smith et al., 2007; stabilizer, blocked the development of mechanical allodynia and
Zuo et al., 2003). It should be further noted that similar observations inhibited mast cell degranulation in mice with oxaliplatin‐induced
have been described for the closely related H3 receptor. mechanical allodynia pain. The H1 and H4 receptors are likely molecu-
The H4 receptor is known to activate the MAPK signalling pathway lar players in this process (Sakamoto, Andoh, & Kuraishi, 2016). For
in mast cells (Desai & Thurmond, 2011). Interestingly, Sanna et al. example, genetically silencing the H4 receptor inhibited the production
(2015, 2018) also identified the effect of H4 receptor stimulation on of IL‐1β for human mast cells (Ebenezer, Prasad, Rajan, Thangam, &
the activity of the MAPK signalling pathway in neurons. They demon- Transduction, 2018), and H4 receptor activation was shown to stimu-
strated that modulation of this signalling pathway within the neurons late a number of cytokines, including IL‐6 (Jemima, Prema, & Thangam,
of the DRG, spinal cord, and sciatic nerve underpinned H4 receptor 2014). In addition, while H1 and H2 receptor antagonism reduced
agonist‐induced antiallodynic activity. They also revealed that neuro- hypersensitivity following nerve injury, it is possible that histamine
pathic pain hypersensitivity observed in H4 receptor‐KO mice is asso- released by mast cells contributes to the recruitment of neutrophils
ciated with an overactivation of the spinal ERK–cAMP response and macrophages in neuropathic pain and, acting via these histamine
element‐binding protein pathway in DβH immunoreactive neurons, receptors, contributes to the regulation of hypersensitivity in this type
supporting a potential association between the noradrenergic system of chronic pain (Jaggi et al., 2017; Smith et al., 2007; Zuo et al., 2003).
and H4 receptor‐mediated analgesia. In summary, increasing evidence There is an important aspect associated with H2 receptor antagonism,
arising from H4 receptor KO mice and the use of selective ligands sup- which should be considered for its therapeutic potential in neuro-
port H4 receptor as an interesting neuronal target for the treatment of pathic pain control. In vitro studies using CHO and HEK‐293 cells
chronic, particularly neuropathic, pain. identified time‐ and dose‐dependent up‐regulation of H2 receptors
upon long‐term exposure to H2 receptor antagonists (e.g., ranitidine),
which may underlie the development of tolerance after prolonged
clinical use of these ligands and result in the rebound hypersecretion
4 | H I S T A M I N E A N D NO N ‐ NE UR ON AL
of gastric acid and anaphylaxis that can occur after withdrawal of
CELLS IN NEUROPATHIC PAIN
treatment (Allen, Chazot, & Dixon, 2018; Smit et al., 1996). Thus, side
Following peripheral nerve injury, the immune system seems to play a effects linked to pharmacological tolerance may potentially compro-
vital role in the development of persistent inflammation and chronic mise long‐term efficacy and tolerability of H2 receptor antagonists in
neuropathic pain (Marchand, Perretti, & McMahon, 2005). Non‐ neuropathic pain. Little is known about the role of the H3 receptors
neuronal astrocytes, satellite glia cells, microglia, and mast cells play in non‐neuronal cells in neuropathic pain states.
key roles in communication between the immune system and the Overall, non‐neuronal cells play a key, but poorly, defined role in
CNS via the production of neuroinflammatory mediators, including his- the mechanisms underlying histamine‐mediated neuropathic pain.
tamine, 5‐HT, chemokines, and growth factors (Zhuang, Gerner, Woolf, We propose that neuronal H1 and H4 receptors (Ferreira et al.,
& Ji, 2005). The neuroimmune interactions between these two systems 2012) may orchestrate these mechanisms, with IL‐6 and IL‐1β cyto-
may reflect distinct roles in the development of chronic neuropathic kines as common denominator mediators. Further complications arise
pain (Zhao et al., 2017). Stimulation of H1 receptors via a from the recent observation that activated mast cells trigger microglial
PKC/MAPK/MEK1 signalling pathway has recently been shown to activation (Zhang, Wang, Dong, Xu, & Zhang, 2016). These cell types
elicit release of the key pro‐inflammatory cytokines IL‐1β and IL‐6 with, and their interactions may potentially go some way to explain the par-
subsequent, regulation of nerve growth factor release from astrocytes adoxical effects of histamine ligands, particularly for the H4 receptor,
(reviewed recently in Jurič, Kržan, & Lipnik‐Stangelj, 2016). Satellite seen in animal pain models.
glial cells, prominent in the PNS, including the DRGs, with active roles
in persistent neuropathic pain are also known to secrete the cytokine
5 | HISTAMINE AND ITS INTERACTION
IL‐6 in the chronic constriction injury neuropathic pain model, but the
WITH OPIOID SYSTEM IN NEUROPATHIC
identity profile of the histamine receptor in these cells has yet to be
PAIN
established (Dubový, Klusáková, Svíženská, & Brázda, 2010). Activated
microglia also release a myriad of pro‐inflammatory cytokines, including Interestingly, in neuropathic pain, high doses of opioids are required
notably, IL‐6, IL‐1β, and TNF‐α (Kempuraj et al., 2016; Mika, to achieve pain relief, and pharmacological tolerance to analgesic
Zychowska, Popiolek‐Barczyk, Rojewska, & Przewlocka, 2013). H3 effect of opioids develops rapidly (Osikowicz, Mika, Makuch, &
and H4 receptor activation of primary and clonal microglia has been Przewlocka, 2008). This phenomenon significantly restricts the clinical
shown to inhibit these cytokines (Ferreira et al., 2012). usefulness of opioids. In addition, the misuse of and addiction to
OBARA ET AL. 593
BJP
opioids, including prescription pain relievers, morphine and heroin, as To the best of our knowledge, the literature does not provide evi-
well as synthetic opioids, such as fentanyl, is a serious international dence for the mechanisms underlying histamine and opioid system
crisis that affects public health as well as social and economic welfare interactions, in relation to the modulation of morphine analgesic
(Lipman & Webster, 2015). effects. Given that the analgesic effects produced by modulation of
An interaction between histaminergic and opioidergic systems the activity of both the histamine and opioid systems could be associ-
within the CNS was suggested nearly 30 years ago, through an obser- ated with blocking SP release from peripheral nerve terminals (Barnes
vation that morphine administration resulted in the release of hista- et al., 1986; Przewłocki & Przewłocka, 2001), it is possible that an
mine and its increased turnover in the periaqueductal grey interaction that would result in potentiation of analgesic efficacy of
(Nishibori, Oishi, Itoh, & Saeki, 1985), suggesting that analgesia pro- morphine may involve, together with other possible mechanisms, the
duced by opioids may be associated with the stimulation of histamine inhibition of peripheral SP accumulation. Such an outcome may be
receptors at the supraspinal level. There are also data suggesting that useful for the management of neuropathic pain, particularly when
ligands of histamine receptors may modulate the analgesic action of peripheral administration of drugs is possible, thus affording reduction
opioids; however, the site and mode of this interaction differ of the undesired secondary effects associated with opioid administra-
between the spinal or supraspinal level, and depend on the subtype tion and peripheral mechanisms of action (e.g., constipation). How-
of histamine receptor involved (Mobarakeh et al., 2002; Mobarakeh ever, centrally acting drugs administered by peripheral routes should
et al., 2006; Mobarakeh, Takahashi, & Yanai, 2009). Specifically, a be taken into consideration due to the potential serious interactions
series of studies over the last two decades has shown that in H1, related to their pharmacodynamics and central mechanisms of action.
H2, or H3 receptor‐KO mice, morphine‐induced antinociception was For example, chlorpheniramine (a first‐generation H1 receptor antago-
significantly augmented when compared to the wild‐type controls in nist) was reported to potentiate fentanyl‐induced sedation and respi-
models of acute pain. H1 receptor‐KO mice showed a reduced spon- ratory depression after surgery (Anwari & Iqbal, 2003).
taneous nociceptive threshold as they responded to significantly
lower pain stimuli when compared to their controls (Mobarakeh
et al., 2002), while thresholds for pain perception in H2 receptor‐ 6 | CO NC LUSIO NS A ND FU TURE
KO mice were higher when compared to their corresponding controls DIRECTIONS
(Mobarakeh et al., 2006). Intrathecal administration of morphine in H1
and H3 receptor‐KO mice and i.c.v. morphine injection in H2 Findings from the last two decades indicate that selective pharmaco-
receptor‐KO mice produce enhanced analgesic effects (Mobarakeh logical antagonism of neurons expressing H3 receptors could provide
et al., 2002; Mobarakeh et al., 2006). Interestingly, pharmacological important and promising therapeutic approaches for the control of
blockade of H1 and H3 receptors by either intrathecal administration mechanical and cold hypersensitivity in peripheral neuropathies
of the first‐generation antihistamine chlorpheniramine (H1 receptor (Table 1). The analgesic effectiveness of H3 receptor antagonists/
antagonist) or thioperamide (H3 receptor antagonist, H3 /H4 receptor inverse agonists was comparable to gabapentin and pregabalin, first‐
inverse agonist), or H2 receptor antagonism, produced by zolantidine line treatments for neuropathic pain. Importantly, multiple examples
(i.c.v. route), resulted in the potentiation of the morphine analgesic of behavioural, electrophysiological, and molecular evidence strongly
effect (Mobarakeh et al., 2002; Mobarakeh et al., 2006). These support the rationale for this neuropathic pain strategy, particularly
behavioural studies, in both KO mice and involving pharmacological given their ability to modulate histamine levels as well as several
interventions, clearly demonstrated that blocking H1, H2 and H3 neurotransmitters critical for chronic pain processing. Moreover, the
receptors in combination with morphine may have beneficial effects recent registered approval of pitolisant (Wakix™), an antagonist/
on analgesia and suggested that endogenous histamine may exert inverse agonist of H3 receptors, for the treatment of narcolepsy in
an inhibitory effect on morphine‐induced analgesia acting via H1 patients, has opened the door for the potential use of H3 receptor
and H3 receptors at the spinal cord level and via H2 receptors at ligands for other conditions, including chronic neuropathic pain. How-
the supraspinal level. ever, due to the wide presynaptic and postsynaptic distribution of H3
Importantly, the observations observed with H3 receptor‐KO mice receptors throughout the CNS and PNS, more research is certainly
are consistent with a pharmacological study using a preclinical model needed to clarify the involvement of peripheral, spinal, and brain H3
of neuropathic pain induced by chronic constriction injury of the sci- receptors in various pain states, before determining their full potential
atic nerve. Here, Popiolek‐Barczyk et al. (2018) showed that blockade in neuropathic pain.
of H3 receptors by a selective antagonist (E‐162) significantly Recent findings also suggest the use of centrally permeable H2
enhanced morphine antinociception assessed with both mechanical receptor antagonists as promising new drug candidates for the treat-
and cold stimuli. Pharmacological analysis of these effects revealed ment of neuropathic pain, in view of their analgesic effects and meta-
an additive effect. Interestingly, Popiolek‐Barczyk et al. (2018) also bolic stability. Interestingly, however, despite the discovery of the
showed that TR‐7, a selective H4 receptor antagonist, significantly most recently discovered histamine receptor, the role of the H4 recep-
enhanced morphine antinociception in neuropathic pain. This latter tor in neuropathic pain transmission is still controversial after nearly
study is the first demonstration of the involvement of H4 receptors 20 years, with apparent confounding effects of both agonists and
in the regulation of morphine efficacy in chronic pain. antagonists on hypersensitivity associated with neuropathic pain. This
594 OBARA ET AL.
BJP
may be due to biased signalling of histamine and H4 receptor agonist ORCID

ligands and differential effects on multiple signalling pathways in cen- Ilona Obara https://orcid.org/0000-0002-8906-2771
tral and peripheral parts of the sensory nervous system. Furthermore,
the paucity of detailed mechanistic definitions of histamine‐mediated RE FE RE NC ES
analgesia, and the additive effects with the opioid system, requires Alexander, S. P. H., Christopoulos, A., Davenport, A. P., Kelly, E., Marrion,
attention to provide a rationale to the field of histamine and develop- N. V., Peters, J. A., … CGTP Collaborators. (2017). The Concise Guide
ment of neuropathic pain control therapeutics. to PHARMACOLOGY 2017/18: G protein‐coupled receptors. British
Journal of Pharmacology, 174, S17–S129. https://doi.org/10.1111/
A better understanding of the interaction between histaminergic
bph.13878
signalling pathway molecules (Figures 1–3) and histamine receptors
Alexander, S. P. H., Fabbro, D., Kelly, E., Marrion, N. V., Peters, J. A.,
may result in the identification of further novel pharmacological
Faccenda, E., … CGTP Collaborators (2017). THE CONCISE GUIDE
targets to improve neuropathic pain control. The literature available TO PHARMACOLOGY 2017/18: Enzymes. British Journal of Pharma-
provides some evidence for potential pharmacological target mole- cology, 174(S1), S272–S359. https://doi.org/10.1111/bph.13877
cules. One potential strategy exploits the role of Ca2+ channels in Alexander, S. P. H., Peters, J. A., Kelly, E., Marrion, N. V., Faccenda, E.,
the regulation of cellular excitability associated with nociception Harding, S. D., … CGTP Collaborators (2017). THE CONCISE GUIDE
TO PHARMACOLOGY 2017/18: Ligand‐gated ion channels. British
(e.g., N‐type Ca2+ channels). Evidence has shown that Ca2+ channel
Journal of Pharmacology, 174(S1), S130–S159. https://doi.org/
blockers (e.g., ω‐conotoxin‐MVIIA/Prialt) offer interesting analgesic 10.1111/bph.13879
potential in treating neuropathic pain (Vanegas & Schaible, 2000). Alexander, S. P. H., Striessnig, J., Kelly, E., Marrion, N. V., Peters, J. A.,
However, there is no evidence for the effect produced by a combina- Faccenda, E., … CGTP Collaborators (2017). THE CONCISE GUIDE
tion of Ca2+ channel blockers and histamine receptor ligands, and we TO PHARMACOLOGY 2017/18: Voltage‐gated ion channels. British
Journal of Pharmacology, 174(S1), S160–S194. https://doi.org/
propose that their interaction should be taken into consideration.
10.1111/bph.13884
Another potential target involves the contribution of the
Allen, S. J., Chazot, P. L., & Dixon, C. (2018). Can H2‐receptor upregulation
MAPK/ERK signalling pathway to the regulation of pain hypersensi-
and raised histamine explain an anaphylactoid reaction on cessation
tivity. Recently, Sanna et al. (2015) showed that H4 receptor stimula- of ranitidine in a 19‐year‐old female? A case report. British Journal of
tion, which led to analgesic activity in neuropathic pain, was Clinical Pharmacology, 84, 1611–1616. https://doi.org/10.1111/
modulated by MAPK/ERK signalling in the neurons of the DRG, bcp.13578
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… Shayo, C. (2013). Cross‐desensitization and cointernalization of H1
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Current Neuropharmacology, 2018, 16, 415-425 415
REVIEW ARTICLE
New Insights on Neuronal Nicotinic Acetylcholine Receptors as Targets for

Pain and Inflammation: A Focus on α7 nAChRs
Deniz Bagdasa,b,*, Mine S. Gurunc, Pamela Floodd, Roger L. Papkee and M. Imad Damaja
a
Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia, USA;
b
Experimental Animals Breeding and Research Center, Faculty of Medicine, Uludag University, Bursa, Turkey;
c
Department of Pharmacology, Faculty of Medicine, Uludag University, Bursa, Turkey; dDepartment of Anesthesiology,
Perioperative and Pain Medicine, Stanford University, Palo Alto, California, USA; eDepartment of Pharmacology and
Therapeutics, University of Florida, Gainesville, Florida, USA
Abstract: Background: Nicotine and nicotinic acetylcholine receptors (nAChRs) have been ex-
plored for the past three decades as targets for pain control. The aim of this review is to introduce
readers particularly to α7 nAChRs in a perspective of pain and its modulation.
A R T I C L E H I S T O R Y
Methods: Developments for α7 nAChR modulators and recent animal studies related to pain are
reviewed.
Received: May 04, 2017
Revised: June 20, 2017 Results: Accumulating evidences suggest that selective ligands for α7 nAChRs hold promise in the
Accepted: August 16, 2017
treatment of chronic pain conditions as they lack many of side effects associated with other nico-
DOI: tinic receptor types.
10.2174/1570159X15666170818102108
Conclusion: This review provides the reader recent insights on α7 nAChRs from structure and
function to the latest findings on the pharmacology and therapeutic targeting of these receptors for
the treatment of pain and inflammation.
Keywords: α7 nicotinic acetylcholine receptors, nicotine, pain, inflammation, modulators, anti-inflammatory, agonists.
1. INTRODUCTION 1.1. Alpha7 Nicotinic Acetylcholine Receptor Structure

and Modulation
Nicotine and other ligands that act on nicotinic acetyl-
choline receptors (nAChR) have been explored for the past The nAChRs are pentameric assemblies of subunits con-
three decades as a strategy for pain control. These receptors figured around a central ion channel pore that can be gated
are widely expressed throughout the central and peripheral by the binding of ligands (orthosteric agonists) to sites in the
nervous system as well as on immune cells. Despite encour- N-terminal extracellular domains at the interface between
aging results with many selective α4β2* agonists in animal subunits. The first nAChR to be studied and isolated were
models of pain, human studies showed a narrow therapeutic those of the neuromuscular junction and the homologous
window between analgesic efficacy and toxicity associated electric organ of the Torpedo ray. These receptors evolved to
with the use of these agonists as analgesics (For a recent rapidly and efficiently activate when stimulated with a large
chapter see [1]). However, several recent developments have increase in acetylcholine (ACh) concentration [3]. These
potentially opened new windows of opportunity in the use of muscle-type receptors were found to have four different
nicotinic agents for analgesia. α7 nAChR agonists have types of subunits, referred to as α1, β1, γ and δ, with two α1
shown to be beneficial for central nervous system disorders subunits per receptor. For the most part, each subunit had a
characterized by cognitive deficits, such as Alzheimer’s relatively well-conserved structure, with three transmem-
disease and schizophrenia [2]. In addition, accumulating brane domains following the N-terminal extracellular do-
evidences suggest that agonists and modulators of nicotinic main, a more variable intracellular domain [4], followed by a
receptors that contain the α7 subunit hold promise for the fourth transmembrane domain. The α1 subunits contained a
treatment of chronic inflammatory pain conditions. pair of disulfide-linked vicinal cysteines, and all of the
subunits contained a disulfide-constrained loop in a region
later shown to be at the interface between the extracellular
*Address correspondence to this author at the Department of Pharmacology and transmembrane domains. This hallmark "Cys-loop" was
and Toxicology, Medical College of Virginia Campus, Virginia Common-
wealth University, Richmond, VA 23298-0613; Tel/Fax: +1-804-828-9256;
ultimately found in all nAChR subunits subsequently cloned,
E-mail: dbagdas@vcu.edu as well as in homologous receptors activated by GABA, gly-
1570-159X/18 $58.00+.00 ©2018 Bentham Science Publishers

416 Current Neuropharmacology, 2018, Vol. 16, No. 4 Bagdas et al.
cine, or serotonin, identifying the Cys-loop superfamily of non-neuronal cells, where no α7-mediated ionic currents can
ligand-gated ion channels (LGIC). be detected [13-15].
The family of nAChR subunits was ultimately expanded The identification of cholinergic anti-inflammatory activ-
to include one additional alternative muscle subunit, epsilon, ity mediated by α7 nAChR in cells of the immune system
and twelve additional subunits cloned from neuronal tissues. [16-18] has drawn attention to the likelihood that ligand-
The neuronal subunits were classified as alpha subunits (α2 - induced conformational changes of α7 receptors are global
α10, although α8 is found only in birds) if they contained and apparently encompass changes in signaling associated
vicinal cysteines homologous to those in α1, or as β subunits with the receptor's interactome [19] and potentially with G-
(β2 - β4) if they did not. Each subunit has a unique pattern of protein-mediated signals [20]. While the prokaryotic Cys-
expression associated with diverse functions for nAChR in loop receptor homologs lack any intracellular domains, the
different tissues and brain structures [5]. Most nAChR sub- vertebrate nAChR subunits show remarkable diversity and
types are heteromers containing both α and non-alpha specialization in their intracellular domains, and the unique
subunits with two orthosteric agonist-binding sites contrib- intracellular domain of α7 receptors has been especially well
uted by α subunits in each receptor, as in the muscle-type conserved throughout the evolution of vertebrates [4].
receptor. In the rodent brain the predominant nAChRs con-
As noted above, the unique configuration of the α7 or-
tain combinations of α4 and β2 subunits, and these receptors
thosteric agonist binding between pairs of identical, rather
were shown to bind radiolabeled ACh or nicotine with high
affinity. It was also shown that a second class of nAChR is than specialized subunits, allows for these receptors to be
effectively targeted by multiple classes of selective agonists
present in brain that did not bind ACh or nicotine with high
[21]. The presence of five potential agonist-binding sites per
affinity but did bind the neuromuscular receptor blocker α-
receptor also permits multiple types of ligand-induced con-
bungarotoxin [6]. These were subsequently shown to be as-
formational states based on the level of binding site occu-
sociated with the expression of the α7 subunit, which could
pancy. Data suggest that only relatively low levels of binding
form receptors without the co-expression of a beta subunit
[7]. site occupancy effectively promote channel activation (albeit
with low probability) [22-24] and that higher levels of ago-
Homomeric α7 nAChR lacks the specializations for syn- nist occupancy preferentially induce nonconducting (i.e. de-
aptic function that are present in muscle-type and ganglionic sensitized) states that are far more stable than the open chan-
nAChR [3]. The absence of non-alpha subunits modifies the nel state. The existence of these unique nonconducting states
agonist binding site, reducing high-affinity agonist binding has been confirmed by the activity of α7-selective positive
to desensitized states and increasing sensitivity to the ubiqui- allosteric modulators (PAMs), which, at least in the case of
tous ACh precursor choline. In regard to functioning as a efficacious type II PAMs like PNU-120596 [25], can desta-
homopentamer, α7 nAChR is like the proton-gated channels bilize the nonconducting conformations and couple them to
Gloeobacter ligand-gated ion channel (GLIC) [8] and Er- unique open-channel states [11, 26, 27]. The identification of
winia ligand-gated ion channel (ELIC) [9] found in bacteria, diverse classes of α7-selective PAMs [25] opened up a new
which are believed to be homologs of the vertebrate Cys- avenue for α7-directed drug development [28] and ultimately
loop LGIC. led to the identification of another novel class of α7-channel
activators, ago-PAMs, including 4-BP-TQS [29], its active
The unique cellular and subcellular α7 expression pat-
isomer GAT107 [30], and more recently, the structurally
terns indicate special roles for this receptor subtype. This
unrelated B-973 [31], that function both as PAMs and allos-
unique pattern includes wide spread expression in non-
teric agonists. The allosteric agonism has been hypothesized
neuronal cells, including cells of the immune system where
to be associated with a specific allosteric activation site in
α7 has been uniquely implicated in regulating the cholinergic
the extracellular domain [32].
anti-inflammatory pathway. Additionally, due to its ability to
be activated by choline in addition to ACh and its rapid con- Agonists, PAMs and ago-PAMs are all capable, to vary-
centration-dependent desensitization of ion channel currents, ing degrees, of activating α7 ion channel currents, although
α7 nAChR will respond in a very different way from other for a PAM that activation is conditional on the coincident
nAChR to endogenous cholinergic signals, including presence of an agonist. All of these currents can be antago-
paracrine signals in peripheral tissues. nized by both competitive and non-competitive antagonists,
for example MLA and mecamylamine, respectively. How-
Open α7 ion channels have high calcium permeability ever, the conducting states activated in the presence of a
[7]. While the high calcium permeability of NMDA-type
PAM differ subtly in their sensitivity to specific antagonists
glutamate receptors is responsible for their key role in synap-
[27] than those activated by orthosteric agonist in the ab-
tic plasticity, it has been implicated to lead to the potential
sence of a PAM. Additionally, molecules have been identi-
for excitotoxic activation, in the case of α7. This feature is
fies which specifically antagonize the effects of PAMs with
offset by the fact that normally the open probability of the α7
little or no effects on activation by ordinary orthosteric ago-
receptor channel is extremely low compared to other nAChR nists [33]. Conversely, silent agonists [34], which produce
[10,11]. Changes in intracellular calcium concentration sub-
channel activation in the presence of a PAM, behave as very
sequent to α7 stimulation are typically more due to release of
low efficacy partial agonists in the absence of a PAM.
calcium from intracellular stores rather than calcium influx
through the α7 channels [12], suggesting a metabotropic-like While the primary focus for the use of α7-PAMs has
function for α7 nAChR. This may especially be the case for been as tools to overcome the intrinsic limitations to α7
New Insights on Neuronal Nicotinic Acetylcholine Receptors as Targets Current Neuropharmacology, 2018, Vol. 16, No. 4 417
channel activation, they have also been used to investigate attenuation of pro-inflammatory cytokine production and
the induction of nonconducting conformational states of the inhibition of the inflammatory process via activation of α7
receptor that may play important roles in the metabotropic nAChRs [15, 66]. This neurophysiological mechanism de-
functions of α7 receptors. As noted above, while the open- creases inflammation by reducing cytokine synthesis via
channel state of α7 is very unstable, the nonconducting states release of ACh in organs of the reticulo-endothelial system,
that can be converted to conducting states by the activity of such as the lungs, spleen, liver, kidneys and gastrointestinal
PAMs are far more stable. This has allowed the use of PAMs tract [67]. It has been revealed that α7 nAChRs are impli-
to identify "silent agonists", ligands that bind to a site that cated in modulating tumor necrosis factor (TNF-α), interleu-
overlaps the agonist binding site [34], do not produce sig- kin-1 (IL-1), interleukin-6 (IL-6), interleukin-18 (IL-18),
nificant activation when applied alone, but will activate cur- high mobility group box 1 (HMGB1) and some other pro-
rents in the presence of a PAM. NS6740 was the first silent inflammatory cytokines without affecting the anti-
agonist introduced in the literature. It was reported to be an inflammatory cytokine interleukin-10 (IL-10) [39,66,68-70].
α7 ligand that required a PAM to produce channel activation. Indeed, a critical role for α7 nAChR as a peripheral compo-
NS6740 was later shown to be very effective at inducing nent in cholinergic anti-inflammatory pathway has been
stable nonconducting states of the receptor and also to be demonstrated using α7 subunit knockout (KO) mice [17].
very effective at reducing the behavioral manifestations of
Intraplantar complete Freund’s adjuvant or inactivated and
pain in several animal models. While NS6740 was ineffec-
dried mycobacteria, which induces chronic inflammation and
tive in an animal model of cognition, it was subsequently
inflammatory pain, injection increased more edema, hyper-
shown that both NS6740 and GTS-21, a low-efficacy α7-
algesia and allodynia in the α7 KO mice compared with the
selective partial agonist, were effective modulators of im-
mune signaling in microglia [35]. Interestingly, the α7 ago- wild-type (WT) littermates [71]. ACh and nicotine pre-
PAM GAT107, which also induces stable nonconducting treatment inhibited lipopolysaccharide (LPS)-induced TNF-α
states, had a profile similar to that of NS6740 in the same release in murine-derived microglial cells through α7
pain models [36]. The long-term effects of NS6740 on ACh- nAChR activation [60]. Another study also showed that pre-
evoked responses are desensitizing [37], while those of treatment with ACh inhibited LPS-induced matrix metallo-
GAT107 are potentiating [30], suggesting that the anti- proteinase 9 (MMP-9) production and macrophage migration
inflammatory activity reducing the manifestations of pain in vitro [72]. It has been reported that activation of these
may be specifically associated with the nonconducting states receptors by an agonist attenuated TNF-α and IL-1β levels in
of the receptor. human whole blood activated by exposure to endotoxin [50]
and microglial α7 nAChR activation reduced TNF-α release
1.2. Alpha7 Nicotinic Acetylcholine Receptor Expression but not IL-1β [58]. Choline has also been shown to modulate
TNF-α release via α7 nAChR-mediated signaling [73]. CDP-
α7 nAChR is present in supraspinal and spinal pain- choline is an endogenously synthesized nucleotide which
transmission pathways [7, 38-42]. Autoradiographic analyses
rapidly metabolizes to choline and cytidine/uridine. Consis-
showed that α7 nAChR binding sites were numerous within
tently, exogenous administration of CDP-choline results in
the substantia gelatinosa in rat [43] and human [40] spinal
elevations in plasma and tissue levels of choline [74, 75].
cord, and these sites were reduced following dorsal
When CDP-choline, also a source for the synthesis of the
rhizotomy [44]. α7 nAChRs have been shown to be ex-
pressed on immune and non-immune cytokine-producing cholinergic neurotransmitter ACh, was locally administered,
cells such as macrophages [17, 45, 46], microglia and kerati- it reduced the production of the TNF-α, reduced edema and
nocytes [17, 39, 47-49]. They are also expressed in mono- reversed the mechanical hyperalgesia through α7 nAChR,
cytes [50-52], T-cells [53, 54], and B-cells [55, 56]. α7 suggesting that the local application of α7 nAChR activators
nAChRs that are expressed on macrophages, which are key may provide a tool to reduce the local inflammation and pain
immune cells involved in the initiation, maintenance, resolu- [76]. Nuclear translocation of nuclear factor-κB (NF-κB) is
tion and modulation of inflammatory processes [15, 39, 45]. the main factor of immune cell activation and proinflamma-
For example, they regulate cytokine release [17, 57]. An- tory cytokines expression [77]. It has been shown that α7
other important expression localization for these receptors is nAChR activation decreases NF-κB activity and reduces
microglia, which can remain in the activated state for a proinflammatory response [17, 51]. It has been suggested that
longed period of time to evoke secretion of various inflam- different mechanisms effect α7 nAChR activation on NF-κB
matory factors [58-60]. activity [51, 78]. One possible mechanism is the recruitment
of Janus kinase-2 (Jak2) to the α7 nAChR and activation of
Both RIC-3, and a newly identified accessory protein Jak2, thereby initiating the anti-inflammatory signal trans-
NACHO [61] have been shown to be crucial regulators of α7 ducer and activator of transcription 3 (STAT3) and suppres-
functional expression on both cellular and subcellular levels. sor of cytokine signaling 3 (SOCS3) signaling cascade.
While the only known function for NACHO has been as a
Vagus nerve stimulation has been shown to attenuate macro-
regulator of α7, RIC-3 has been more broadly implications as
phage activation through activating the Jak2-STAT3 signal-
a regulator of other cys-loop receptors [61-65].
ing pathway and STAT3 phosphorylation, a potential nega-
1.3. Anti-inflammatory Signaling of alpha7 Nicotinic tive regulator of inflammatory responses, depends on α7
Acetylcholine Receptors nAChR activation [78, 79]. In addition, α7 nAChR activation
leads to suppression of the phosphorylation of I kappa B
α7 nAChR endogenous ligands, ACh and choline, are (I-κB) which results in inhibition of transcriptional activity
closely associated with controlling immune cell functions, of NF-κB and inhibition of proinflammatory mediators [51].
Another possible mechanism may result from an interaction modulate nerve injury- and chemotherapy-induced neuro-
between α7 nAChRs and toll-like receptor 4 (TLR4) through pathic pain through α7 nAChRs [94, 95]. The partial agonist
myeloid differentiation factor 88 (MyD88) and NF-κB. GTS-21 has been reported to have antinociceptive properties
MyD88, a key intracellular reactive protein for TLR4, could in a mouse model of postoperative pain [50]. Stimulation of
activate I-κB kinases (IKK) and induce NF-κB translocation cholinergic anti-inflammatory pathways resulted in attenua-
[80]. Nicotine has been shown to decrease the expression of tion of neuroinflammation in a tibial fracture model of rats
MyD88 and to interfere NF-κB transcription in an α7 through α7 nAChR [96]. Choline has been also reported to
nAChR antagonist α-bungarotoxin dependent manner. Con- attenuate a model of postoperative pain in mice [97]. Choline
sequently, nicotine reduced TNF-α expression through α7 and nicotine have been shown to enhance inhibitory synaptic
nAChR/MyD88/NF-κB pathway [81]. Many other down- transmission in the dorsal horn neurons of spinals cords of
stream pathways may involve α7 nAChRs and TLR4 interac- rats [98]. However, there are some limitations to the devel-
tion, such as overexpression of IL-1 receptor-associated opment α7 nAChR agonists for the treatment of pain, such as
kinase M (IRAK-M), a negative regulator of TLR4, in receptor selectivity issues (cross-reactivity with 5-HT3 re-
macrophages resulted in decreased TNF-α production ceptors, which have high homology with α7 nAChRs, and
through a Jak2-STAT3 signaling pathway by α7 nAChR possible related adverse effects), overactivation and possible
activation [82]. HMGB1, a cytokine mediator of inflamma- desensitization of the receptor. In addition, while α7 nAChR
tion, has been reported to upregulate α7 nAChR expression agonists have shown beneficial effects in chronic pain mod-
[83]. In addition, vagus nerve stimulation or cholinergic re- els in some studies, this effect was not consistently seen in
ceptor agonists have been suggested to inhibit the activation others [99]. Efficacy of various α7 nAChRs ligands in vari-
of NACHT, LRR, PYD domains-containing protein 3 ous animal models of pain is shown in Table 1. Therefore,
(NLRP3) inflammasome, a key modulator of innate immu- alternative approaches to overcome these limitations associ-
nity, whereas genetic deletion of α7 nAChR enhances in- ated with direct nAChR agonists were developed in the last
flammasome activation. Mitochondrial α7 nAChR activation decade.
suppressed the release of mitochondrial DNA, an NLRP3
In addition to agonists, α7 nAChRs PAMs also provide a
ligand, as a result IL-1β and HMGB1 expressions were sup-
unique strategy for rational drug design and discovery. As
pressed [84]. It has also been shown that α7 nAChR gene
discussed above, PAMs for α7 nAChRs increase the potency
expression was increased in septic patients' peripheral blood
and/or maximal efficacy of endogenous (ACh and choline)
mononuclear cells and these patients responded dynamically
or exogenous agonists for the α7 nAChRs without directly
to the inflammatory challenge; besides a group of patients
activating α7 nAChRs. Recently, several structurally distinct
with low levels of α7 expression had a defective response to
and selective α7 nAChR PAMs were identified and classified
infection and showed worst clinical outcome with respect to
as type I and type II based on their electrophysiological
inflammation [85]. This is also suggestive that there might
properties [100, 101]. Type I PAMs increase agonist re-
be bilateral interactions between α7 nAChR and inflamma- sponse with little or no effect on desensitization of α7
tory mediators. With regard to these results, α7 gene expres- nAChRs, whereas type II PAMs increase agonist response
sion level in septic patients might be used to assess choliner- and slow down the apparent desensitization profile of the
gic anti-inflammatory pathway activity as a clinical marker. agonist response [28]. It has been suggested that PAMs may
Furthermore, ligands for α7 nAChRs might open new strate- modulate conformational states for both channel activity and
gies to monitor and cure inflammatory diseases such as ul- ion channel-independent signaling [27]. Importantly, it has
cerative colitis, sepsis, acute pancreatitis and asthma. There- been reported that in contrast to α7 agonists, α7 nAChR
fore, these receptors present an alternative therapeutic strat- PAMs do not induce upregulation of α7 nAChR expression
egy for modulation of inflammation-based syndromes. in the brain in vivo [102].
1.4. Effects of alpha7 Nicotinic Acetylcholine Receptors Both PAM types have been recently tested in vivo for
Agonists and Modulators in Rodents Pain Models their efficacy in animal models of inflammatory and neuro-
pathic pain. Type II but not type I PAMs were shown to be
Animal studies have shown that α7 nAChR agonists such efficacious in these models [92, 103]. This is different from
as choline, compound B [(R)-N-(1- azabicyclo[2.2.2]oct-3- rodent models of cognition and memory, where both type I
yl)(5-(2-pyridyl)thiopene-2-carboxamide)], JN403, PHA- and type II PAMs for the α7 nAChRs showed cognitive en-
543613 and AR-R17779 exhibit anti-inflammatory effects in hancement. There are several examples of antinociceptive,
various inflammatory pain models in rodents [86-90]. In antiallodynic and antihyperalgesic activities of type II
addition, they were found to be active in chronic neuropathic PAMs. PNU-120596, an α7 nAChR type II PAM, has well
pain models [91-93]. For example, repeated administration described activity in tonic, inflammatory and chronic pain
of a selective α7 agonist decreases allodynia in a chronic models in rodents [92, 103, 104]. Recently, another α7
neuropathic pain model in the rat and reverses signs of neu- nAChR type II PAM, 2,4,2’,5’-tetrahydroxychalcone, has been
roinflammation and neurodegeneration (macrophage infiltra- found active against complete Freund’s adjuvant-induced
tion, decreases in axon compactness and diameter with a inflammatory pain in rats [105]. In addition, 3-furan-2-yl-N-
significant loss of myelin sheaths) [93]. Not limited to direct p-tolyl-acrylamide, an α7 nAChR type II PAM agent reduces
α7 nAChR agonists, CDP-choline which is an intermediate neuropathic and inflammatory pain outcomes in mice in a
in the biosynthesis of phosphatidylcholine and leads to an dose and time dependent manner [92,106]. Different mecha-
increase in choline levels, has been reported as effective to nisms may underlie PAM-induced anti-inflammatory effects.
Table 1. Efficacy of α7 neuronal nicotinic acetylcholine receptor ligands in various animal models of pain.
Compound Ligand Animal Model Animal Response Refs.

Type
Choline Agonist Murine endotoxemia and Mice Reduced systemic TNF levels, suppressed HMGB1 release [73]
sepsis
Choline Agonist Tail flick test Mice Antinociceptive [87]
Choline Agonist Incisional postoperative pain Mice Reduced thermal hyperalgesia and mechanical allodynia [97]
CDP-choline Source of Carrageenan Rats Reduced mechanical hyperalgesia [76]

choline decreased TNF-α in the paw
CDP-choline Source of Oxaliplatin-induced CIPN Rats Reduced mechanical hyperalgesia [94]

choline
CDP-choline Source of CCI Rats Reduced mechanical hyperalgesia [95]

choline
JN403 Agonist CFA and partial sciatic nerve Rats Reduced mechanical hyperalgesia [86]
ligation models
Tail flick test Lack of effect
DMXB and Partial Tail flick test Mice Lack of effect by itself [87]
4-OH-DMXB agonist blocked choline-induced antinociception
Compound-B Agonist CFA Rats, Reduced mechanical hyperalgesia [88]

mice
Compound-B Agonist Carrageenan and CFA Rats Reduced mechanical hyperalgesia [104]
Formalin models Attenuated pain behavior in formalin test
Nicotine Agonist CIA model Mice Ameliorated arthritis and reduced synovial inflammation [89]
AR-R17779 Agonist CIA model Mice Ameliorated arthritis and reduced synovial inflammation [89]
TC-7020 Agonist CCI Rats Reduced mechanical allodynia [91]
PHA-543613 Agonist CCI Mice Reduced mechanical allodynia [92]
PNU-282987 Agonist CCI Rats Reduced mechanical hyperalgesia [93]
NS1738 Type I Carrageenan Mice Reduced thermal hyperalgesia [92]

PAM CCI Lack of effect
NS1738 Type I Hot plate and tail-flick tests Mice Lack of effect [103]
PAM Formalin model Lack of effect
PNU-120596 Type II Carrageenan Mice Reduced thermal hyperalgesia [92]

PAM CCI reduced thermal hyperalgesia and mechanical allodynia
potentiated effects of PHA-543613
PNU-120596 Type II Carrageenan and CFA Rats Reduced mechanical hyperalgesia and weight-bearing [104]
PAM Formalin model deficit
lack of effect
PNU-120596 Type II Hot plate and tail-flick tests Mice Lack of effect [103]
PAM formalin model Attenuated pain behavior in formalin test
2,4,2’,5’- Type II CFA Rats Lack of effect on thermal hyperalgesia reduced mechanical [105]
tetrahydroxychalcone PAM hyperalgesia
3-furan-2-yl-N-p- Type II Carrageenan Mice Reduced mechanical allodynia by itself and potentiated [106]
tolyl-acrylamide PAM antiallodynic effect of choline
CFA Reduced thermal hyperalgesia
CCI Reduced mechanical allodynia
CPA Reversed negative affective behaviors
(Table 1). contd….
Compound Ligand Animal Model Animal Response Refs.

Type
GAT107 Ago- Tail flick and hot plate tests mice Lack of effect [36]
PAM Formalin model antinociceptive
(allosteric CFA and CCI models reduced thermal hyperalgesia and mechanical allodynia
agonist LPS reduced mechanical allodynia
and type Acetic acid induced visceral decreased stretching behavior and reversed negative
II PAM) stretching and CPA affective behaviors
NS6740 Silent Formalin model mice Attenuated pain behavior in formalin test [37]
agonist CCI reduced mechanical allodynia
CPA reversed negative affective behaviors
PMP-072 Silent CIA mice Reduced arthritis [109]

agonist
CCI—Chronic constrictive nerve injury, CFA—Complete Freund's adjuvant, CIA—collagen-induced arthritis, CIPN—chemotherapy-induced peripheral neuropathy, CPA—
Conditioned place aversion, PAM—positive allosteric modulator, PSNL—Partial sciatic nerve ligation, SNL—Spinal nerve ligation.
PNU120596 reduced TNF- α and IL-6 within the hind paw orthosteric binding site of the receptor, but preferentially
edema in a rat model of the carrageenan test [104]. The acti- induce a nonconducting state associated with desensitization
vation of spinal extracellular signal-regulated kinase-1/2 that can none the less modulate signal transduction [108,
(ERK1/2) pathways are the likely one possible post receptor 109]. Recent studies showed that α7 nAChR-selective silent
signaling mechanism for the antinociceptive effect of PNU- agonism may provide a novel strategy for pain management.
120596 in the formalin test [92]. Although, α7 nAChR NS6740, α7 silent agonist, induces nonconducting states of
modulation attenuates inflammatory and neuropathic pain, the receptor. NS6740 modulates the inflammatory responses
they are mostly lack of efficacy for acute pain [36, 37, 103]. of microglia cells in vitro and it is effective in both chronic
Additional studies to fully clarify the analgesic-like proper- constriction nerve injury-induced neuropathic pain and for-
ties of α7 nAChR are necessary in animal models of chronic malin model of tonic inflammatory pain [37]. Another α7
pain. nAChR silent agonist PMP-072 has also been shown to have
anti-inflammatory effects in collagen-induced arthritis in
An alternative therapeutic approach involves allosteric mice [109]. The behavioral and pharmacological profile of
agonist and allosteric modulators called ago-PAMs. While PMP-072 and NS6740 in these models are consistent with
PAMs are thought to bind to a distinct binding site from the the induction of nonconducting conformational states of the
orthosteric site and thus lack intrinsic agonist activation, receptor which confirmed in vitro studies. Indeed, data ob-
recent studies have reported that some molecules show dual tained from α7 nAChR silent agonist R-47 (another name of
activity as allosteric modulators and allosteric agonists compound PMP-072) support the hypothesis that the anti-
[33,107]. GAT107 is a potent α7 nAChR type II PAM with inflammatory effects of silent agonism was mediated by a
intrinsic allosteric agonist activities suggesting it is an ago- signal transduction pathway that was independent of ion cur-
PAM for α7 nAChRs [107]. In several mouse models of rent [110]. α7 nAChRs are downregulated in dorsal root
chronic inflammatory and neuropathic pain, GAT107 attenu- ganglion and spinal cord of rats by oxaliplatin treatment
ated pain behavior and showed anti-inflammatory effects. [111] suggesting that α7 nAChRs might have modulatory
Furthermore, intrathecal, but not intraplantar, injections of role in chemotherapy-induced neuropathic pain. In support
GAT107 reversed nociception in the complete Freund’s ad- of this hypothesis, another study showed that α7 nAChR
juvant-induced inflammatory pain model, suggesting a spinal activation protected neurons from oxaliplatin-induced toxic-
mechanism of action. Immunohistochemical evaluation of ity through a mechanism related to the neuroprotective func-
the lumbar spinal cord dorsal horn of mice treated with com- tions of astrocytes [112]. Ligands that favor non-conducting
plete Freund’s adjuvant showed an increase in the expression states are favorable compared to α7 full agonists that en-
of astrocyte-specific glial fibrillary acidic protein (GFAP) hanced tumor growth in small cell lung cancer cell lines
and phosphorylated p38-mitogen-activated-kinase (p- [113], and in mouse model of lung cancer [114]. Because of
p38MAPK). Treatment with intrathecal GAT107 robustly these considerations, α7 agonists have limited potential for
attenuated these inflammatory changes. These findings sug- the treatment of chemotherapy-induced neuropathic pain.
gest that α7 ago-PAMs represent a novel and effective phar- Targeting alternative conformational states of α7 nAChRs,
macological strategy for treating inflammatory and neuro- such as silent agonism, may provide a better therapeutic ap-
pathic pain in mice [36]. proach for this common type of neuropathic pain. The recent
characterization of α7 nAChR silent agonists has created
An additional new class of selective ligand has been new opportunities for targeting these receptors as analgesic
identified for α7 nAChR. While direct α7 nAChR agonists agents for cancer and chemotherapy-induced peripheral neu-
bind to the receptor on the orthosteric binding site to activate ropathy. It is very possible that α7 silent agonists may be
the channel, ligands called “silent agonists” also bind the more efficacious to prevent or attenuate the chemotherapy-
induced neuropathic pain. Further pre-clinical behavioral SOCS3 = Suppressor of cytokine signaling 3
studies will clarify the effects of silent agonists potentially
I-κB = I kappa B
influencing their future clinical development.
TLR4 = Toll-like receptor 4
Pain has been described as a multi-dimensional state
composed of sensory, affective, and cognitive components MyD88 = Myeloid differentiation factor 88
[115-117]. As such, pain states that require clinical interven-
tion are often accompanied by changes in affective behaviors IKK = I-κB kinases
that have complex interplay with the nociceptive aspects of IRAK-M = IL-1 receptor-associated kinase M
the pain response [118-121]. A positive feature of α7 nAChR
ligands is that in contrast to opioids, they appear to improve NLRP3 = NACHT, LRR, PYD domains-
pain related affective behaviors in preclinical models. The containing protein 3
conditioned place aversion model [122] was used to test the ERK1/2 = Extracellular signal-regulated kinase-
activities of several α7 nAChR. The type II PAM 3-furan-2- 1/2
yl-N-p-tolyl-acrylamide, silent agonist NS6740 and ago-
PAM GAT107 were effective to reverse negative affective GFAP = Astrocyte-specific glial fibrillary
behaviors associated with visceral pain induced by acetic acidic protein
acid in mice [36, 37, 106]. These findings suggest that α7 p-p38MAPK = Phosphorylated p38-mitogen-activated-
nAChR modulation may serve an efficacious strategy to treat kinase
both the sensory and affective components of pain.
CONSENT FOR PUBLICATION
CONCLUSION
Not applicable.
In summary, many of immune cells and non-immune
neuronal cells have been reported to express α7 nAChR sub- CONFLICT OF INTEREST
type. This receptor subtype affords a critical role on the ini-
tiation, maintenance and modulation inflammation in addi- The authors declare no conflict of interest, financial or
tion to direct neuronal signaling. Exploring different path- otherwise.
ways to the activation and/or modulation of α7 nAChRs and
ACKNOWLEDGEMENTS
determining downstream signaling pathways will provide
data critical to develop beneficial α7 nAChR ligands for This work was supported by grants from National Insti-
translational research on pain and inflammation. tute of Health [R01-CA206028] to MID, [GM57481] to RLP
and from Research Fund of Uludag University [KUAP (T)
LIST OF ABBREVIATIONS 2016/15 and 2016/16) to MSG.
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REVIEW
published: 21 August 2020
doi: 10.3389/fncel.2020.00256
Expression and Function of GABA

Receptors in Myelinating Cells
Mari Paz Serrano-Regal 1,2,3† , Laura Bayón-Cordero 1,2,3 , Rainald Pablo Ordaz 4 ,
Edith Garay 4 , Agenor Limon 5 , Rogelio O. Arellano 4 , Carlos Matute 1,2,3 * and
María Victoria Sánchez-Gómez 1,2,3 *
1
Laboratory of Neurobiology, Achucarro Basque Center for Neuroscience, Leioa, Spain, 2 Department of Neurosciences,
University of the Basque Country (UPV/EHU), Leioa, Spain, 3 Centro de Investigación Biomédica en Red de Enfermedades
Neurodegenerativas (CIBERNED), Leioa, Spain, 4 Laboratorio de Neurofisiología Celular, Instituto de Neurobiología,
Universidad Nacional Autónoma de México, Juriquilla, Mexico, 5 Department of Neurology, Mitchell Center for
Neurodegenerative Diseases, University of Texas Medical Branch, Galveston, TX, United States
Edited by:
Nicola B. Hamilton-Whitaker,
Myelin facilitates the fast transmission of nerve impulses and provides metabolic support
King’s College London,
United Kingdom to axons. Differentiation of oligodendrocyte progenitor cells (OPCs) and Schwann cell
Reviewed by: (SC) precursors is critical for myelination during development and myelin repair in
Åsa Fex-Svenningsen, demyelinating disorders. Myelination is tightly controlled by neuron-glia communication
University of Southern Denmark,
Denmark
and requires the participation of a wide repertoire of signals, including neurotransmitters
Beatriz Garcia-Diaz, such as glutamate, ATP, adenosine, or γ-aminobutyric acid (GABA). GABA is the main
INSERM U1127 Institut du Cerveau
inhibitory neurotransmitter in the central nervous system (CNS) and it is also present in
et de la Moelle épinière (ICM), France
Maria Cecilia Angulo, the peripheral nervous system (PNS). The composition and function of GABA receptors
Centre National de la Recherche (GABARs) are well studied in neurons, while their nature and role in glial cells are still
Scientifique (CNRS), France
incipient. Recent studies demonstrate that GABA-mediated signaling mechanisms play
*Correspondence:
Carlos Matute
relevant roles in OPC and SC precursor development and function, and stand out the
carlos.matute@ehu.eus implication of GABARs in oligodendrocyte (OL) and SC maturation and myelination.
María Victoria Sánchez-Gómez In this review, we highlight the evidence supporting the novel role of GABA with an
vicky.sanchez@ehu.eus
emphasis on the molecular identity of the receptors expressed in these glial cells
†
ORCID:
and the possible signaling pathways involved in their actions. GABAergic signaling
Mari Paz Serrano-Regal
orcid.org/0000-0002-1133-7261 in myelinating cells may have potential implications for developing novel reparative
Specialty section: therapies in demyelinating diseases.
This article was submitted to
Non-Neuronal Cells, Keywords: GABA, GABA receptor, oligodendrocyte, Schwann cell, differentiation, myelination
a section of the journal
Frontiers in Cellular Neuroscience
INTRODUCTION
Received: 25 May 2020
Accepted: 24 July 2020 Glial cells express a vast repertoire of receptors and transporters for neurotransmitters and
Published: 21 August 2020 neuromodulators and respond to axonal signals, being key and active elements of the nervous
Citation: system (Allen and Lyons, 2018). In vertebrates, oligodendrocytes (OLs) and Schwann cells
Serrano-Regal MP, Bayón-Cordero L, (SCs) are the myelin-forming glia of the central nervous system (CNS) and peripheral
Ordaz RP, Garay E, Limon A, nervous system (PNS), respectively. These cells are responsible for myelin building and
Arellano RO, Matute C and
maintenance, a function highly regulated by neuronal activity (Gibson et al., 2014; Mitew
Sánchez-Gómez MV
(2020) Expression and Function of
et al., 2018). Myelin speeds up nerve impulse propagation and provides metabolic and
GABA Receptors in Myelinating Cells. trophic support to axons (Nave and Trapp, 2008; Kidd et al., 2013; Philips and Rothstein,
Front. Cell. Neurosci. 14:256. 2017). Thus, myelination represents the major function of these cells, although they carry
doi: 10.3389/fncel.2020.00256 it out with some differences; while OLs can myelinate multiple axons simultaneously, each SC
Frontiers in Cellular Neuroscience | www.frontiersin.org 1 August 2020 | Volume 14 | Article 256

Serrano-Regal et al. GABA Receptors in Myelinating Cells
wraps one single axon (Jessen and Mirsky, 2005; Nave and Trapp, include 6 α (α1-α6), 3 β (β1-β3), 3 γ (γ1-γ3), 3 ρ (ρ1-
2008). Regarding their specific characteristics, oligodendroglial ρ3), and 1 gene of the respective δ, ε, θ, and π subunits
cells represent a highly diverse and specialized cell population (Sieghart and Savić, 2018). This diversity results in different
(Marques et al., 2016). Mature myelinating OLs develop homomeric or heteromeric subunit combinations that may have
from glial precursors named oligodendrocyte progenitor cells specific locations in the CNS, particular pharmacology, and,
(OPCs), which constitute the main proliferating cell type in consequently, distinctive functional characteristics (Vogt, 2015).
the adult CNS (Dawson et al., 2003). On the other hand, SCs The subunit profile that forms GABAA Rs depends on several
derive from SC precursors, which differentiate into immature factors including brain region, cell type, developmental stage, and
SCs. These immature SCs can generate both myelinating physiological or pathophysiological conditions (Levitan et al.,
and non-myelinating SCs (or Remak glia) according to PNS 1988; Seeburg et al., 1990; Waldvogel and Faull, 2015). Currently,
requirements, like the presence of specific signals in the 11 GABAA R subtypes with different subunit combinations have
microenvironment and the diameter of axons in their vicinity been identified, being most of them heteromeric receptors
(Jessen and Mirsky, 2005, 2019; Kidd et al., 2013). formed by αxβxγx or αxβxδ (where x represents any subtype
Differentiation of OPCs and SC precursors is necessary for of a given subunit; Figure 1A), whereas others are homomeric
remyelination in demyelinating diseases like multiple sclerosis receptors formed by ρ subunits (Barnard et al., 1998).
(MS) and myelin formation in dysmyelinating diseases such as
leukodystrophies in the CNS or Charcot-Marie Tooth in the PNS. Oligodendroglial Cells
In this regard, understanding the mechanisms of action involved Activation of GABAA Rs is relevant for the modulation of
in this complex neuron-glia crosstalk will help us in the search myelinating cell physiology (Magnaghi, 2007; Vélez-Fort et al.,
for new therapeutic approaches in these pathologies. 2012), however, the specific subunit composition of GABAA Rs
Neuronal activity and several signals such as transcriptional expressed in these cells remains unknown. Electrophysiological
and growth factors, axonal ligands, hormones, extracellular recordings in OPCs and OLs reveal differences between the
matrix components or neurotransmitters regulate OPC/SC response of the GABAA R expressed in these cells and those
precursor differentiation and myelination. Among them, expressed in neurons and astrocytes (Table 1), suggesting
GABAergic signaling has attracted great interest in the last years the presence of a novel GABAA R subtype with unique
(Procacci et al., 2013; Zonouzi et al., 2015; Arellano et al., 2016; stoichiometry in the oligodendroglial lineage (von Blankenfeld
Hamilton et al., 2017; Serrano-Regal et al., 2020). et al., 1991; Williamson et al., 1998; Vélez-Fort et al., 2012;
GABA, which is present both in the CNS and PNS, exerts Arellano et al., 2016).
an excitatory role during development to modulate neuronal von Blankenfeld et al. (1991) suggested that GABAA Rs in
growth and synapse formation (Ben-Ari, 2002). It acts mostly murine OPCs and OLs carry a γ subunit required to form the
through ionotropic GABAA (GABAA Rs) and metabotropic benzodiazepine binding site, as they observed potentiation of
GABAB receptors (GABAB Rs) that are well described in neurons the GABA response in these cells with classic benzodiazepines.
but not yet fully characterized in myelinating cells. Although the Moreover, the inverse agonist β-carboline methyl 4-ethyl-6,7-
expression of GABA receptors (GABARs) in OL/SC precursor dimethoxy-9H-β-carboline-3-carboxylate (DMCM) reduced the
lineages is widely documented (von Blankenfeld et al., 1991; GABA-induced current responses in oligodendroglial cells,
Williamson et al., 1998; Magnaghi et al., 2004; Luyt et al., 2007; unlike what happens in astrocytes. Contrary to that, Williamson
Arellano et al., 2016; Serrano-Regal et al., 2020), their role in et al. (1998) reported no influence of flunitrazepam or DMCM in
differentiation and myelination is a matter of ongoing research. the response elicited by GABA in rat-derived OPCs, indicating
In this review, we recapitulate recent evidence about an absence of the γ subunit in the GABAA Rs expressed by
GABAA and GABAB receptor expression and function in these cells. This observation was supported by the inhibitory
oligodendroglial and SCs, together with the implication of effect of Zn2+ , which is characteristic of receptors that lack the
the GABAergic signaling in OPC/SC differentiation and γ2 subunit. RT-PCR analyses conducted in the same study did
myelination. Furthermore, we discuss possible signaling not find expression of γ2, α1, α6, and δ subunit mRNAs in OPCs.
pathways involved in these events and their relevance Although amplification of other subunits was demonstrated, the
to develop new therapies to treat demyelinating and results were interpreted with caution since the preparation was
dysmyelinating diseases. 85% pure for OPCs and the presence of GABAA Rs from other
cell types could not be excluded. In a third study conducted by
EXPRESSION OF GABARs IN Bronstein et al. (1998), the GABA response of an immortalized
OLIGODENDROGLIAL AND SCHWANN murine glial cell line that expresses mature myelin proteins was
CELLS insensitive to diazepam and sensitive to Zn2+ , reinforcing the
idea of γ-subunit absence.
GABAA Receptors Later, Passlick et al. (2013) reported the expression of two
GABAA Rs are integral membrane ion channels—permeable types of GABAA Rs in hippocampal NG2 cells from juvenile
to Cl− and HCO3 − anions—composed of five subunits that mice by functional and pharmacological analyses and single-cell
mediate the major form of fast inhibitory neurotransmission RT-PCR. NG2 cells from this brain area express, on the one
in the CNS (Olsen and Sieghart, 2008; Doyon et al., 2016). hand, postsynaptic GABAA Rs comprised of a combination of
There are, at least, 19 distinct GABAA R subunit genes, which α1, α2, β3, γ1, and γ2 subunits and, on the other hand, they

FIGURE 1 | GABAA R expression in myelinating cells. (A) There are 11 different GABAA R subtypes described, mainly heteromeric receptors with αxβxγx or αxβxδ
(where x represents any subtype of a given subunit) stoichiometry (Olsen and Sieghart, 2008). Among them, a single type of GABAA R appears to be present in
oligodendrocytes though its molecular nature remains elusive. (B) Recent evidence using gene expression and pharmacology shows that GABAA Rs in
oligodendroglia are heterogeneous and may change in subunit composition during differentiation. Thus, at least two novel subtypes were identified: one formed by a
combination of α1 or α2, β3, and γ2 subunits (Passlick et al., 2013) and another made up by α3, β2 or β3, and γ1 or γ3 subunits (Arellano et al., 2016). (C) In turn,
studies using RT-PCR and/or immunohistochemistry conclude that SCs express high levels of α2, α3, β1, β2, and β3, while α1, γ1, and γ2 levels are relatively low in
these cells (Magnaghi et al., 2006).
have extrasynaptic GABAA Rs mostly lacking the γ2 subunit sensitivity to GABA (Karim et al., 2013), as it is the case of OLs
(Figure 1B). (EC50 between 70 and 100 µM).
Our studies of GABAA R responses conducted in cultured Together, these pharmacological studies suggest that
immature OLs from the rat forebrain and mature OLs from the composition of the GABAA R expressed in rat-derived
the optic nerve showed that these cells are diazepam-sensitive, oligodendroglial cells is a combination of the α3, β2 or β3,
suggesting once more the presence of a γ subunit (Arellano and γ1 or γ3 subunits (Figure 1B). Also, we confirmed the
et al., 2016). This positive modulation by benzodiazepines expression of the α3 subunit by immunocytochemistry in
was observed when using low GABA concentrations (≤EC30 ), cultured OLs (Arellano et al., 2016). These observations are
which may explain the discrepancies with previous studies. supported by previous functional genomic analyses performed
Concerning the specific subtype of γ subunit, two observations in OLs (Cahoy et al., 2008). Moreover, RNA sequencing (RNA-
indicate that γ2 may not contribute to oligodendroglial Seq) transcriptional analyses of purified NG2 cells obtained from
GABAA Rs: (1) Zn2+ blocks GABA responses; and (2) indiplon, P17 mice revealed that the α3 subunit is the most expressed
a positive allosteric modulator acting on γ2 subunit-containing α-subtype, while among β subunits, β3 and β2 are much more
receptors, does not modulate GABA currents. Therefore, abundant than β1. Lastly, γ1 is expressed at much higher levels
these receptors likely contain either γ1 or γ3 subunit than γ2 and γ3 (Larson et al., 2016).
(Arellano et al., 2016). An important factor for the diversity of GABAA Rs
Regarding β subunits, potentiation of the GABA response expressed and the subunits involved in their conformation
by loreclezole suggests the presence of β2 or β3 subunits, since will undoubtedly be the species. For example, regarding
β1 subunit-containing receptors are insensitive to this drug humans, a recent study of the GABAA R-subunit expression
(Arellano et al., 2016). Finally, concerning α subunits, α3 is in OPCs isolated from the middle temporal gyrus of healthy
the most likely candidate because it forms receptors with low adults—based on the single-nucleus RNA-Seq analysis by Hodge

TABLE 1 | Pharmacological properties of neuronal and oligodendroglial GABAA Rs.
Neurons
Drug Synaptic (αxβxγ2) Extrasynaptic (αxβxδ) Oligodendroglial cells
GABA Low EC50 1–30 µM Low EC50 0.5 nM–10 µM High EC50 70–100 µM
(Gibbs et al., 1996; Baur and Sigel, (Brown et al., 2002; Wallner et al., (Williamson et al., 1998; Arellano
2003; Mortensen et al., 2012) 2003; Mortensen et al., 2012) et al., 2016)
THIP No effect + No effect
(Mortensen et al., 2010) (Brown et al., 2002; Meera et al., (Arellano et al., 2016)
2011)
Zn2+ Low or No effect − −
(Hosie et al., 2003) (Carver et al., 2016) (Bronstein et al., 1998; Passlick
et al., 2013; Arellano et al., 2016)
β-CCB − or No effect No effect +
(Peña et al., 1986; (Jiménez-González et al., 2011) (Arellano et al., 2016;
Cisneros-Mejorado et al., 2020) Cisneros-Mejorado et al., 2020)
DMCM − − −
(Peña et al., 1986) (Brown et al., 2002) (von Blankenfeld et al., 1991;
Arellano et al., 2016)
Diazepam + No effect +
(Walters et al., 2000; Goodkin and (Goodkin and Kapur, 2009) (von Blankenfeld et al., 1991;
Kapur, 2009) Passlick et al., 2013; Arellano et al.,
2016)
Indiplon + No effect No effect
(Petroski et al., 2006) (Michelsen et al., 2007) (Arellano et al., 2016)
Flunitrazepam + No effect +
(Goodkin and Kapur, 2009) (Goodkin and Kapur, 2009) (von Blankenfeld et al., 1991;
Arellano et al., 2016)
Loreclezole +* +* +
(Wingrove et al., 1994) (Wingrove et al., 1994) (Arellano et al., 2016)
(+) Potentiator/agonist; (−) Inhibitor; (*) β2 or β3 subunit required.
et al. (2019)—showed that OPCs from this brain area express transmission. They were first described pharmacologically
high mRNA levels of α3, all β subunits, γ2 and, interestingly, as bicuculline-insensitive metabotropic receptors that were
the ε subunit (Figure 2). These mRNAs, if translated and activated by the GABA analog baclofen (Bowery and Hudson,
incorporated into functional receptors, would increase the 1979; Hill and Bowery, 1981). Functional GABAB Rs are
variety of potential configurations and may have important heterodimers constituted by two receptor subunits, GABAB1 and
functional and pharmacological consequences (Jones and GABAB2 , that cooperate to perform signal activation (Kaupmann
Henderson, 2007; Bollan et al., 2008; Belujon et al., 2009). et al., 1998; Kuner et al., 1999). GABAB1 is responsible for ligand
binding, while GABAB2 contains binding sites for allosteric
Schwann Cells
modulators (Galvez et al., 2001; Binet et al., 2004), couples with
GABAA -type receptors are relevant to SC physiology (Magnaghi
Gi/o -protein, and is necessary for trafficking the heterodimer to
et al., 2001, 2006). However, their pharmacological and
the cell surface, where the receptor becomes active (Calver et al.,
functional properties, as well as their molecular identity, are not
2000; Couve et al., 2000). Among the effector elements involved
clear. GABAA R subunit composition in rat-derived cultured SCs
in GABAB R signaling pathways in neurons are voltage-gated
includes α2 and α3, as well as the three β subunits, while mRNAs
Ca2+ channels (VGCC), inwardly-rectifying potassium channels
of α1 and γ2 subunits have been found at much lower levels
(Kir) and adenylyl cyclase (AC; Bowery et al., 2002; Bettler et al.,
(Magnaghi et al., 2006; Figure 1C). Moreover, the presence of α2,
2004; Figure 3). However, the specific coupling of GABAB Rs to
α3, and β3 proteins were confirmed by immunocytochemistry.
the molecular effector may differ depending on the cell type and
Alpha-2 and β3 subunits are also expressed in SC-like adult
region analyzed (Booker et al., 2018).
stem cells derived from bone marrow or adipose tissue, as
their levels are upregulated following SC differentiation in vitro
(Faroni et al., 2012). Also, GABAA R stimulation with muscimol Oligodendroglial Cells
increases the proliferation rate of SCs, meaning that GABAergic Myelinating cells express both subunits of GABAB Rs, which are
signaling has an important role in these cells (Magnaghi et al., negatively-coupled to AC (Magnaghi et al., 2004; Luyt et al.,
2006). However, despite these important findings, there is still 2007). However, their functional characteristics are not as well
little knowledge about the specific composition of GABAA Rs known as in the case of neurons. Regarding oligodendroglial
expressed in differentiated SCs. cells, we recently confirmed by immunocytochemistry and
RT-qPCR the expression of GABAB1 and GABAB2 subunits
GABAB Receptors in OPCs and OLs from the rat cerebral cortex and in OLs
GABAB Rs are G-protein coupled receptors (GPCRs) responsible from the optic nerve (Serrano-Regal et al., 2020). We also
for the slower and prolonged GABA-mediated inhibitory performed calcium imaging assays and electrophysiological

the mouse periventricular white matter, while the expression

of GABAB2 remained constant. As they reported changes
in GABAB1 /GABAB2 ratios in mature OLs, they raised the
possibility that one subunit alone or in combination with another
protein could make GABAB R functional in different cell types
(Calver et al., 2000; Luyt et al., 2007). In contrast, our recent
results show that cultured oligodendroglial cells from the rat
forebrain and mature OLs from the optic nerve express GABAB1
and GABAB2 subunits at different stages of maturation, as well as
mature OLs from the juvenile and adult rodent corpus callosum
in vivo (Serrano-Regal et al., 2020). These discrepancies can be
explained by the different regions analyzed, as GABAB1 /GABAB2
expression exhibits important regional variations (Luyt et al.,
2007). Since oligodendroglial cells are extremely diversified, the
different cells targeted in these studies may correspond to distinct
oligodendroglial and OPC subpopulations (Marques et al., 2016;
Spitzer et al., 2019; Marisca et al., 2020).
Schwann Cells
SCs also express different isoforms of the GABAB R, such as -1a,
-1b, -1c, and -2 (Magnaghi et al., 2004, 2006).
Downregulation of GABAB R expression occurs in pre-
and non-myelinating SCs (Corell et al., 2015). Neurosteroids
modulate the expression of GABAB R subunits in cultured SCs
and, as GABAB R is downregulated with age in the PNS, the
FIGURE 2 | The fractional contribution (FC) of GABAA R subunits in human
GABA synthesized in the adult sciatic nerve acts through
oligodendrocyte progenitor cells (OPCs). Normalized gene expression levels the ionotropic GABAA R, present both in neurons and SCs
for all 19 GABAA R subunits expressed as a percentage (mean ± S.E.M.) of (Magnaghi et al., 2006). Interestingly, the conditional knockout
the total available pool of mRNA for GABAA Rs in OPCs (PDGFRα+ cells) from of the GABAB1 subunit in SCs changes the expression of
human brains, estimated from publicly available datasets (Hodge et al., 2019; GABAA R subunits α3, α4, β1, and δ (Faroni et al., 2019),
https://celltypes.brain-map.org/rnaseq). The single-nucleus analysis used
normalized RNA-Seq datasets from the middle temporal gyrus isolated from
suggesting that GABAB Rs in these cells regulate somehow the
six subjects with no known neuropsychiatry or neuropathological history expression of GABAA Rs and/or their subunits.
(three males and three females; 35–66 years old). Gene expression level in Overall, more detailed analyses such as single-cell RNA-seq
each dataset was transformed into FC (Sequeira et al., 2019). FC is defined would help to better figure out the expression of GABAB1 and
as the percentage of the expression level of each subunit gene (signaled in
GABAB2 subunits along the oligodendroglial and SC lineages.
the “y” axis) to the sum of the 19 genes for GABAA Rs subunits within each
human/cell. Detailed demographic characteristics, as well as technical white
papers for data processing and quality control, can be downloaded from the POTENTIAL GABA SYNTHESIS AND
same site. Confirmatory analysis of OPC markers enrichment and lack of
neuronal markers were performed for all datasets.
RELEASE IN MYELINATING CELLS
Although GABAergic neurons are the main source of GABA
recordings in these cells and observed that baclofen does not (especially in the CNS), GABA synthesis also occurs in glial cells
modify their response to KCl 50 mM, calcium influx, and (Seiler et al., 1979; Angulo et al., 2008; Héja et al., 2012). Two
Kir currents. These results strongly suggest that GABAB Rs in potential pathways for GABA synthesis have been described in
oligodendroglial cells are not coupled to Ca2+ and Kir channels brain-derived glial cells. GABA is mainly produced through the
in the same manner as in other cell types (Serrano-Regal et al., classical pathway as a result of glutamate decarboxylation by
2020). Likewise, GABAB Rs from CA1 somatostatin interneurons, the glutamic acid decarboxylase (GAD) enzymes (Roberts and
unlike pyramidal neurons, are not coupled to the canonical Frankel, 1950). In neurons, the two isoforms of GAD—GAD65
Kir3 signaling cascade (Booker et al., 2018), which suggests a and GAD67 —differ in their catalytic and kinetic properties
functional diversity of downstream effectors depending on cell and their subcellular distribution (Kaufman et al., 1991).
type and location, and warrants the need of exploring these Also, GABA can be synthesized from the monoacetylation
features in OLs as well as in SCs. of putrescine with the participation of the monoamine
Charles et al. (2003) did not find any colocalization of oxidase B (MAOB ) enzyme in the non-classical pathway
GABAB1 subunit and MBP expression in myelinating OLs (Seiler et al., 1973).
in the white matter of the rat spinal cord and suggested Consistent with an RNA-seq transcriptome and splicing
that GABAB R expression in developing OLs decreases during database (Zhang et al., 2014), oligodendroglial cells express
differentiation. Following this, Luyt et al. (2007) observed gad1, gad2, and maob mRNAs. GAD67 mRNA (gad1) is greatly
downregulation of the GABAB1 subunit in mature OLs from expressed by OPCs, although its levels decrease notably as they

FIGURE 3 | Possible signaling pathways downstream GABARs leading to myelination. Activation of the Gi/o-linked GABAB R may reduce CREB phosphorylation as
it is negatively coupled to adenylyl cyclase (AC; Luyt et al., 2007). Alternatively, it may also induce CREB phosphorylation possibly via activation of PLCβ /FAK/PKC or
MAPK cascades, as observed in neurons (Carlezon et al., 2005; Zhang et al., 2015). Moreover, activation of GABAB R leads to Src phosphorylation (Serrano-Regal
et al., 2020) that could ultimately induce CREB activation. Phosphorylation of Src may also lead to Akt phosphorylation via PI3K as observed earlier (Barati et al.,
2015), and contribute to a positive feedback loop with p38MAPK (Mugabe et al., 2010; Lin et al., 2015), which is involved in myelination (Fragoso et al., 2003, 2007).
On the other hand, the GABAB1 subunit of GABAB R might be sequestered by phosphatases like PP2A (as occurs in some neurons), a mechanism that blocks its
activity and can be reverted by high intracellular Ca2+ levels (Li et al., 2020). Finally, the GABAA R allosteric modulator ALLO regulates Schwann cell (SC) myelination
via Src-FAK signaling, involving cytoskeleton reorganization (Melfi et al., 2017).
differentiate into mature myelinating OLs. However, gad2 is oligodendroglial cells at 1, 3, and 6 days in vitro (Serrano-Regal
expressed to a lesser extent throughout the oligodendroglial et al., 2020). Similarly, we verified the expression of the MAOB
lineage. Regarding maob, OPCs and myelinating OLs express enzyme in these cells at the same time points. These results
higher levels than newly formed or immature OLs (Zhang et al., indicate that cultured oligodendroglial cells may synthesize
2014). Accordingly, we confirmed the presence of GAD65/67 by GABA by the two alternative pathways mentioned above. Indeed,
immunocytochemistry and western blot in rat-derived cortical we found GABA immunostaining in cortical and optic-nerve

derived oligodendroglial cells at different stages of maturation Ca2+ in the cytosol may regulate OPC proliferation, migration
(Serrano-Regal et al., 2020). Possibly, GABA is synthesized by and maturation and, consequently, OL (re)myelination (Cheli
one pathway or another depending on the stage of maturation et al., 2016; Santiago-González et al., 2017; Baraban et al., 2018;
of the cells, as GABA may have different roles in OPCs vs. Krasnow et al., 2018; Marisca et al., 2020).
mature OLs. As oligodendroglial cells constitute a highly dynamic
GAD67 is present in SCs and its levels increase in the and heterogeneous population, the expression of GABAA Rs
presence of the progesterone metabolite allopregnanolone and their different subunits changes as these cells progress
(ALLO; Magnaghi et al., 2010). Moreover, Corell et al. along their lineage, as occurs with the expression of certain
(2015) demonstrated the presence of GABA and GAD65/67 in ion-channels (Spitzer et al., 2019), and these changes can
premyelinating and non-myelinating SCs. These findings show affect their intercellular relationship and differentiation. In
that SCs both produce and store GABA. line with this, we observed that oligodendroglial GABAA R
Together, these observations indicate that OLs and SCs expression in vitro is dependent on the close interaction
synthesize and store GABA, which could be released by between axons and OLs, as OLs cultured alone lose GABA
reversal operation of GABA transporters including GAT-1 and responses with differentiation (Arellano et al., 2016). Moreover,
GAT-3, that are expressed by OLs (Fattorini et al., 2017; the presence of the γ2 subunit, which is associated with a
Serrano-Regal et al., 2020). Also, SCs are capable to take possible role in neuron-OPC synapse formation, decreases with
up ambient GABA at concentrations above 1 µM (Brown age along with the density of GABAergic synaptic contacts
et al., 1979), though the nature of the transporters involved is in cortical NG2 cells of mature mice (Balia et al., 2015).
still unknown. Thus, NG2 cells may switch the expression of GABAA Rs
from synaptic (with γ2 subunit) to extrasynaptic (without
GABA RECEPTORS IN OPC/SC γ2 subunit) during development (Vélez-Fort et al., 2010).
PRECURSOR DIFFERENTIATION AND Surprisingly, genetic inactivation of oligodendroglial γ2 does
MYELINATION not affect OPC proliferation and differentiation, while it
causes progressive and specific depletion of the OPC pool
In the CNS, OPCs are the main (but not unique) source of that lacks γ2-mediated synaptic activity without affecting
remyelination, since they respond to white matter injury, migrate the oligodendrocyte production (Balia et al., 2017). These
to the lesioned area and differentiate into mature OLs to produce observations indicate that GABAergic communication in
new myelin sheaths (Franklin et al., 1997; Nait-Oumesmar et al., cortical OPCs through γ2-containing GABAA Rs does not
1999; Hesp et al., 2015). Moreover, surviving mature OLs are play a role in oligodendrogenesis but rather modulates
also a source of remyelination (Duncan et al., 2018). SCs may OPC maintenance.
also participate, although to a lesser extent, in restoring myelin GABAergic signaling regulates OPC population and OPC
in the CNS (Zawadzka et al., 2010; García-Díaz and Baron-Van differentiation and myelination in the cerebellar white matter
Evercooren, 2020). in vivo (Zonouzi et al., 2015). In turn, hypoxia causes a strong
Differentiation of OPCs/SCs and myelination are exquisitely downregulation of the GABAergic synaptic input from local
coordinated processes mediated by a deep dialogue between interneurons to OPCs (NG2 cells) as well as an increase in the
neuronal and glial cells that entail the participation of a variety proliferation of these cells and a delay in their maturation, which
of signals and intercellular communication systems, including limits myelination. These effects are mimicked in control animals
ATP, glutamate or GABA neurotransmitter signaling (Li et al., when blocking GABAA Rs with their antagonist bicuculline.
2013; Faroni et al., 2014; Salzer, 2015; Zonouzi et al., 2015; However, they are reverted when applying tiagabine, a selective
Arellano et al., 2016; Hamilton et al., 2017; Serrano-Regal et al., inhibitor of the GABA transporter GAT-1 that increases GABA
2020), as well as neuronal activity (Wake et al., 2011; Gibson availability in the extracellular space. Treatment with tiagabine
et al., 2014; Fannon et al., 2015). Specifically, GABAergic neurons results in a decrease of NG2 cell proliferation and an increase of
establish direct synapses with OPCs throughout the CNS, myelinating OLs, reverting the hypomyelinating effect caused by
indicating that this communication may control proliferation, perinatal hypoxia (Zonouzi et al., 2015). These findings strongly
migration, differentiation, the establishment of axonal contacts suggest that GABAergic signaling (either neuronal activity-
and their wrapping, OPC survival in the adult brain or myelin dependent or independent) influences OPC development
maintenance (Lin and Bergles, 2004; Kukley et al., 2008; Vélez- and differentiation and, therefore, it may help to develop
Fort et al., 2010; Orduz et al., 2015; Zonouzi et al., 2015; novel therapies to improve OPC differentiation into damaged
Balia et al., 2017; Mount et al., 2019). However, the precise brain areas.
function of GABA in OPC differentiation and myelination GABAergic signaling through GABAA Rs may also be relevant
remains controversial. for the stronger remyelination that occurs following focal
demyelination in the corpus callosum of late pregnant rats
GABAA Receptors compared to virgin and postpartum ones (Kalakh and Mouihate,
Contrary to mature neurons, activation of GABAA Rs in OPCs 2019). This pregnancy-associated promyelinating effect was lost
leads to depolarization and an increase in cytosolic Ca2+ levels when either the GABAA R was blocked or when 5α-reductase,
(Kirchhoff and Kettenmann, 1992), resulting from Ca2+ influx the rate-limiting enzyme for the endogenous GABAA R activator
through activated VGCC (Paez and Lyons, 2020). Thus, a rise in ALLO, was inhibited (Kalakh and Mouihate, 2019). Moreover,

N-butyl-β-carboline-3-carboxylate (β-CCB), a selective drug approaches to specifically target the different GABAA R subunits
activating preferentially oligodendroglial GABAA Rs, promotes expressed in these cells.
remyelination in a model of gliotoxin-induced demyelination
in the rat cerebellar caudal peduncle as assessed using GABAB Receptors
magnetic resonance imaging (MRI) together with myelin An early study suggests that GABAB Rs may be relevant for OPC
staining (Cisneros-Mejorado et al., 2020). Together, these results development as baclofen increases migration and proliferation in
strongly suggest that GABAA R-mediated signaling promotes cultured OPCs derived from periventricular white matter (Luyt
myelination and remyelination in OLs either directly or et al., 2007). However, more recent studies could not confirm
indirectly. However, at odds with these data, activation of those findings as baclofen did not change OPC proliferation
GABAA Rs by endogenous GABA in cortical organotypic cultures or total OPC number in dissociated and organotypic cultures
reduces the number of oligodendroglial cells and myelination derived from the cortex (Hamilton et al., 2017; Serrano-Regal
whereas enlarges internode length, influencing the velocity of et al., 2020). This discrepancy could reflect the different brain
the nerve impulse propagation (Hamilton et al., 2017). These areas studied. Thus, different subpopulations of OPCs may
effects may be due to GABA released by glial cells. However, exist in white and gray matter that behave differently or have
we could not assess this idea as gabazine treatment of OPC different responses to baclofen, as proposed by Luyt et al. (2007).
cultures had no significant effect on myelin protein production In contrast, baclofen reduced cell proliferation of SC cultures
(Serrano-Regal et al., 2020). (Magnaghi et al., 2004). However, in dissociated developing DRG
Neurosteroid therapy is another pharmacological approach primary cultures, in which SCs proliferate spontaneously in vitro,
to modulate GABAA R activity in the nervous system, as they baclofen did not affect (Corell et al., 2015).
act as allosteric modulators of these receptors in the nanomolar On the other hand, GABA and baclofen modulate OPC
concentration range (Lambert et al., 2009). Indeed, progestin differentiation, as well as the myelination capacity of mature OLs
ALLO increases myelin basic protein (MBP) production in cultured with DRG neurons, pointing out GABAB Rs as relevant
rat-derived cerebellar organotypic slices, an effect that requires modulators of OL maturation and myelination (Serrano-Regal
GABAA Rs (Ghoumari et al., 2003). This observation points et al., 2020). Consistent with these observations, GABAB Rs
to these receptors as mediators of myelination. The effects of also regulate SC differentiation both in the myelinating
neurosteroids are of special interest in postnatal development, and non-myelinating phenotypes. Thus, forskolin-induced
as they may help to prevent neurodevelopmental disorders SC differentiation in vitro correlates with a redistribution
associated with preterm birth (Shaw et al., 2019). ALLO, which is of GABAB1 and GABAB2 subunits of GABAB Rs. Indeed,
mainly synthesized in the placenta, has an important role during in the cytoskeleton rearrangement that takes place during
nervous system development. In premature neonates, ALLO differentiation, GABAB Rs colocalize with f-actin on the SC
concentration decreases abruptly and this decrease is associated, elongated processes (Procacci et al., 2013).
in part, with hypomyelination (Shaw et al., 2015). Consequently, Apart from being essential for SC commitment to a
experimental administration of the ALLO analog ganaxolone non-myelinating phenotype during development, GABAB Rs
as replacement therapy in guinea pig-preterm neonates showed are key modulators of neuronal-SC interactions regarding
positive effects on myelination, through its interaction with myelination, as GABAB1 receptor total null mice showed altered
GABAA Rs. Therefore, neurosteroid replacement could be a good levels of PMP22 and myelin protein zero (P0) as well as thinner
therapeutic option to improve myelination in this condition myelin sheaths. These mice also presented fiber alterations,
(Shaw et al., 2019). which causes changes in pain behavior, gait abnormalities,
Neurosteroids may also enhance GABAA R function in and motor coordination disturbances (Magnaghi et al., 2008).
SCs. Thus, ALLO acting via GABAA receptor can influence Together, these findings suggest a role for GABAB Rs in the
peripheral myelin protein 22 (PMP22) synthesis (Magnaghi et al., control of SC myelination. Moreover, both GABAB R subunits
2006). Moreover, ALLO modulates SC morphology, motility, in addition to GABA and GAD65/67 were found at the node of
and myelination in SC/dorsal root ganglia neuron (DRG) Ranvier in a sub-population of myelinated sensory fibers (Corell
co-cultures via the Src/focal adhesion kinase (FAK) pathway, et al., 2015). Surprisingly, GABAB R expression is upregulated in
a signaling cascade that involves GABAA Rs and relies on actin SCs of injured nerves, which may be interpreted as an adaptive
rearrangements (Melfi et al., 2017). Therefore, neurosteroids response for stimulating the neighboring axons to re-grow
represent a promising molecular approach for the treatment distally to the injury (Corell et al., 2015).
of peripheric pathologies. Together, the studies discussed Finally, conditional deletion of the GABAB1 subunit in SCs
in this section connect GABAA R signaling with OPC/SC altered their proliferation, migration, and myelination capacities,
differentiation and/or myelination using pharmacological as well as reduced neurite length of co-cultured DRGs (Faroni
approaches. However, the results observed cannot be solely et al., 2019). Furthermore, molecular and transcriptomic changes
attributed to the action of GABA on GABAA Rs. Although were also observed both in SCs and DRGs derived from mice
pharmacology may be a good strategy to enhance OPC/SC lacking GABAB1 subunit in SCs (P0-GABA-B1fl/fl ). Interestingly,
differentiation and myelination in pathological conditions, the expression of some GABAA R subunits by SCs and DRGs
potential side-effects must also be considered. To minimize was also altered, indicating a possible role of GABAB Rs in
them, it would be of great interest to use more specific drugs regulating the expression of GABAA Rs in these cells (Faroni et al.,
acting on myelinating cell GABAA Rs and/or to use genetic 2019). Similar studies using conditional deletion of GABAB R

subunits in oligodendroglia will help to understand the role of Also, conditional knockout of p38 in oligodendroglial cells leads
these receptors and their impact on myelination and pain and to defects in myelination early in development (Chung et al.,
motor behavior. 2015). At odds with those findings, deletion of p38 in the same
mouse model increases remyelination after cuprizone-induced
POSSIBLE SIGNALING PATHWAYS demyelination (Chung et al., 2015), while selective deletion of
DOWNSTREAM GABARs RELATED TO p38α MAPK in OLs did not compromise myelination in a mouse
MYELINATION model of periventricular leukomalacia (PVL; Chung et al., 2018).
These conflicting pieces of evidence indicate that the precise role
Myelination, either by OLs or SCs, involves the participation of p38 MAPK in SC/OL differentiation and myelination and its
of several intracellular signaling pathways. Indeed, some relation with GABARs remains to be elucidated (Figure 3).
of those pathways are common in the CNS and PNS. For Since GABAB Rs couple negatively to AC in OLs (Luyt
instance, binding of neuregulins (NRGs) to ErbB receptors et al., 2007), activation of CREB downstream these receptors
activates a sequence of canonical intracellular pathways is not expected due to decreased cAMP levels. However, other
downstream from many receptor tyrosine kinases (RTKs), such intracellular signaling cascades activated downstream G-protein
as phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target coupled receptors, such as MAPK cascades, may phosphorylate
of rapamycin (mTOR) or mitogen-activated protein kinases CREB (see Carlezon et al., 2005). Thus, GABAB R stimulation
(MAPK; Newbern and Birchmeier, 2010). in cultured mouse cerebellar granule neurons with baclofen
Intracellular 30 ,50 -cyclic adenosine monophosphate (cAMP) activates CREB via PLCβ/FAK/PKC (Zhang et al., 2015). Further
induces cell differentiation and myelination requiring the clarification of the link between GABAB R activation and CREB
participation of the cAMP response element-binding protein specifically in myelinating cells is a matter of ongoing study and
(CREB). CREB mediates the stimulation of MBP expression by could contribute to a better understanding of the signaling routes
cAMP in OLs (Afshari et al., 2001). Moreover, in mouse-derived that control myelination and remyelination (Figure 3).
cultured SCs, the combined action of cAMP/NRG1 increases Finally, GABAB R function may be modulated by its direct
the expression of myelin proteins Krox-20 and P0, through a association to protein phosphatase 2A (PP2A), as observed in
mechanism that relies on the activity of transcription factors GABAergic neurons from the rodent ventral tegmental area (Li
from the CREB family (Arthur-Farraj et al., 2011). et al., 2020). Therefore, PP2A-GABAB R interaction results in
The Src family kinases (SFKs) are nonreceptor tyrosine an increase of GABAB R dephosphorylation and its subsequent
kinases that integrate external signals from both integrin and internalization, an effect reverted with high intracellular Ca2+
growth factor receptors and transduce signals related to OL and levels. Again, it is worth exploring if these mechanisms also occur
SC development and myelination (Colognato et al., 2004; Melfi in myelinating cells and whether they are relevant to myelin
et al., 2017). In particular, signaling pathways downstream the pathology (Figure 3).
Src-family member Fyn regulate morphological differentiation
of OLs, the recruitment of cytoskeleton components, and DISCUSSION
local translation of MBP (see White and Krämer-Albers, 2014;
Quintela-López et al., 2019). GABAB R specific activation with GABA is among the signals that drive OLs and SCs to axon
baclofen induces Akt phosphorylation, which is dependent on interactions. The fact that GABA acts mainly on two different
PI3K and Src kinases, promoting chemotaxis and cytoskeletal types of receptors—ionotropic GABAA Rs and metabotropic
rearrangement in rat basophilic leukemic cells (Barati et al., GABAB Rs—makes it difficult to understand the role of this
2015). Accordingly, we found that Src-family kinases inhibition neurotransmitter in myelinating cell physiology. Moreover, OLs
abrogates GABAB R-induced OL differentiation (Serrano-Regal and SCs are highly dynamic cell lineages with different stages
et al., 2020). This observation corroborates the role of the Src of maturation.
family in OL differentiation through a mechanism dependent on While the molecular composition of both GABARs and
GABAB R activation (Figure 3). their mechanisms of action are well described in neurons,
GABAA Rs are also linked to Src and FAK signaling. their properties in myelinating glial cells remain elusive. Native
The modulation of SC development and myelination by the GABAA Rs are composed of multiple subunit combinations
neurosteroid ALLO in SC/DRG co-cultures occurs via Src and with diverse pharmacology, both of which vary regionally,
FAK signaling activation, which depends on GABAA Rs and actin adding a huge heterogeneity to their properties and functions.
reorganization (Melfi et al., 2017). Besides, Src-family members This diversity is also reflected somehow in GABAA Rs in
can interact reciprocally with kinases from the MAPK family, like OLs and SCs. Thus, SCs express extra-synaptic subunits (in
the serine/threonine-protein kinase p38 MAPK, as c-Src elicits particular the δ subunit, which is key for neurosteroid affinity),
p38 MAPK phosphorylation and the opposite (Mugabe et al., while OLs express subunits commonly found at postsynaptic
2010; Lin et al., 2015; Wu et al., 2015). Therefore, it would densities, meaning that GABAA Rs of OLs and SCs have
be of great interest to investigate this kind of interactions in different subunit composition and, consequently, different
myelinating cells, as p38 MAPK is a key element in the initial pharmacological profiles and functional behaviors (Faroni et al.,
steps of myelination in SCs (Fragoso et al., 2003), as well as 2019). Also, there is a switch in the expression of synaptic to
in OL maturation and myelination since specific p38 inhibitors extrasynaptic GABAA Rs as OPCs progress in the lineage (Vélez-
block in vitro myelination of DRGs by OLs (Fragoso et al., 2007). Fort et al., 2010; Balia et al., 2017).

FIGURE 4 | Effects of GABAergic signaling in oligodendroglial differentiation and myelination. OPCs express GABAA and GABAB Rs. Activation of GABAA Rs causes
depolarization in these cells (Kirchhoff and Kettenmann, 1992; Baraban et al., 2018). In absence of axons (top), they lose GABAA R expression as they differentiate
into mature OLs (Berger et al., 1992; Arellano et al., 2016); however, expression of GABAB Rs is largely stable over time (Serrano-Regal et al., 2020). In the presence
of axons (low), GABAA R expression is modulated by neurons, as OPCs maintain their expression towards the myelinating stage (Arellano et al., 2016). Moreover,
oligodendroglial cells express GAT-1 transporter and synthesize GABA, which may be released by reverse GAT-1 operation and activate GABA receptors (GABARs)
in an autocrine manner. Exogenous GABA or baclofen promotes oligodendroglial differentiation and myelination in vitro (Serrano-Regal et al., 2020).
In oligodendroglial cells, GABAA R expression goes down modulate their differentiation and/or their myelination
as they mature and acquire a myelinating phenotype (Berger capacity (Figure 4). Interestingly, this mechanism operates
et al., 1992; Arellano et al., 2016). In contrast, GABAB R for instance in polysialylated forms of neural cell-adhesion
expression is quite stable at all stages (Serrano-Regal et al., molecule (PSA-NCAM) progenitor cells in the CNS, which
2020). Sustained GABAA R expression in oligodendroglial cells eventually differentiate into glial cells. Thus, autocrine/paracrine
depends on the presence of axons, though the mechanisms loops involving neurosteroids and GABA signaling in these
driving GABAA R stabilization remain still unknown. Thus, it progenitors modulate their proliferation and differentiation
is likely that molecules released from neurons in an activity- (Gago et al., 2004). Synthesis of neurosteroids occurs in
independent manner may drive GABAA R expression (Arellano SCs (Chan et al., 2000), and may stimulate GABA synthesis
et al., 2016; Hamilton et al., 2017; Figure 4). in these cells via a rapid protein kinase A (PKA)-dependent
Also, OLs and SCs can synthesize and store GABA, and autocrine loop (Magnaghi et al., 2010). In this way, neurosteroids
to take it up from the extracellular fluid through specific provide the specific ligand for GABAA R activation (Magnaghi
GABA transporters (Fattorini et al., 2017). It is therefore et al., 2010). As neurosteroids are involved in promoting SC
conceivable that these cells may release GABA by mechanisms differentiation and myelination acting through GABAA Rs,
including reverse functioning of the transporters, as observed a possible paracrine/autocrine mechanism could underlie
in other glial cell types (Barakat and Bordey, 2002). Early these processes.
experiments demonstrated that cultured satellite glial cells from GABAA and GABAB receptors may exert opposite roles
DRG can release [3 H]GABA in response to a depolarizing on myelinating cells, as proposed for SCs in pathological
stimulus (Minchin and Iversen, 1974). Thus, GABA released conditions (Faroni and Magnaghi, 2011). Both central and
by myelinating cells might act in a paracrine or autocrine peripheral myelinating cells express GABAA and GABAB
way (as suggested by Magnaghi, 2007) to, ultimately, receptors, however, this expression depends on the presence

of surrounding axons and, as occurs with other receptors and and the mechanisms that mediate these responses. To that
transporters, may vary along the lineage or even depending on aim, it will be important to specifically target GABAA Rs and
the nervous system area (Marques et al., 2016; Spitzer et al., GABAB Rs either at the progenitor stage or the more mature
2019). Thus, depending on the developmental stage of these stages of the myelinating cells. Drugs preferentially acting on
cells and the GABAR involved, the neurotransmitter GABA may GABARs in OLs and SCs will certainly help to successfully tackle
play different physiological functions. Moreover, GABAergic these tasks.
signaling could potentially regulate specific subgroups of cells
from the OL/SC lineages in different ways, either action- AUTHOR CONTRIBUTIONS
potential dependently or independently. Thus, it appears that
in OPCs GABA acts mostly through GABAA Rs to carry out MS-R and RA wrote the manuscript, designed its content, and
some important functions at the progenitor level (as regulation prepared the figures and table. LB-C, RO, and AL wrote the
of the population size, OPC maintenance, axon-glia recognition, manuscript and prepared the figures and table. EG wrote the
differentiation or myelination initiation; Zonouzi et al., 2015; manuscript. CM and MS-G wrote the manuscript and designed
Arellano et al., 2016; Balia et al., 2017; Hamilton et al., 2017; its content.
Marisca et al., 2020).
Finally, GABAB Rs may contribute to myelin maintenance FUNDING
(Figure 4). In this regard, GABARs may parallel somehow the
various functions played by glutamate receptors, as AMPARs This work was supported by CIBERNED (CB06/05/0076;
are crucial for the early stages of remyelination while NMDARs CM) and by grants from the Ministry of Economy and
are relevant for myelin maintenance and to fuel axonal function Competitiveness, Government of Spain (SAF2016-75292-R and
(Lundgaard et al., 2013; Gautier et al., 2015; Saab et al., 2016). PID2019-109724RB-I00; CM), Basque Government (IT1203-
In sum, understanding the contribution of the GABAergic 19; CM), CONACYT-México (No. 252121; RA), PAPIIT-
signaling to OL and SC physiology may be critical to find UNAM-México (IN203519; RA) and NIH (R21AG053740 and
therapeutic tools to improve remyelination in demyelinating R21MH113177; AL). MS-R was hired thanks to the Gangoiti
diseases. Meanwhile, it is necessary to clarify in detail the Foundation (Bilbao). LB-C and RO hold fellowships from Basque
role of GABA in OL and SC differentiation and myelination, Government and CONACYT-México, respectively.
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RE VIEW
Structure, function, and allosteric modulation of

NMDA receptors
Kasper B. Hansen1, Feng Yi1, Riley E. Perszyk2, Hiro Furukawa3, Lonnie P. Wollmuth4, Alasdair J. Gibb5, and Stephen F. Traynelis2
NMDA-type glutamate receptors are ligand-gated ion channels that mediate a Ca2+-permeable component of excitatory
neurotransmission in the central nervous system (CNS). They are expressed throughout the CNS and play key physiological
roles in synaptic function, such as synaptic plasticity, learning, and memory. NMDA receptors are also implicated in the
pathophysiology of several CNS disorders and more recently have been identified as a locus for disease-associated genomic
Downloaded from http://rupress.org/jgp/article-pdf/150/8/1081/1235977/jgp_201812032.pdf by guest on 10 November 2020

variation. NMDA receptors exist as a diverse array of subtypes formed by variation in assembly of seven subunits (GluN1,
GluN2A-D, and GluN3A-B) into tetrameric receptor complexes. These NMDA receptor subtypes show unique structural
features that account for their distinct functional and pharmacological properties allowing precise tuning of their
physiological roles. Here, we review the relationship between NMDA receptor structure and function with an emphasis on
emerging atomic resolution structures, which begin to explain unique features of this receptor.
Introduction aptic neurons. These excitatory postsynaptic currents (EPSCs)

The vast majority of the excitatory neurotransmission in the can be described primarily by two temporally distinct compo-
central nervous system (CNS) is mediated by vesicular release of nents corresponding to activation of AMPA and NMDA receptors.
glutamate, which activates both pre and postsynaptic G-protein– AMPA receptors mediate a synaptic current with rapid rise time
coupled metabotropic glutamate receptors and ionotropic gluta- and decay, whereas NMDA receptor activation mediates a current
mate receptors (iGluRs). iGluRs are ligand-gated cation channels that activates more slowly with a time course that endures for
that are divided into three major structurally distinct functional tens to hundreds of milliseconds (Hestrin et al., 1990; Sah et al.,
classes: the α-amino-3-hydroxy-5-methyl-4-isoxasolepropionic 1990; Trussell et al., 1993; Geiger et al., 1997; Fig. 1 B). At rest, the
acid (AMPA) receptors, kainate receptors, and NMDA receptors NMDA receptor pore is strongly blocked in a voltage-dependent
(Traynelis et al., 2010; Fig. 1 A). The nomenclature for these func- manner by extracellular Mg2+, but this block can be released by
tional classes was initially based on the activating agonist, and the depolarization that accompanies rapid activation of AMPA
subsequent molecular cloning revealed cDNAs encoding mul- receptors, particularly when there is a series of closely spaced
tiple subunits within the three classes of iGluRs. An intriguing synaptic events (Fig. 1 C). Thus, the current mediated by NMDA
fourth class of iGluRs (GluD1-2) have structural resemblance to receptors is dependent on both the membrane potential and
AMPA and kainate receptors but do not function as ion channels frequency of synaptic release, rendering these receptors coinci-
under normal circumstances (Yuzaki and Aricescu, 2017). Sev- dence detectors that respond uniquely to simultaneous presyn-
eral unique properties distinguish NMDA receptors from other aptic release of glutamate and postsynaptic depolarization with
glutamate receptors, including voltage-dependent block by extra- a slow synaptic current that allows substantial influx of external
cellular Mg2+, high permeability to Ca2+, and the requirement for Ca2+ into the dendritic spine (Bourne and Nicoll, 1993; Seeburg
binding of two coagonists, glutamate and glycine (or d-serine), for et al., 1995; Nevian and Sakmann, 2004). This increase in intra-
channel activation (Traynelis et al., 2010). These features have a cellular Ca2+ serves as a signal that leads to multiple changes in
profound impact on the physiological roles of NMDA receptors the postsynaptic neuron, including changes that ultimately pro-
and have therefore been the topic of intense investigation. duce either short-term or long-term changes in synaptic strength
At central synapses, glutamate release activates iGluRs that (Lau and Zukin, 2007; Holtmaat and Svoboda, 2009; Traynelis et
mediate an inward current and thereby depolarize the postsyn- al., 2010; Higley and Sabatini, 2012; Zorumski and Izumi, 2012;
1Department of Biomedical and Pharmaceutical Sciences and Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, MT; 2Department of
Pharmacology, Emory University School of Medicine, Atlanta, GA; 3Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; 4Departments of Neurobiology & Behavior
and Biochemistry & Cell Biology, Stony Brook University, Stony Brook, NY ; 5Department of Neuroscience, Physiology and Pharmacology, University College London,
London, UK.
Correspondence to Stephen F. Traynelis: strayne@emory.edu.
© 2018 Hansen et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the
publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0
International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).
Rockefeller University Press https://doi.org/10.1085/jgp.201812032 1081

J. Gen. Physiol. 2018 Vol. 150 No. 8 1081–1105
Figure 1. Functional classes of iGluRs. (A) iGluRs are divided into AMPA, kainate, and NMDA receptors with multiple subunits cloned in each of these func-
tional classes. (B) EPSCs from central synapses can be divided into fast AMPA or slow NMDA receptor–mediated components in the absence of Mg2+ using the
AMPA receptor antagonist CNQX or the NMDA receptor antagonist AP5. The figure is adapted from Traynelis et al. (2010). (C) The relationships between NMDA
receptor current response and membrane potential (i.e., holding potential) in the presence and absence of 100 µM extracellular Mg2+ reveal the voltage-depen-

dent Mg2+ block, which is relieved as the membrane potential approaches 0 mV (i.e., with depolarization). Data are from Yi et al. (2018).
Paoletti et al., 2013; Volianskis et al., 2015). The nature of these two GRIN3 genes encode GluN3A-B (Traynelis et al., 2010). All
changes (e.g., increased or decreased synaptic strength) depends known NMDA receptors are heterotetrameric assemblies of
on the frequency and duration of synaptic NMDA receptor ac- subunits, which together form a central ion channel pore with
tivation (Citri and Malenka, 2008; Granger and Nicoll, 2013), striking similarity to an inverted potassium channel. The stoi-
thereby providing the brain with a mechanism for encoding in- chiometry of the NMDA receptor has been definitively shown to
formation (Hunt and Castillo, 2012; Morris, 2013). be two glycine-binding GluN1 and two glutamate-binding GluN2
NMDA receptors are unique among synaptic receptors in their subunits (i.e., GluN1/2 receptors; Ulbrich and Isacoff, 2007;
requirement for the binding of two agonists, glutamate and glycine Karakas and Furukawa, 2014; Lee et al., 2014; Fig. 2). However,
(or d-serine; Johnson and Ascher, 1987; Kleckner and Dingledine, subunit assembly and physiological roles of the glycine-bind-
1988; Benveniste and Mayer, 1991; Clements and Westbrook, 1991, ing GluN3 subunits remain elusive, and the GluN3 subunits will
1994). Synaptic NMDA receptors are temporally controlled by the not be considered in this review (Cavara and Hollmann, 2008;
synaptic release of glutamate for activation, because extracellular Henson et al., 2010; Low and Wee, 2010; Pachernegg et al., 2012;
glycine (or d-serine) is thought to be continuously present at fairly Kehoe et al., 2013; Pérez-Otaño et al., 2016).
constant concentration. The distinction of glycine or d-serine ap- When glutamate is released into the synaptic cleft, it reaches
pears to depend on brain region in addition to the subcellular lo- a high concentration (∼1.1 mM) for a brief duration of time, de-
calization of the receptor (Wolosker, 2007; Oliet and Mothet, 2009; caying with a time constant of ∼1.2 ms (Clements et al., 1992) as
Mothet et al., 2015). For example, some data suggested that d-ser- a result of diffusion and active removal of glutamate from the
ine is the dominant coagonist at synapses, with glycine being more synaptic cleft by excitatory amino acid transporters (i.e., gluta-
important at extrasynaptic sites (Papouin et al., 2012). Although mate transporter; Divito and Underhill, 2014). In the synaptic
this is an intriguing subdivision, more work will be required to cleft, glutamate will bind to AMPA (and/or kainate) and NMDA
confirm this idea as a general principle. Furthermore, glycine and receptors, inducing the necessary conformational changes that
d-serine are unlikely to be present at concentrations that saturate trigger opening of the ion channel pore, a process referred to as
the coagonist binding site (Berger et al., 1998; Bergeron et al., 1998; gating. The NMDA receptor–mediated component of the EPSC
Billups and Attwell, 2003). Thus, the requirement for a coagonist continues to pass current for tens to hundreds of milliseconds
enables an additional layer of regulation of NMDA receptor func- after synaptic glutamate is removed (Lester et al., 1990), which
tion, in which synaptic activation can be modulated by changes is in part a reflection of agonist binding affinity but also because
in the ambient levels of glycine/d-serine (Ahmadi et al., 2003; the receptor activation mechanism involves pregating steps as
Sullivan and Miller, 2012; Meunier et al., 2017). well as repeated transitions between glutamate-bound open and
There is a rapidly increasing body of data from crystal or closed conformational states until glutamate eventually unbinds
cryo-EM structures of intact NMDA receptors or individual do- and the EPSC is terminated (Lester et al., 1990; Lester and Jahr,
mains, which provides a structural framework in which to consider 1992; Erreger et al., 2005a; Zhang et al., 2008). The functional
biophysical properties of the receptors and allosteric modulation. consequences of the gating reaction mechanism are strongly
In this review, we will focus on how emerging structural under- dependent on the identity of the GluN2 subunit (Monyer et al.,
standing has provided functional insight into key properties of the 1992, 1994; Vicini et al., 1998; Wyllie et al., 1998). The four dif-
NMDA receptor that are relevant to its roles in the CNS. ferent GluN2 subunits thus create substantial diversity among
NMDA receptors, and assembly of receptors that contain differ-
Subunit composition of NMDA receptors ent GluN2 subunits with distinct properties allows tuning of the
Seven genes encode the NMDA receptor subunits: a single GRIN1 synaptic response time course and variation in parameters that
gene encodes GluN1, four GRIN2 genes encode GluN2A-D, and control synaptic strength and plasticity. This diversity exerts
Hansen et al. Journal of General Physiology 1082

Structure and function of NMDA receptors https://doi.org/10.1085/jgp.201812032
virtually eliminates potentiation by extracellular polyamines
(Durand et al., 1992, 1993; Zhang et al., 1994; Traynelis et al., 1995,
1998; Pahk and Williams, 1997; Mott et al., 1998; Rumbaugh et al.,
2000; Yi et al., 2018).
Alternative splicing of exons 21 and 22 changes the amino
acid composition of the intracellular GluN1 CTD, which interacts
with PSD-95, calmodulin, and the neurofilament subunit NF-L
(Traynelis et al., 2010). These proteins are involved in surface
trafficking and anchoring of receptors at synaptic sites, and
alternative splicing of exons 21 and 22 influences cell surface
distribution of NMDA receptors (Scott et al., 2001, 2003; Mu et
al., 2003; Wenthold et al., 2003). The CTD of GluN1 is a binding
site for calmodulin (Ehlers et al., 1996; Iacobucci and Popescu,
2017b), as well as a target of kinases and phosphatases (Tingley
et al., 1993, 1997). The relationships between functional roles and
structural features of the residues encoded by GluN1 exon 21 and

Figure 2. Subunit stoichiometry and subunit arrangement of GluN1/2
NMDA receptors. The crystal structure of the intact GluN1/2B NMDA recep- 22 are not yet fully understood.
tor (the intracellular CTD omitted from structure; Protein Data Bank accession
no. 4PE5; Karakas and Furukawa, 2014) definitively demonstrated that GluN1 The glutamate-binding GluN2 subunits
and GluN2 subunits assemble as heterotetramers with an alternating pattern
The four glutamate-binding GluN2A-D subunits provide the
(i.e., 1-2-1-2). The NMDA receptor is therefore comprised of two glycine-bind-
ing GluN1 and two glutamate-binding GluN2 subunits (i.e., GluN1/2 receptors) CNS with a means of controlling NMDA receptor properties as
that form a central cation-permeable channel pore. a function of developmental period and brain region (Fig. 3 A).
Many studies have described the variation in expression profiles
of these subunits, which ultimately control important features
many effects on neuronal function, circuit properties, and ner- of the synaptic NMDA receptor component (Monyer et al., 1992,
vous system development. 1994; Watanabe et al., 1992; Ishii et al., 1993; Akazawa et al., 1994;
Zhong et al., 1995). Attempts to pharmacologically control spe-
The glycine-binding GluN1 subunit cific NMDA receptor subtypes have, not surprisingly, focused
The GluN1 subunit, which binds glycine and d-serine, is an oblig- on the development of small molecules that can distinguish be-
atory subunit in all functional NMDA receptors and is therefore tween the GluN2 subunits (Ogden and Traynelis, 2011; Strong et
widely expressed in virtually all central neurons. Three exons in al., 2014; Vyklicky et al., 2014; Zhu and Paoletti, 2015; Hackos and
the GluN1 subunit can be alternatively spliced to produce eight Hanson, 2017; Burnell et al., 2018). These efforts are driven in
different isoforms (Durand et al., 1992; Nakanishi et al., 1992; part by the hope that GluN2 subunit–selective pharmacological
Sugihara et al., 1992; Hollmann et al., 1993). Exon 5 encodes 21 probes will allow targeting of unique circuits at specific devel-
amino acids in the GluN1 amino-terminal domain (ATD), exon 21 opmental periods to bring about a desired therapeutically ben-
encodes 37 amino acids in the carboxyl-terminal domain (CTD), eficial effect.
and exon 22 encodes 38 amino acids in the CTD. Deletion of exon Among the many differences in functional properties gov-
22 eliminates a stop codon and produces a frameshift, which re- erned by the GluN2 subunit, several are particularly noteworthy
sults in the inclusion of 22 alternative amino acids in the mature (Erreger et al., 2004; Traynelis et al., 2010; Paoletti et al., 2013;
polypeptide chain. The GluN1 splice variants show variation in Wyllie et al., 2013; Glasgow et al., 2015). The potency of gluta-
regional and developmental profiles (Laurie and Seeburg, 1994; mate is influenced by the GluN2 subunits. For example, the EC50
Zhong et al., 1995; Paupard et al., 1997) and endow the receptor for glutamate-activating NMDA receptors containing two GluN1
with unique function and pharmacology (see below). and two GluN2D subunits is more than fivefold lower (i.e., more
One important property of NMDA receptors containing GluN1 potent) than that for GluN1/2A, whereas GluN1/2B and GluN1/2C
with residues encoded by exon 5 (e.g., GluN1-1b) is reduced ag- receptors show intermediate EC50 values (Erreger et al., 2007;
onist potency (i.e., increased EC50, the concentration that pro- Chen et al., 2008; Hansen et al., 2008). The time course of deac-
duces a half-maximal response; Traynelis et al., 1995, 1998). tivation after removal of glutamate, which controls the duration
Consistent with the effect on agonist potency, the GluN1-1b splice of the synaptic EPSC (Lester et al., 1990), varies over 100-fold
variant accelerates deactivation of the NMDA receptor response for the different GluN2 subunits (Fig. 3 B). The time constants
after removal of glutamate, resulting in EPSCs with a shorter describing the exponential deactivation time course (τdecay)
duration (Rumbaugh et al., 2000; Vance et al., 2012; Swanger et are ∼40–50 ms for GluN1/2A, ∼300–400 ms for GluN1/2B and
al., 2015; Yi et al., 2018). These actions may reflect interactions GluN1/2C, and ∼4 s for GluN1/2D (Monyer et al., 1992; Vicini et
between the ATD and both the GluN1 and GluN2 agonist-binding al., 1998; Wyllie et al., 1998; Yuan et al., 2009). Interestingly, in-
domains (ABDs) created by residues encoded by exon 5 (Regan trareceptor allosteric interactions render the potency of glycine
et al., 2018). In addition, GluN1-1b alleviates inhibition of NMDA and d-serine at the GluN1 subunit sensitive to the identity of the
receptor function by GluN2B-selective antagonists, such as ifen- GluN2 subunit (Sheinin et al., 2001; Chen et al., 2008; Dravid et
prodil, reduces inhibition by extracellular Zn2+ and protons, and al., 2010; Jessen et al., 2017; Maolanon et al., 2017). For example,

the potency of glycine at GluN1/2A receptors is ∼10-fold less than
at GluN1/2D receptors (Chen et al., 2008).
Multiple biophysical properties are also controlled by the
GluN2 subunit. GluN1/2A and GluN1/2B have higher single-chan-
nel conductance than GluN1/2C and GluN1/2D receptors (Erreger
et al., 2004; Traynelis et al., 2010; Paoletti et al., 2013; Wyllie et
al., 2013; Glasgow et al., 2015; Fig. 3 C). GluN1/2A and GluN1/2B
also show higher Ca2+ permeability and are more sensitive to
Mg2+ block than GluN1/2C and GluN1/2D (Monyer et al., 1992,
1994; Burnashev et al., 1995; Kuner and Schoepfer, 1996; Qian et
al., 2005; Siegler Retchless et al., 2012). These biophysical dif-
ferences are important, as the sensitivity to voltage-dependent
Mg2+ block can influence the temporal window for spike timing–
dependent plasticity (Nevian and Sakmann, 2004, 2006; Carter
and Jahr, 2016). Furthermore, the probability that the channel
will be open when all agonist-binding sites are occupied by ago-

nists (i.e., the open probability) is strongly dependent on GluN2
identity (Fig. 3 C). The open probability is ∼0.5 for recombi-
nant GluN1/2A, ∼0.1 for GluN1/2B, and <0.02 for GluN1/2C and
GluN1/2D (Erreger et al., 2004; Traynelis et al., 2010; Paoletti et
al., 2013; Wyllie et al., 2013; Glasgow et al., 2015). The GluN2 sub-
units also control inhibition of NMDA receptors by endogenous
modulators, such as protons and extracellular Zn2+ (Traynelis et
al., 1995, 1998; Paoletti et al., 1997).
The amino acid sequence of the intracellular CTD is highly
variable among GluN2 subunits, thereby producing pronounced
differences in interaction sites for phosphatases, kinases, and
proteins responsible for anchoring at synaptic sites and surface
trafficking (Wenthold et al., 2003; Traynelis et al., 2010; Sanz-
Clemente et al., 2013; Aman et al., 2014; Lussier et al., 2015). As a
result of this variation, the GluN2 subunits therefore influence
cell-surface expression, subcellular localization, and recycling/
degradation of NMDA receptor subtypes.
Diheteromeric and triheteromeric NMDA receptors

The NMDA receptor subunits assemble into receptors with vary-
ing composition and distinct functional properties and roles in
the CNS. At least two different GluN2 subunits are expressed in
most neurons, and thus virtually all neurons have the oppor-
tunity to signal through triheteromeric NMDA receptors that
contain two GluN1 and two different GluN2 subunits (Chazot et
al., 1994; Sheng et al., 1994; Chazot and Stephenson, 1997; Luo
et al., 1997; Cathala et al., 2000; Piña-Crespo and Gibb, 2002;
Brickley et al., 2003; Jones and Gibb, 2005; Al-Hallaq et al., 2007;
Rauner and Köhr, 2011; Tovar et al., 2013; Huang and Gibb, 2014;
Swanger et al., 2015). Multiple lines of evidence support the ex-
pression of triheteromeric NMDA receptors of the GluN1/2A/2B,
Figure 3. Expression and functional properties of NMDA receptor sub-

types determined by the GluN2 subunit. (A) Autoradiograms obtained by open tip current in the upper trace. Fig. 3 B is adapted from Vicini et al. (1998)
in situ hybridizations of oligonucleotide probes to parasagittal sections of rat with permission from the Journal of Neurophysiology. (C) Single-channel
brain at indicated postnatal (P) days reveal distinct regional and developmen- recordings of currents from outside-out membrane patches obtained from
tal expression of GluN2 subunits. Fig. 3 A is modified from Akazawa et al., 1994 HEK293 cells expressing recombinant NMDA receptor subtypes. The sin-
with permission from the Journal of Comparative Neurology. (B) Whole-cell gle-channel recordings demonstrate distinct open probabilities and channel
patch-clamp recordings of responses from recombinant diheteromeric NMDA conductances depending on the GluN2 subunit in the diheteromeric NMDA
receptor subtypes expressed in HEK293 cells. The receptors are activated by receptor. Highlights of individual openings are shown on the left. Adapted
a brief application of glutamate (1 ms of 1 mM glutamate) indicated by the from Yuan et al. (2008).

GluN1/2A/2C, and GluN1/2B/2D subtypes in neurons (Chazot et ABD, a pore-forming transmembrane domain (TMD), and an
al., 1994; Sheng et al., 1994; Takahashi et al., 1996; Chazot and intracellular CTD (Fig. 4 A). The TMD is formed by three trans-
Stephenson, 1997; Luo et al., 1997; Sundström et al., 1997; Dunah membrane helices (M1, M3, and M4) and a reentrant loop (M2).
et al., 1998; Tovar and Westbrook, 1999; Piña-Crespo and Gibb, In iGluRs, the reentrant loop lines the intracellular portion of the
2002; Brickley et al., 2003; Dunah and Standaert, 2003; Jones ion channel pore, whereas elements of the third transmembrane
and Gibb, 2005; Lu et al., 2006; Al-Hallaq et al., 2007; Brothwell segment (M3) form the extracellular region of the pore. Among
et al., 2008; Gray et al., 2011; Rauner and Köhr, 2011; Tovar et al., NMDA receptor subtypes, the residues in the pore region, which
2013; Huang and Gibb, 2014; Swanger et al., 2015, 2018). Many influence ion permeation, are highly conserved. A key determi-
important properties of triheteromeric NMDA receptors in the nant of ion permeation, which controls divalent ion permeabil-
CNS are still poorly understood, and the combinations of GluN2 ity and Mg2+ block, resides at the apex of the reentrant M2 loop
subunits that can form triheteromeric receptors have not been and is often referred to as the Q/R/N site on the basis of amino
fully established. This knowledge gap persists because trihetero- acids at this position in AMPA, kainate, and NMDA receptors
meric NMDA receptors have been difficult to study in isolation (Wollmuth, 2018).
(Chazot et al., 1994; Brimecombe et al., 1997; Vicini et al., 1998; The ATDs from each subunit adopt bilobed structures formed
Tovar and Westbrook, 1999; Hatton and Paoletti, 2005; Hansen by the first ∼350 amino acids that associate as back-to-side het-
et al., 2014; Stroebel et al., 2014). That is, coexpression of GluN1 erodimers between GluN1 and GluN2. The ATDs play important

with two different GluN2 subunits (e.g., GluN2A and GluN2B) roles in assembly and strongly modulate NMDA receptor func-
will produce three populations of functional NMDA receptors, tion (Atlason et al., 2007; Gielen et al., 2009; Yuan et al., 2009;
including diheteromeric GluN1/2A and GluN1/2B as well as tri- Farina et al., 2011). Furthermore, the ATDs create binding sites
heteromeric GluN1/2A/2B receptors (Brimecombe et al., 1997; for allosteric modulators, including extracellular Zn2+ and a di-
Vicini et al., 1998; Hatton and Paoletti, 2005; Hansen et al., 2014; verse series of GluN2B-selective antagonists (exemplified by if-
Stroebel et al., 2014). A wealth of information exists describing enprodil; Karakas et al., 2009, 2011; Romero-Hernandez et al.,
the function, pharmacology, and regulation of recombinant di- 2016; Tajima et al., 2016; Fig. 4 B).
heteromeric NMDA receptors that contain two copies each of The ABD is formed by the S1 and S2 segments of the polypep-
GluN1 and a single type of GluN2 (e.g., GluN1/GluN2A). In con- tide chain, which are separated by the M1, M2, and M3 segments.
trast, relatively little is known about how the coassembly of two The ABDs form kidney-shaped bilobed structures that contain
different GluN2 subunits affects receptor properties, including an upper lobe (D1) and a lower lobe (D2) with the agonist-bind-
the deactivation time course, concentration dependence, and ing site residing in the cleft between these two lobes (Fig. 4 A).
voltage dependence of Mg2+ block and the sensitivity to sub- The ABD structure, intra- and intersubunit interactions, and its
unit-selective allosteric modulators. Similarly, phosphoryla- influence on receptor function have been studied for more than
tion sites and trafficking properties of the intracellular GluN2 two decades. More recently, crystallographic and cryo-EM data
CTDs have been extensively studied in diheteromeric receptors, have provided the first glimpses of the domain organization of
whereas the regulation of triheteromeric NMDA receptors that inactive and active GluN1/2B NMDA receptors, providing mech-
possess two distinct GluN2 CTDs remains elusive (Tang et al., anistic hypotheses by which the different domains and their cog-
2010). Knowledge of the key NMDA receptor properties is an nate ligands influence receptor function (Karakas and Furukawa,
essential step to understand the roles of triheteromeric recep- 2014; Lee et al., 2014; Tajima et al., 2016; Zhu et al., 2016; Regan
tors in the brain. One recent advance that has enabled a deter- et al., 2018; Song et al., 2018). We will consider each of these do-
mination of the functional and pharmacological properties of mains in more detail below.
some triheteromeric NMDA receptors has been to control cell
surface expression of receptors with known GluN2 subunit com- Structure and function of GluN1 and GluN2 ABDs
position (Hansen et al., 2014; Yi et al., 2017, 2018). This method Keinänen and colleagues were the first to demonstrate that re-
has provided information about the properties of triheteromeric combinant glutamate receptor ABDs can be generated as soluble
GluN1/2A/2B receptors, which are distinct from the properties proteins by linking the S1 and S2 polypeptide sequences with
of the diheteromeric receptors that contain composite subunits an artificial peptide linker (Kuusinen et al., 1995; Arvola and
(Hansen et al., 2014; Stroebel et al., 2014; Cheriyan et al., 2016; Keinänen, 1996). Subsequent work by Gouaux and colleagues
Hackos et al., 2016; Serraz et al., 2016; Yi et al., 2016, 2018). Im- resulted in the first crystal structures of glutamate receptor
portantly, these properties are not simply the average of the ABDs (Armstrong et al., 1998; Armstrong and Gouaux, 2000).
respective diheteromeric NMDA receptor properties. This new The water-soluble ABD proteins produced by this approach re-
approach should allow new opportunities to develop therapeutic tain ligand-binding activities comparable to those in full-length
agents that target disease-relevant triheteromeric NMDA recep- glutamate receptors, indicating that structural integrity and
tors (Khatri et al., 2014; Yuan et al., 2014; Hackos et al., 2016; characteristics of the agonist-binding pocket are retained in iso-
Serraz et al., 2016; Yi et al., 2016; Swanger et al., 2018). lated ABDs. ABD structures for GluN1, GluN2, and GluN3 sub-
units have been solved in complex with agonists, antagonists, and
NMDA receptor structure and function allosteric modulators (Furukawa and Gouaux, 2003; Furukawa
All glutamate receptor subunits share a similar architecture that et al., 2005; Inanobe et al., 2005; Yao and Mayer, 2006; Yao et
comprises four domains: a large extracellular ATD, a bilobed al., 2008, 2013; Vance et al., 2011; Hansen et al., 2013; Kvist et

Figure 4. Domain organization and ligand-binding sites in NMDA receptors. (A) The linear representation of the polypeptide chain illustrates the segments
that form the four semiautonomous subunit domains shown in the cartoon, which are the extracellular ATD, the ABD, the TMD formed by three transmem-
brane helices (M1, M2, and M4) and a membrane reentrant loop (M2), and the intracellular CTD. The ABD is formed by two polypeptide segments (S1 and S2)
that fold into a bilobed structure with an upper lobe (D1) and lower lobe (D2). The agonist-binding site is located in the cleft between the two lobes. (B) The
crystal structure of the GluN1/2B NMDA receptor (Protein Data Bank accession no. 4PE5; Karakas and Furukawa, 2014) shows the subunit arrangement and
the layered domain organization. The binding sites for agonists (and competitive antagonists) as well as predicted and known binding sites for PAMs and NAMs
are highlighted. The figure is adapted from Hansen et al. (2017).
al., 2013; Jespersen et al., 2014; Hackos et al., 2016; Volgraf et al., tional binding pocket that competitive antagonists can exploit in
2016; Yi et al., 2016; Lind et al., 2017; Romero-Hernandez and a GluN2-dependent manner (Fig. 5 B).
Furukawa, 2017). In addition to NMDA receptor subunits, nu- The residues at the heterodimer interface between the GluN1
merous crystal structures for AMPA and kainate receptor sub- and GluN2 ABDs modulate receptor function in several import-
units (Pøhlsgaard et al., 2011; Kumar and Mayer, 2013; Karakas et ant ways. Three separate areas of contact between GluN1 and
al., 2015) have provided insight into the mechanism underlying GluN2A can be seen in the ABD heterodimer crystal structures
full and partial agonism, suggested molecular determinants of (referred to as sites I, II, and III; Furukawa et al., 2005; Fig. 5 A).
subunit selectivity, and demonstrated mechanism and binding Sites I and III consist of hydrophobic residues from both GluN1
pose for competitive antagonists. and GluN2, and nonpolar interactions between these residues
The GluN2A ABD in complex with the GluN1 ABD provided mediate ABD heterodimerization (Furukawa et al., 2005). The
the first structural information about a GluN1/GluN2 subunit heterodimeric arrangement of GluN1 and GluN2A ABDs is sim-
interface within the NMDA receptor complex, in addition to the ilar to the homodimeric arrangement found in some AMPA and
binding mode for glutamate and glycine between the two lobes kainate receptors. In AMPA receptors, allosteric modulators such
(D1 and D2) of GluN2A and GluN1, respectively (Furukawa et al., as cyclothiazide and aniracetam bind to sites equivalent to site
2005; Fig. 5 A). Multiple water molecules reside in close prox- I + III and site II, respectively, resulting in block of desensiti-
imity to the agonists, and some form a hydrogen-bonding net- zation and slowing of deactivation speeds (Sun et al., 2002; Jin
work that interacts with the ligand. The glycine-binding pocket et al., 2005). The aromatic ring of Tyr535 in GluN1 has a posi-
in GluN1 is considerably smaller and more hydrophobic than tional overlap with that of aniracetam bound in AMPA receptors,
the glutamate-binding pocket in GluN2 (Furukawa and Gouaux, therefore acting like a natural “tethered ligand” incorporated
2003; Furukawa et al., 2005; Inanobe et al., 2005; Yao et al., in the primary sequence (Furukawa et al., 2005). Consistently,
2008, 2013). Residues within the glutamate-binding pocket that mutations of Tyr535 in GluN1 alters deactivation time course of
make atomic contacts with agonists or competitive antagonists NMDA receptors, suggesting that the heterodimer interface can
are mostly conserved in the GluN2 subunits, and it has there- influence factors controlling deactivation, such as agonist disso-
fore proven difficult to identify ligands that bind to this site with ciation or channel open time (Furukawa et al., 2005; Borschel et
strong selectivity between the different NMDA receptor sub- al., 2015). Recent crystallographic studies have shown that site
types. However, recent crystallographic data have revealed the II of the GluN1/2A ABD heterodimer contains the binding sites
structural basis for binding of antagonists with modest selectiv- for both positive and negative allosteric modulators with strong
ity (Lind et al., 2017; Romero-Hernandez and Furukawa, 2017). selectivity for GluN2A (Hackos et al., 2016; Volgraf et al., 2016; Yi
Selectivity in these cases is driven by space outside the conven- et al., 2016). Together, these results identify the heterodimer ABD

Figure 5. Crystal structures of NMDA receptor ABDs. (A) Structures of the soluble GluN1/2 ABD heterodimers reveal the subunit interface and back-to-
back dimer arrangement of the ABDs. The structure shown here is for the GluN1/2A ABD heterodimer with bound glutamate and glycine shown as spheres
(Protein Data Bank accession no. 5I57; Yi et al., 2016). The top view of the structure highlights sites I–III at the subunit interface. (B) Overlay of crystal structures
of GluN1/2A ABD heterodimers in complex with glycine and either glutamate agonist (Protein Data Bank accession no. 5I57; Yi et al., 2016) or a competitive
glutamate site antagonist (Protein Data Bank accession no. 5U8C; Romero-Hernandez and Furukawa, 2017). Activation of NMDA receptors requires agonist-in-
duced ABD closure. Competitive antagonists bind the ABD without inducing domain closure, thereby preventing receptor activation. (C) Magnified views of the
glutamate-binding site with bound GluN2A-preferring antagonists NVP-AAM077 (Protein Data Bank accession no. 5U8C; Romero-Hernandez and Furukawa,
2017) or ST3 (Protein Data Bank accession no. 5VII; Lind et al., 2017). Schild analyses demonstrated that NVP-AAM077 has 11-fold and ST3 has 15-fold pref-
erence for GluN1/2A over GluN1/2B receptors (data adapted from Lind et al., 2017). The crystal structures reveal a binding mode in which NVP-AAM077 and
ST3 occupy a cavity that extends toward GluN1 at the subunit interface, and mutational analyses show that the GluN2A preference of these antagonists is
primarily mediated by four nonconserved residues (Lys738, Tyr754, Ile755, and Thr758) that do not directly contact the ligand but are positioned within 12 Å

interface as an important locus for modulation of NMDA receptor al., 2005; Vance et al., 2011; Hansen et al., 2013). However, these
function (Fig. 5 C). crystal structures capture only one conformation of the isolated
NMDA receptors are sensitive to the redox potential, and ABDs, which may be influenced by the lack of interacting do-
reducing conditions can enhance NMDA receptor–mediated mains (ATD and TMD) and is further stabilized by contacts in
current responses (Aizenman et al., 1989; Tang and Aizenman, the crystal lattice. This caveat to crystal structures of the isolated
1993; Köhr et al., 1994; Choi and Lipton, 2000). This sensitivity ABDs is highlighted by recent single-molecule FRET and molec-
appears to be mediated by a pair of conserved cysteine residues ular dynamics studies that provide insight into the dynamic be-
(C744 and C798) within the GluN1 subunit (Sullivan et al., 1994; havior of the NMDA receptor ABDs (Yao et al., 2013; Dai et al.,
Choi et al., 2001). These two residues interact as a disulfide bond 2015; Dai and Zhou, 2015; Dolino et al., 2015, 2016). These studies
in the GluN1/2A ABD heterodimer structure, and reduction of suggest that the ABDs fluctuate between open and closed cleft
this conformational constraint in GluN1, but not GluN2, en- conformations even in the absence of agonist (i.e., the apo state).
hances NMDA receptor function (Sullivan et al., 1994; Talukder However, binding of full agonist changes the energy landscape
et al., 2011). Several other disulfide bonds exist in ABD crystal for ABD conformations to strongly favor a fully closed confor-
structures of both GluN1 and GluN2 subunits, but functional ef- mation, whereas binding of partial agonists is less efficient in
fects of their reduction or oxidation have not yet been described changing this landscape, thereby enabling the ABD to adopt con-
(Takahashi et al., 2015). formations with intermediate domain closure more frequently

Structures of the GluN1-GluN2A ABD heterodimer in com- than full agonists. Hence, a conformational selection mechanism
plex with various agonists, partial agonists, and antagonists have is likely to account for partial agonism in NMDA receptors de-
suggested a structural basis for their modes of action (Furukawa spite the lack of crystallographic data showing intermediate do-
and Gouaux, 2003; Furukawa et al., 2005; Inanobe et al., 2005; main closure for partial agonists.
Vance et al., 2011; Hansen et al., 2013; Yao et al., 2013; Jespersen
et al., 2014). Binding of glycine and glutamate to GluN1 and Structures of intact tetrameric NMDA receptors
GluN2 ABDs, respectively, produces a rapid ABD rearrange- The first structures of intact NMDA receptors (GluN1/2B extra-
ment that involves reduction of the angle between the D1 and D2 cellular domains and TMDs) confirmed the hypothesized domain
lobes, producing a clamshell-like closure of the bilobed domain organization and showed that GluN1 and GluN2B subunits exist in
(Fig. 5 B). This agonist-mediated ABD closure triggers formation an alternating pattern (i.e., 1-2-1-2) within the tetrameric assem-
of hydrogen bonds between residues from the upper and lower bly (Karakas and Furukawa, 2014; Lee et al., 2014; Fig. 6). These
lobes, which are hypothesized to stabilize the agonist-bound ABD studies also confirmed that the NMDA receptor structure shares
structure (Kalbaugh et al., 2004; Paganelli et al., 2013). The en- certain characteristics with AMPA and kainate receptors. First,
ergy provided by agonist binding and ABD closure triggers the the receptor subunits adopt a layered structure, with one layer
receptor to undergo a series of conformational changes that ulti- formed by TMDs and two extracellular layers formed by ABD het-
mately open the ion channel pore. Thus, ABD closure that results erodimers and ATD heterodimers. Second, the TMDs have a qua-
from agonist binding is the initial conformational change that si-fourfold symmetry, whereas the extracellular portion shows
ultimately triggers the process of ion channel gating. Binding twofold symmetry between the two ABD heterodimers and ATD
of competitive antagonists, such as the glycine site antagonist heterodimers in a dimer-of-dimer arrangement. Thus, there is
DCKA and the glutamate site antagonist D-AP5, stabilizes an a symmetry mismatch between the TMD layer and the extracel-
open cleft conformation that is incapable of triggering channel lular layers of the receptor. Third, there is a remarkable subunit
gating (Fig. 5 B). crossover between the ABD layer and the ATD layer (Fig. 6). In ad-
The stabilization of the NMDA receptor ABDs in a closed cleft dition, the NMDA receptor has several unique structural features
conformation by agonist binding and in an open cleft conforma- when compared with AMPA and kainate receptors (Karakas and
tion by competitive antagonist binding is similar to that found Furukawa, 2014; Lee et al., 2014). For example, there are exten-
for the AMPA and kainate receptor ABDs (Pøhlsgaard et al., 2011; sive contacts between the two GluN1/2 ABD heterodimers that
Kumar and Mayer, 2013; Karakas et al., 2015). However, one no- are not present in AMPA and kainate receptor structures. These
table difference exists. Multiple structures of AMPA receptor contacts may provide the structural basis of the GluN2 subunit
ABDs in complex with partial agonists show partial domain clo- dependence of glycine potency. In addition, the NMDA receptor
sure that correlates with their efficacy (Pøhlsgaard et al., 2011). In ATDs show a different arrangement, leading to distinct subunit
contrast, multiple structures of ABD with bound partial agonists, interfaces compared with AMPA and kainate receptors. Impor-
such as d-cycloserine, ACPC, and ACBC in GluN1 and NMDA and tantly, the ATDs form extensive contacts with the upper lobe
Pr-NHP5G in GluN2 show virtually identical degrees of domain of the ABD whereas the ATD–ABD interactions are minimal in
closure compared with structures with full agonists (Inanobe et AMPA and kainate receptors. These interactions give the NMDA
of the glutamate-binding site. (D) Structure of the agonist-bound GluN1/2A ABD heterodimer with the NAM MPX-007 bound at site II in the subunit interface
(Protein Data Bank accession no. 5I59; Yi et al., 2016). (E) Magnified views of site II in GluN1/2A ABD heterodimer with bound MPX-007 (NAM; Protein Data Bank
accession no. 5I59; Yi et al., 2016) or PAM GNE-8324 (Protein Data Bank accession no. 5H8Q; Hackos et al., 2016). The overlay illustrates the distinct effects of
NAM and PAM binding on Val783 in GluN2A and Tyr535 in GluN1. The GluN2A selectivity of the NAMs and PAMs binding at this modulatory site is mediated by
Val783 in GluN2A, which is nonconserved among GluN2 subunits (Phe in GluN2B and Leu in GluN2C/GluN2D).

Figure 6. Structure of the intact NMDA
receptors. (A) Structure of the glycine- and
glutamate-bound GluN1-1b/2B NMDA recep-
tor without CTDs (Protein Data Bank accession
no. 5FXI; Tajima et al., 2016). (B) The GluN1 (1)
and GluN2 (2) subunits are arranged as a dimer
of heterodimers at the ATD and ABD layers in a
1-2-1-2 fashion. Note that the heterodimer pairs
are interchanged between the ATD and ABD lay-
ers (i.e., subunit crossover). In the TMD layer,
the GluN1 and GluN2 subunits are arranged as
a tetramer with pseudo-fourfold symmetry. (C)
Comparison of the two major conformational
states observed in the presence of glycine and
glutamate by cryo-EM/single-particle analysis.
Shown in spheres are the Cα of the gating ring
residues, GluN1-1b Arg684 and GluN2B Glu658,
which are adjacent to the pore-forming M3
transmembrane helices. In the nonactive (Pro-

tein Data Bank accession no. 5FXI; Tajima et al.,
2016) and active (Protein Data Bank accession
no. 5FXG; Tajima et al., 2016) conformations, the
distances between the two GluN2B Glu658 Cα
atoms are ∼29 Å and ∼45 Å, respectively, indi-
cating that degrees of tension in the ABD–TMD
loops are different.
receptor a more compact “hot air balloon–like” appearance, multiple conformations in the extracellular region, providing
which is distinct from the more Y-shaped AMPA and kainate the first dynamic pictures of NMDA receptor conformational
receptors. The ABD–ATD interactions also create a protein–pro- changes and insight into the structural mechanism of receptor
tein interface at which modulators can bind (Khatri et al., 2014; activation and allosteric modulation (Tajima et al., 2016; Zhu et
Kaiser et al., 2018). A recent study also showed that the motif al., 2016). Unfortunately, the TMDs for the active and antago-
encoded by exon 5, which controls pH sensitivity, deactivation nist-bound states are not well resolved in the cryo-EM structures,
time course, and agonist potency, is also located at the ABD–ATD limiting mechanistic insights into gating and antagonism. How-
interface and modulates the ABD–ATD interaction (Regan et al., ever, in the active conformation, distances between the residues
2018). The structure of the NMDA receptor thus reveals unique that are in proximity of the TMDs increase as much as ∼20 Å in
intra- and interdomain contacts that provide a framework for the context of the heterotetramer (Fig. 6 C). This “dilation” of the
understanding allosteric interactions between subunits, as well gating ring likely generates sufficient tensions in the ABD–TMD
as allosteric modulation by small-molecule ligands. linker for rearrangement of the helices that form the gate (Kazi
Although the crystallographic structures of intact NMDA re- et al., 2014; Twomey and Sobolevsky, 2018). This tension can
ceptors advance our understanding of the structure–function lead to reorientation of the M3 helix in AMPA receptors as well
relationship, they nevertheless capture only a low energy confor- as a kink at an alanine residue that appears to serve as a hinge.
mation among the many conformations that the NMDA receptor Whether or not gating of NMDA receptor ion channels involves
moves through en route to activation. In these crystal structures, similar conformational alterations of the ABD–TMD linker and
glycine and glutamate were bound to GluN1 and GluN2B, respec- the TMD demonstrated in the recent AMPA receptor structures
tively, and the GluN2B-selective negative allosteric modulator if- (Twomey and Sobolevsky, 2018) remains to be seen, although the
enprodil was bound to the interface between GluN1 and GluN2B gating motifs are highly conserved. Nevertheless, given that the
ATDs (Karakas and Furukawa, 2014; Lee et al., 2014). The struc- relative orientation of the ABDs and TMDs is distinct in NMDA
tures represent the agonist-bound, inhibited receptor with the receptors, structural data are required to evaluate whether these
ion channel closed. However, recent cryo-EM data have described ideas transfer between AMPA and NMDA receptors.

Control of NMDA receptor function by the ATD Functional and structural studies have converged on a struc-
The ATD adopts a bilobed structure, which is unrelated to the tural model for NMDA receptor modulation by Zn2+ and ifen-
ABD, with R1 and R2 referring to upper and lower lobes, respec- prodil, where modulator binding regulates receptor function
tively (Karakas et al., 2009, 2011). Furthermore, there is a unique through rearrangement of the ATD layer and GluN2 ATD clam-
dimer-of-dimer arrangement of the NMDA receptor ATDs com- shell opening and closing (Sirrieh et al., 2013, 2015a). In GluN1/2B,
pared with the ATDs in AMPA and kainate receptors (Karakas opening of the ATD bilobes robustly alters inter-GluN1/GluN2
and Furukawa, 2014; Lee et al., 2014; Meyerson et al., 2014; subunit arrangement within the ATD, which results in a ∼13°
Sobolevsky, 2015; Tajima et al., 2016; Zhu et al., 2016). This ar- rotation between the GluN1/2B ABD dimers and dilation of the
rangement, which is revealed in crystal and cryo-EM structures gating ring (Tajima et al., 2016). NAMs such as ifenprodil and
of intact iGluRs, is characterized by a protein–protein interface zinc favor the closure of the bilobed GluN2B ATD thereby “lock-
formed by the upper R2 lobes from the GluN1 and GluN2 sub- ing” the subunit arrangement in a way that prevents dilation of
units, whereas the lower R1 lobes, which connect to the ABDs, are the gating ring. Interestingly, the zinc-bound GluN2A ATD is ∼13°
almost completely separated. more open compared with the zinc-bound GluN2B ATD (Karakas
Many of the GluN2-specific differences between NMDA re- et al., 2009; Romero-Hernandez et al., 2016). This may explain in
ceptor subtypes are caused by sequence variation in the GluN2 part the observation that the extent of zinc inhibition is smaller
ATDs (Gielen et al., 2009; Yuan et al., 2009). Consistent with in GluN2A than GluN2B (Rachline et al., 2005).

this idea, chimeric GluN2 subunits that swap the ATD between NMDA receptors containing GluN1 with exon 5 (e.g., the
GluN2A and GluN2D shift the open probability, deactivation time GluN1-1b splice variant) have reduced sensitivity to all three al-
course, and agonist potency toward that of the subunit contrib- losteric modulators (Zn2+, ifenprodil, and spermine; Traynelis
uting the ATD (Gielen et al., 2009; Yuan et al., 2009). Although it et al., 1995; Mott et al., 1998; Yi et al., 2018). In recent cryo-EM
remains unclear how the ATD controls NMDA receptor function, structures, the 21 amino acids encoded by exon 5 are placed just
the mechanism likely involves intra- and intersubunit allosteric above the GluN1-GluN2 ABD heterodimer interface between the
interactions between the ATDs and ABDs that influence the con- ATD and ABD layers, positioned to influence allosteric interac-
figuration of the GluN1/GluN2 ABD heterodimer and thereby tions between GluN2 ATD clamshell motions and GluN1-GluN2
impact channel activation (Gielen et al., 2008; Zhu et al., 2013; ABDs (Regan et al., 2018). Furthermore, GluN2C residues from
Tajima et al., 2016). Thus, some GluN2-specific functional and both the ATD and ABD that influenced the activity of PYD-106, a
pharmacological NMDA receptor properties are presumably GluN2C-selective positive allosteric modulator (PAM), have been
controlled by distinct conformations adopted by the ATDs in a identified and molecular modeling proposed a modulatory bind-
GluN2-specific manner (Hansen et al., 2014; Zhu et al., 2014; ing site located in a pocket at the ATD–ABD interface of GluN2C
Sirrieh et al., 2015b; Lü et al., 2017; Sun et al., 2017). (Khatri et al., 2014; Kaiser et al., 2018). These studies all point to
the ATD as the major structural determinant of GluN2-specific
Ligand binding to the ATD variation among NMDA receptor subtypes. For this reason, al-
Crystal structures have demonstrated that GluN2B-selective neg- losteric modulation of NMDA receptors by the ATD is intensely
ative allosteric modulators (NAMs), such as ifenprodil and Ro 25– investigated, and drug discovery studies are poised to identify
6981, bind to a modulatory site located at the subunit interface novel ATD ligands with therapeutic potential.
between GluN1 and GluN2B ATDs (Karakas et al., 2011; Karakas
and Furukawa, 2014; Lee et al., 2014; Stroebel et al., 2016). These Channel gating in NMDA receptors
crystal structures revealed that only one residue in this modula- All three transmembrane helices (M1, M3, and M4) and the
tory site is different between GluN2A and GluN2B subunits, but membrane-reentrant pore-forming loop (M2) are involved in the
sensitivity to ifenprodil is not introduced by converting this or process of pore opening (i.e., channel gating; Schneggenburger
other residues in GluN2A to that in GluN2B (Karakas et al., 2011; and Ascher, 1997; Krupp et al., 1998; Villarroel et al., 1998; Ren
Burger et al., 2012). This stems from the fact that the intersubunit et al., 2003; Talukder et al., 2010; Kazi et al., 2013; Ogden and
arrangements in GluN1/2A and GluN1/2B ATD heterodimers are Traynelis, 2013; Alsaloum et al., 2016). The transmembrane helix
distinct from each other, as demonstrated in the recent crystal M3 forms a helical bundle crossing that physically occludes the
structure of the GluN1/2A ATD heterodimer (Romero-Hernandez pore, and thus M3 helices must change their position before ions
et al., 2016). Specifically, the “pocket” in the GluN1/GluN2 sub- can pass through the channel pore (Jones et al., 2002; Yuan et
unit interface is ideally sized to accommodate ifenprodil ana- al., 2005; Chang and Kuo, 2008). The M3 transmembrane helix
logues in GluN1/2B, whereas such a pocket is absent in GluN1/2A contains nine amino acids (SYTANLAAF) that are almost fully
because of the different subunit arrangement characterized by a conserved in iGluRs throughout the animal kingdom. Multiple
∼10° rotation compared with GluN1/2B. Multiple lines of investi- structural and functional studies suggest that these residues
gation, including cryo-EM structures of intact NMDA receptors, comprise the activation gate and that dilation of the M3 helical
functional studies, and computational analyses, suggest that if- bundle crossing is thought to be the key change that allows ion
enprodil inhibition involves closure of the GluN2B ATD bilobes conduction (Beck et al., 1999; Sobolevsky et al., 2002a; Chang
with accompanying changes in the arrangement of the GluN1/2B and Kuo, 2008).
ATD heterodimers (Burger et al., 2012; Tajima et al., 2016), in- What sequence of events leads to M3 rearrangement? Ag-
dicating that both clamshell conformation and subunit arrange- onist binding to the bilobed ABDs involves a clamshell closure
ment are coupled to function of the NMDA receptor ion channel. around the ligands that must be the first step in a sequence of

Figure 7. Single-channel recordings of NMDA
receptor gating. Recording of receptor activation
(i.e., channel gating or pore dilation) in an excised
outside-out membrane patch containing a single
GluN1/2B receptor exposed to 1 mM glutamate plus
30 µM glycine for 1 ms as indicated. In this exam-
ple, receptor activation results in a characteristically
long burst of channel openings and closings (dura-
tion 128 ms). Evaluation of closed periods within the
GluN1/2B activation suggests that two kinetically dis-
tinct pregating steps exist (i.e., fast and slow steps;
see Fig. 8 D for model). Some (but not all) closures
within the activation will reflect reversal of pore dila-
tion, reversal of a single pregating step, followed by
forward movement back through the pregating step
and pore dilation. Two possible closures that might
reflect the slow and fast pregating components
are highlighted in red. Data are from Banke and
Traynelis (2003).

conformational changes that lead to gating. These are followed by 2005a; Dravid et al., 2008; Kussius and Popescu, 2009; Amico-
multiple short-lived, intermediate conformations that precede a Ruvio and Popescu, 2010; Vance et al., 2012).
rapid transition from the closed to the open state of the ion chan-
nel, inferred by brief, kinetically distinguishable closed states in Kinetic models for NMDA receptor activation
the single-channel record and the relatively slow time course The sequence of protein conformational changes that trigger
for receptor activation by supersaturating agonist (Banke and channel gating can be described as reaction schemes (i.e., ki-
Traynelis, 2003; Popescu et al., 2004; Auerbach and Zhou, 2005; netic models) with agonist binding steps and transitions be-
Erreger et al., 2005a; Schorge et al., 2005; Kussius and Popescu, tween different conformational states of the receptor (Fig. 8).
2009; Fig. 7). However, there is poor understanding of the pro- The first widely applied kinetic model for NMDA receptor gating
tein conformations that represent the rate limiting steps en route was solely designed to account for the time course of the mac-
to channel opening. Moreover, the lifetimes of some of these in- roscopic current response and consisted of two identical but
termediate conformations are brief, suggesting they are unlikely independent glutamate binding steps, one desensitized state,
to be captured in crystal structures or cryo-EM studies, leaving one closed state, and one open state (Lester and Jahr, 1992).
functional experiments as the most feasible (yet imperfect) way This simple kinetic model appeared to effectively capture key
to glean clues as to how these changes might control channel features of macroscopic NMDA receptor responses but was not
opening. Recent functional studies have built explicit models of intended to describe the complexity observed in single-channel
channel activation in which specific conformations are hypoth- recordings (Ascher et al., 1988; Howe et al., 1991; Traynelis and
esized for each of the four subunits (Gibb et al., 2018). Moreover, Cull-Candy, 1991; Gibb and Colquhoun, 1992). Furthermore, the
work with disease-causing mutations identified in human pa- usefulness of the Lester and Jahr model was limited by the lack
tients has provided key insights into the elements that comprise of glycine-binding steps required for receptor activation. Kinetic
the gating control mechanism. Residues in the region connecting models that account for both glutamate- and glycine-binding
the S1 segment of the ABD with the M1 transmembrane helix (i.e., steps as well as intersubunit interactions between the glutamate
the pre-M1 linker) are invariant in the healthy population, and and glycine ABDs have also been developed (Benveniste et al.,
a locus for disease-associated mutations in various neurological 1990), and these models could capture additional features of the
diseases (Ogden et al., 2017). In addition, the region connecting time course of NMDA receptors, including glycine-dependent
the S2 segment of the ABD with the M4 transmembrane helix desensitization (see below).
(i.e., the pre-M4 linker) also appears to be implicated in patients Newer, more complex kinetic models have been proposed
with NMDA receptor missense mutations, and both the pre-M1 that better describe single-channel data by incorporating mul-
and pre-M4 linkers are close enough to be in contact with the tiple steps between binding and gating (Banke and Traynelis,
conserved SYTANLAAF motif in the M3 helical bundle crossing. 2003; Popescu et al., 2004; Auerbach and Zhou, 2005; Erreger
These three elements (pre-M1, SYTANLAAF, and pre-M4) ap- et al., 2005a; Schorge et al., 2005). Investigations of macro-
pear positioned to form a gating control mechanism (Chen et al., scopic and single-channel responses to partial and full agonists
2017), and it is possible that kinetically distinct conformational suggest that agonist binding to either GluN1 or GluN2 controls
states may be the result of rearrangements of this triad of in- distinct steps in the kinetic model (Banke and Traynelis, 2003;
teracting regions. Moreover, the different amino acid sequences Auerbach and Zhou, 2005; Erreger et al., 2005a; Schorge et al.,
for pre-M1 and pre-M4 that exist for GluN1 and GluN2 as well as 2005; Fig. 8), although it has also been suggested that partial
different positions of these elements in relation to the gating ring agonists can impact all pregating steps irrespective of the sub-
could lead to distinct lifetimes for intermediate conformations unit they bind to (Kussius and Popescu, 2009; Kussius et al.,
that must be traversed before rapid pore dilation (Erreger et al., 2010). In some of these models, the actions of allosteric modu-

Figure 8. Application of a gating reaction mechanism of NMDA receptors. (A) Individual responses from a recombinant GluN1/2B channel in an excised
outside-out patch activated by 1 ms application of maximally effective glutamate and glycine (indicated by the gray vertical bar and the open tip recording above
the channel recordings). The patch contained a single active channel, which allowed analysis of the variable delay before channel opening. NMDA receptors
bind agonist rapidly and subsequently open after a multimillisecond delay that reflects transition through kinetically distinct protein conformations before
pore dilation (i.e., channel gating). Note that although application of maximal glutamate and glycine always produces a binding event, not all binding events
lead to channel opening. Reproduced from Erreger et al. (2005a). (B) The cumulative plot of latency to opening after application of 1 mM glutamate for 1 ms.
(C) The average of all individual recordings of single activations produced a macroscopic waveform with a characteristic rise time. (D) Evaluation of closed
periods within the GluN1/2B activation suggested a model where two pregating steps can occur in any order and explosive opening of the pore, which occurs
faster than the resolution of the recordings, is assumed to happen instantaneously once both pregating steps have been traversed. (E) Simulation of a single
activation for a GluN1/2B channel (using the model in D) illustrates how brief gaps can contain information about forward rates for the fast kinetically distinct
pregating step. The color above the simulation indicates occupancy in the corresponding closed state of the model in D. The slow step often reverses again
through the fast state (green) before reopening.
lators are accounted for by explicitly representing the modulator subunits to trigger channel gating and that these structural
tor bound and unbound receptor as independent states (Banke changes can occur in any order to arrive at an intermediate
et al., 2005; Erreger and Traynelis, 2008; Amico-Ruvio et al., state that can subsequently transition to the open state of the
2011). Other models for channel blockers and other use-depen- ion channel (Banke and Traynelis, 2003; Auerbach and Zhou,
dent modulators have been described that exclusively allow 2005; Erreger et al., 2005a,b; Schorge et al., 2005). Other mod-
modulators to bind to the open state (Huettner and Bean, 1988; els account for macroscopic and single-channel responses by
MacDonald et al., 1991; Blanpied et al., 1997, 2005; Dravid et including a few sequential gating steps in a linear kinetic model
al., 2007; Kussius et al., 2009; Paganelli and Popescu, 2015; with an implicit order for slow and fast gating steps (Popescu et
Glasgow et al., 2017). al., 2004; Kussius and Popescu, 2009). These models have been
The ability of AMPA receptor subunits to operate semi-inde- used to explore the kinetic aspects of modal gating (Zhang et al.,
pendently (Rosenmund et al., 1998; Jin et al., 2003; Kristensen 2008; Iacobucci and Popescu, 2017a), an intriguing phenome-
et al., 2011) and the modular domain architecture of glutamate non that is readily apparent in cell-attached recordings from
receptor structures raise the possibility that independent con- GluN1/GluN2A receptors. Gating modes are defined by different
formational changes in different subunits may progress within open probabilities and open times and have been described for
the sequence of steps leading to channel opening (Gibb et al., GluN1/2A and GluN1/2B receptors (Popescu and Auerbach, 2003;
2018). Some kinetic models suggest that such subunit-specific Popescu et al., 2004; Amico-Ruvio and Popescu, 2010; Popescu,
conformational changes are required in all four NMDA recep- 2012) but are rarely observed in GluN1/2D (Vance et al., 2013).

Figure 9. General pore structure of NMDA receptors. (A) Pore-lining elements contributed by the GluN1 subunit (blue; Protein Data Bank accession no.
5UN1; Song et al., 2018). The M3 transmembrane segment lines the extracellular part of the permeation pathway, whereas the M2 pore loop lines the intracel-

lular part with the N site asparagine (red circle) positioned at the tip of the M2 pore loop. The channel is in the closed conformation. (B) The narrow constriction
is formed by nonhomologous asparagine residues, the GluN1 N site and the GluN2 N+1 site (Wollmuth et al., 1996; Song et al., 2018). The GluN2B subunit is
colored orange. For both GluN1 and GluN2, the N site asparagine residue is positioned at the tip of the M2 loop.
Although the mechanism remains elusive, mode switching has the apexes of M2 in GluN1 and GluN2 are slightly staggered
been proposed to influence the time course of the synaptic cur- (Sobolevsky et al., 2002b). Diheteromeric GluN1/3 receptors
rent (Zhang et al., 2008). have glycine/arginine residues at the Q/R/N and Q/R/N +1 and
All these kinetic models for NMDA receptor gating that faith- show both markedly reduced Ca2+-permeability and Mg2+-block
fully describe both macroscopic responses and single-channel compared with GluN1/2 receptors (Cavara and Hollmann, 2008;
data require both multiple pregating steps and multiple open Henson et al., 2010; Low and Wee, 2010; Pachernegg et al., 2012;
states. The interpretation of this observation is that ion channel Kehoe et al., 2013). Recent data describing the pore of the AMPA
opening in NMDA receptors is not directly coupled to agonist-in- receptor in the open state reinforce the idea that the apex of the
duced ABD closure; instead, the receptor must proceed through a reentrant loops form a constriction that impacts ion permeation
sequence of conformational changes that couple agonist binding (Twomey et al., 2017). The structural basis for this functional
to ion channel gating. asymmetry will require high-resolution images of the NMDA
receptor in the open state.
Structural determinants of ion permeation and channel block
The ion channel pore in NMDA receptors can be divided into Determinants of ion permeation
the intracellular and extracellular vestibules separated by a NMDA receptor ion channels are permeable to the physio-
narrow constriction (Fig. 9). The narrow restriction resides ap- logically relevant Ca2+, Na+, and K+ ions. The different NMDA
proximately halfway across the membrane at the apex of the receptor subtypes display similar permeability to Na+ and K+
membrane reentrant loop M2 (i.e., the Q/R/N site) and is often ions (PK/PNa = 1.14) but are more permeable to Ca2+ relative to
referred to as the selectivity filter because of its role as key de- monovalent ions (PCa/PX = 1.8–4.5), with variation in Ca2+ per-
terminant of Ca2+ permeability, single-channel conductance, meability that depends on the GluN2 subunit (Burnashev et al.,
and channel block (Wollmuth and Sobolevsky, 2004; Traynelis 1995; Schneggenburger, 1996, 1998; Sharma and Stevens, 1996;
et al., 2010; Glasgow et al., 2015). The residue at the Q/R/N site Jatzke et al., 2002). However, NMDA receptors also exhibit block
is asparagine (N) in both GluN1 and GluN2, but the contribu- by external Ca2+, despite being highly Ca2+ permeable, which
tion of this residue to ion permeation is asymmetric between can be observed as a reduction in channel conductance in sin-
GluN1 and GluN2 subunits (Burnashev et al., 1992; Wollmuth gle-channel data (Premkumar and Auerbach, 1996; Wyllie et
et al., 1996, 1998; Sobolevsky et al., 2002b). This is because the al., 1996; Premkumar et al., 1997; Dravid et al., 2008). The con-
narrow constriction is formed by the Q/R/N site asparagine in current block by Ca2+ and the high Ca2+ permeability are not
GluN1 but by the asparagine residue adjacent to the Q/R/N site incompatible properties but are expected if multiple Ca2+-bind-
(i.e., Q/R/N +1 site) in GluN2. The asymmetric contribution by ing sites are located in the ion channel pore of NMDA receptors
GluN1 and GluN2 is revealed by substitutions of the Q/R/N site (Premkumar and Auerbach, 1996; Sharma and Stevens, 1996).
residue in GluN2 that have weak effects on Ca2+ permeability and Studies have suggested that one Ca2+-binding site is located at
dramatically reduce Mg2+ block, whereas the same substitutions the Q/R/N site, whereas another, more external Ca2+-binding
of the Q/R/N site residue in GluN1 dramatically reduce Ca2+ per- site could be formed by a cluster of charged DRPEER residues
meability and have weak effects on Mg2+ block (Burnashev et al., in GluN1 (Watanabe et al., 2002; Karakas and Furukawa, 2014).
1992; Wollmuth et al., 1998). However, mutations at the Q/R/N +1 The external Ca2+-binding site is located at the external entrance
site in GluN2 strongly reduce Mg2+ block (Wollmuth et al., 1998). to the ion channel above the transmembrane helix M3 of GluN1.
Functional data therefore suggest a structural asymmetry, where Although structural elements of Ca2+ permeation in GluN1/N2

subunits have been identified, the mechanism of Ca2+ perme- channel gate (Blanpied et al., 2005; Johnson et al., 2015). Chan-
ation remains unknown. nel blockers proposed to have bifunctionality include nitrome-
mantine derivatives that bind the ion channel pore, facilitating
Determinants of channel block the targeting of a nitro group to a redox-mediated regulatory
GluN1/2A and GluN1/2B channels are more strongly blocked site on the receptor (Takahashi et al., 2015).
by extracellular Mg2+ than GluN1/2C and GluN1/2D channels In general, the open channel blockers are considered nonse-
(Monyer et al., 1994; Kuner and Schoepfer, 1996; Qian et al., lective among NMDA receptor subtypes (Dravid et al., 2007), but
2005; Clarke and Johnson, 2006; Siegler Retchless et al., 2012). some channel blockers, such as ketamine and memantine, display
This channel block is highly dependent on the membrane poten- 5- to 10-fold preference for GluN2C/D-containing receptors over
tial (i.e., voltage dependent), and the IC50 values (the concentra- GluN2A/B-containing receptors in the presence of 1 mM extra-
tions that produce half-maximal inhibition) for block by external cellular Mg2+ (i.e., under physiological conditions; Kotermanski
Mg2+ are 2 µM, 2 µM, 14 µM, and 10 µM for GluN1/2A, GluN1/2B, and Johnson, 2009). NMDA receptor channel blockers have ro-
GluN1/2C, and GluN1/2D, respectively, at a holding potential of bust neuroprotective effects in animal models of CNS disorders
−100 mV (Kuner and Schoepfer, 1996). The dependency of Mg2+ that involve excessive NMDA receptor activation, such as stroke,
block on the GluN2 subunit is influenced by multiple structural epilepsy, and traumatic brain injury. However, clinical trials have
elements, but a main determinant appears to be a single residue not been successful because of dose-limiting side effects, patient

at the S/L site located in the M3 transmembrane helix (Siegler heterogeneity, and a narrow temporal window for intervention
Retchless et al., 2012). The residue at the S/L site, which is a ser- that could have confounded interpretation (Ikonomidou and
ine in GluN2A/B and a leucine in GluN2C/D, is not lining the ion Turski, 2002; Farin and Marshall, 2004; Muir, 2006; see also
channel pore but has been suggested to interact with tryptophan Table S2 in Yuan et al., 2015). NMDA receptor channel blockers
residues in the membrane reentrant loop M2 of GluN1 (Siegler that bind with high affinity, such as ketamine and PCP, are typi-
Retchless et al., 2012). This interaction between GluN1 and the cally dissociative anesthetics, and their clinical use is limited by
GluN2 S/L site also appears to be a key determinant of GluN2 sub- psychomimetic side effects. Nonetheless, there is an intense in-
unit–specific variation in channel conductance and Ca2+ permea- terest in use of ketamine or similar molecules for the treatment
bility (Siegler Retchless et al., 2012). Although important insight of major depressive disorder because of several promising clin-
into the structural mechanism of GluN2 subunit–dependent ical trials in recent years based on the discovery of antidepres-
control of Mg2+ block is still missing, it is possible that structural sant effects for NMDA receptor antagonists (Niciu et al., 2014;
elements, including the GluN2 S/L site, govern Mg2+ block by Abdallah et al., 2015; Zanos et al., 2018).
influencing binding sites for permeant ions in the channel pore Channel blockers such as memantine, which is approved in
(Antonov and Johnson, 1999; Zhu and Auerbach, 2001a,b; Qian et the treatment of moderate to severe Alzheimer’s disease, have
al., 2002; Qian and Johnson, 2006). lower affinity than ketamine and PCP and show faster blocking/
unblocking kinetics (Parsons et al., 1993). These kinetic proper-
Channel block by organic cations ties have been suggested to contribute to an improved therapeu-
The NMDA receptor ion channel pore can be blocked in a volt- tic index with respect to psychomimetic effects, perhaps because
age-dependent manner by a wide range of organic cations with of reduced channel block during normal synaptic transmission
diverse chemical structures (Huettner and Bean, 1988; Brackley (Chen and Lipton, 2006), although the mechanism by which
et al., 1993; Parsons et al., 1995). These compounds almost ex- memantine may contribute to a symptomatic benefit in Alzhei-
clusively block open channels in activated NMDA receptors mer’s disease is not well understood. Interestingly, Glasgow et al.
and are positively charged at physiological pH, a mechanism (2017) have proposed that memantine stabilizes occupancy of a
of channel block termed uncompetitive or use dependent. desensitized state of GluN1/2A receptors, whereas ketamine re-
In general, the open channel blockers can be classified into duces occupancy of a GluN1/2B desensitized state (Glasgow et al.,
three categories based on their interaction with the channel: 2017). Thus, the affinity of these blockers for their binding site in
(1) “foot-in-the-door” or sequential blockers (e.g., aminoac- the channel may be allosterically affected by the conformational
ridine derivatives and tetrapentylammonium) can only bind changes in the receptor protein associated with desensitization.
to the channel when it is open and prevent channel closure Given the prevalence of triheteromeric GluN1/2A/2B receptors
when bound (Benveniste and Mayer, 1995; Sobolevsky, 1999; in the brain, it will be important to evaluate memantine and ket-
Sobolevsky et al., 1999; Bolshakov et al., 2003; Barygin et al., amine block at these triheteromeric receptors.
2009); (2) partial trapping blockers (e.g., amantadine and me-
mantine) obstruct channel closure but are unable to completely Endogenous mechanisms of functional modulation
prevent it (Blanpied et al., 1997, 2005; Chen and Lipton, 1997; NMDA receptors are complex macromolecular membrane-bound
Mealing et al., 1999; Kotermanski et al., 2009; Johnson et al., protein complexes, and their functional properties and mem-
2015); and (3) trapping blockers (e.g., Mg2+, ketamine, phency- brane trafficking can be altered by extracellular ions, phosphor-
clidine [PCP], and MK-801) are trapped inside the channel pore ylation, and intracellular binding proteins. Additionally, the
as it closes, and agonists can unbind while the trapping blocker differences between various diheteromeric and triheteromeric
remains bound (Sobolevsky and Yelshansky, 2000; Poulsen NMDA receptor subtypes create selective actions of many of
et al., 2015). Some channel blockers have also been shown to these types of modulation. Here, we will describe various forms
facilitate channel closure, presumably by interacting with the of endogenous regulation of NMDA receptor function.

Modulation by protons tion that involves a change in the angle between the two lobes
Extracellular protons inhibit NMDA receptor function with an R1 and R2, in addition to twisting motions around the hinge re-
IC50 of ∼50 nM, corresponding to a pH of ∼7.3 (Giffard et al., gion of the bilobed ATD clamshell (Paoletti et al., 2000; Gielen et
1990; Traynelis and Cull-Candy, 1990, 1991; Vyklický et al., 1990). al., 2008; Karakas et al., 2009; Romero-Hernandez et al., 2016).
Thus, neuronal NMDA receptors are tonically inhibited by pro- Binding of Zn2+ stabilizes a conformation of the GluN2 ATD,
tons at physiological pH and are therefore poised to respond to which is presumably accompanied by structural changes at the
small changes in extracellular pH that can occur under physio- GluN1/GluN2 ABD layers that favor channel closure (Gielen et
logical conditions caused by release of protons from acidic synap- al., 2008; Romero-Hernandez et al., 2016). Previous studies have
tic vesicles or movement of protons across the plasma membrane suggested that Zn2+ binding can enhance the proton sensitivity
by pumps (Chesler, 2003). Furthermore, pathological conditions, (Choi and Lipton, 1999). In support of this idea, there is a strong
including seizure and ischemia, produce extracellular acidifica- correlation between mutations that perturb the IC50 of Zn2+ in-
tion, which can decrease pH to levels that strongly inhibit NMDA hibition and their effect on proton IC50 (Traynelis et al., 1998).
receptor function (Chesler, 2003). Furthermore, single-channel analysis can detect changes in the
The sensitivity of the NMDA receptors to inhibition by extra- protonation rates for Zn2+-bound receptors, supporting the idea
cellular protons depends on the GluN2 subunit (Traynelis et al., that Zn2+ alters the equilibrium between NMDA receptors and
1995), with GluN2A-, GluN2B-, and GluN2D-containing NMDA protons at physiological pH (Erreger and Traynelis, 2008). The

receptors showing proton IC50 values near physiological pH (7.0– incomplete inhibition by high-affinity Zn2+ binding is consistent
7.4). In contrast, GluN2C-containing receptors are less sensitive with enhancement of proton sensitivity, because Zn2+ binding
to changes in pH, with an IC50 value near pH 6.0 (Traynelis et produces a leftward shift of the proton inhibition curve, leading
al., 1995; Low et al., 2003). NMDA receptors that include GluN1 to more complete inhibition at acidic pH (Traynelis et al., 1998;
subunits containing the alternatively spliced exon 5 in the ATD Choi and Lipton, 1999; Low et al., 2000; Erreger and Traynelis,
(i.e., GluN1-1b) are notably less sensitive to protons (Traynelis et 2008). Interestingly, triheteromeric GluN1/2A/2B receptors re-
al., 1995). Proton inhibition is voltage independent, and without tain a high-affinity Zn2+ binding, although there is reduced in-
effect on glutamate potency; low pH produces modest shifts in hibition at maximally effective concentrations of Zn2+ (Hatton
the glycine potency (Tang et al., 1990; Traynelis and Cull-Candy, and Paoletti, 2005; Hansen et al., 2014; Stroebel et al., 2014).
1990, 1991; Traynelis et al., 1995). The structural determinants Higher concentrations of Zn2+ can produce a voltage-dependent
underlying proton inhibition are unknown, although mutations channel block (Williams, 1996), but it remains unclear whether
at the ABD interface, linkers to pore-forming elements, and changes in extracellular Zn2+ in brain tissue are large enough
within the M2 reentrant loop can all influence pH sensitivity to produce voltage-dependent channel block (Vogt et al., 2000;
(Low et al., 2003; Gielen et al., 2008). This suggests that NMDA Anderson et al., 2015).
receptor gating is tightly coupled to proton inhibition of the re- The affinity for Zn2+ at the GluN2A ATD is high enough such
ceptor. This idea is consistent with the observation that channel that Zn2+ contamination in physiological levels of salts can pro-
blockers appear to sense the protonation state of the receptor duce significant inhibition (Paoletti et al., 1997). The effects of
(Dravid et al., 2007). contaminant Zn2+ in functional experiments can be removed by
The actions of ATD modulators appear to involve a change inclusion of even low concentrations of divalent ion chelators,
in the pKa of the proton sensor that leads to enhancement or such as 10 µM EDTA. The high affinity of such chelators for Zn2+
reduction of tonic proton inhibition at physiological pH. Thus, means that even low micromolar levels of chelator will bind vir-
both Zn2+ and ifenprodil enhance proton sensitivity, which will tually all of the nanomolar-contaminating Zn2+ ions but exert
increase tonic inhibition at resting pH (Pahk and Williams, 1997; minimal effects on millimolar concentrations of Ca2+ or Mg2+
Mott et al., 1998; Traynelis et al., 1998; Choi and Lipton, 1999; (Anderson et al., 2015).
Erreger and Traynelis, 2008; Bhatt et al., 2013). In contrast, the
binding of extracellular polyamines reduces the sensitivity to ex- Positive and negative allosteric modulation by neurosteroids
tracellular pH, resulting in potentiation due to reduced tonic in- Several endogenous neurosteroids positively and negatively
hibition by physiological levels of protons (Traynelis et al., 1995; modulate NMDA receptor activity (Traynelis et al., 2010), al-
Kashiwagi et al., 1996, 1997). though the actions of these lipophilic molecules are complex.
For instance, pregnenolone sulfate has dual actions on NMDA
Actions of extracellular Zn2+ receptor responses, having both inhibitory and potentiating ac-
Extracellular Zn2+ binds with high affinity to the GluN2A ATD, tivity over a wide range of potencies (Horak et al., 2006). The
with an IC50 value in the nanomolar range at GluN1/GluN2A potentiating actions of pregnenolone sulfate are most prominent
receptors (Williams, 1996; Chen et al., 1997; Paoletti et al., 1997; when applied before receptor activation, whereas inhibitory ac-
Traynelis et al., 1998). In contrast, the IC50 for Zn2+ inhibition of tions arise when applied continuously (Horak et al., 2006). The
GluN1/GluN2B receptors resulting from binding to the GluN2B dual actions of pregnenolone sulfate lead to divergent effects de-
ATD is in the low micromolar range (Rachline et al., 2005). Crys- pending on the GluN2 subunit; when applied during steady-state
tallographic and functional data show that the Zn2+-binding site NMDA receptor responses, GluN1/2A and GluN1/2B are potenti-
is located within the cleft formed by the two lobes R1 and R2 of ated, whereas GluN1/2C and GluN1/2D are inhibited.
the ATD (Karakas et al., 2009; Romero-Hernandez et al., 2016). Other neurosteroid analogues, such as pregnanolone sulfate,
Multiple observations suggest a mechanism of Zn2+ modula- inhibit all NMDA receptor subtypes (i.e., they are pan-inhibi-

tors) in a use-dependent manner through actions that involve binding of glutamate decreases the glycine affinity and vice versa
the extracellular portion of the conserved M3 SYTANLAAF (Benveniste et al., 1990; Lester et al., 1993). Thus, when glutamate
motif (Malayev et al., 2002). For GluN2A, pregnanolone sulfate binds to GluN2 in the absence of high concentrations of glycine,
has been proposed to increase the occupancy of a desensitized the current will initially rise to a peak and then decline to a new
state (Kussius et al., 2009). In contrast, 24(S)-hydroxycholesterol equilibrium as glycine unbinds from the receptor after the al-
and related analogues appear to be pan-potentiators, whereas losteric reduction in glycine affinity. The time course for the
25(S)-hydroxycholesterol may antagonize actions of endoge- desensitization is dictated by glycine unbinding (time constant
nous 24(S)-hydroxycholesterol (Paul et al., 2013; Linsenbardt et ∼0.3 s) and is temporally close to the synaptic NMDA receptor
al., 2014). Some of these neurosteroids and analogues have been time course, raising the possibility that glycine-dependent de-
shown to exhibit agonist dependency, although this property is sensitization could impact synaptic signaling when glycine is
difficult to assess, because neurosteroids can have distinct ac- subsaturating (Berger et al., 1998). Recent structural data for
tions on NMDA receptors, dependent on the timing of modulator NMDA receptors provide plausible models for the negative al-
application (i.e., see pregnenolone sulfate actions above). Addi- losteric coupling between glutamate- and glycine-binding sites,
tionally, steroid derivatives may partition into the membrane given their close proximity (Karakas and Furukawa, 2014; Lee
en route to their active site, which will alter the concentration– et al., 2014; Tajima et al., 2016; Zhu et al., 2016). However, the
response relationship of their actions (Borovska et al., 2012; structural features that enable glycine-dependent desensitiza-

Vyklicky et al., 2015). A recent study reported that cholesterol tion remain poorly understood.
modulates NMDA receptor function and its removal inhibited
receptor activity (Korinek et al., 2015), suggesting that the mem- Zn2+-dependent NMDA receptor desensitization
brane environment influences NMDA receptor activity and may Extracellular Zn2+ inhibits GluN1/GluN2A and GluN1/GluN2B
be an important determinate of neurosteroid action. Although a receptors in a voltage-independent manner through a binding
clear binding site has not been resolved, it seems likely that neu- site located in the ATD (Williams, 1996; Traynelis et al., 1998;
rosteroid derivatives interact directly with the receptor rather Choi and Lipton, 1999; Fayyazuddin et al., 2000; Low et al., 2000;
than simply alter membrane fluidity. A subset of neurosteroid Paoletti et al., 2000; Rachline et al., 2005; Karakas et al., 2009).
inhibitors also have voltage-dependent actions, suggesting that However, NMDA receptors also display a rapid component of de-
they may inhibit NMDA receptors through blocking the channel sensitization in the presence of extracellular Zn2+ that occurs by
(Vyklicky et al., 2015). These findings highlight the complexity a mechanism similar to glycine-dependent desensitization (Chen
associated with neurosteroid activity, and more work is required et al., 1997). This is the result of a positive intrasubunit allosteric
to delineate the mechanism of action of these compounds. interaction between glutamate binding to the GluN2 ABD and
Zn2+ binding to the GluN2A ATD (Zheng et al., 2001; Erreger and
Desensitization of NMDA receptors Traynelis, 2005). As a result, in the presence of subsaturating
The process of desensitization is broadly defined as a decrease in concentrations of Zn2+, the glutamate-induced increase in Zn2+
a response in the continued presence of a stimulus. NMDA recep- affinity will cause a relaxation of the response to a new equilib-
tors exhibit several forms of desensitization, which can be distin- rium as more Zn2+ ions bind and inhibit the receptor. The time
guished on the basis of time course and mechanism, including course for Zn2+-dependent desensitization therefore follows the
glycine-, Zn2+-, and Ca2+-dependent desensitization. Most li- time course for Zn2+ binding.
gand-gated channels can desensitize in the continued presence
of agonist by a mechanism thought to involve a conformational Ca2+-dependent NMDA receptor inactivation
change to a stable and long-lived agonist-bound closed state. NMDA receptors also undergo Ca2+-dependent desensitization
NMDA receptors can also desensitize in the continued presence or inactivation, which requires an increase in intracellular Ca2+
of glutamate and glycine, presumably by this same mechanism, over several seconds (Clark et al., 1990; Legendre et al., 1993;
in a manner that is independent of glycine, Zn2+, and Ca2+. NMDA Vyklický, 1993; Rosenmund et al., 1995). The magnitude of this
receptors exhibit only weak desensitization compared with the form of desensitization varies with different NMDA receptor
relatively strong desensitization of AMPA and kainate receptors. subtypes; it is most prominent for GluN2A-containing NMDA
However, this desensitization is sensitive to intracellular dialy- receptors and more limited for GluN2B- and GluN2C-contain-
sis, being more prominent in excised outside-out membrane ing NMDA receptors (Medina et al., 1995; Krupp et al., 1996). The
patches (Sather et al., 1990, 1992), and is perturbed by mutations proposed mechanism involves an increase in the intracellular
in a wide range of domains, including the conserved M3 gating Ca2+ in the vicinity of the NMDA receptor that triggers uncou-
motif, the pre-M1 linker region, the ion channel pore, the ABD, pling of the receptor from filamentous actin (Rosenmund and
and the TMD–ABD interface (Chen et al., 2004; Hu and Zheng, Westbrook, 1993). In addition, calmodulin binding to the GluN1
2005; Alsaloum et al., 2016). CTD may play a role in this form of desensitization (Ehlers et
al., 1996; Rycroft and Gibb, 2002; Iacobucci and Popescu, 2017b);
Glycine-dependent NMDA receptor desensitization Ca2+-dependent desensitization is absent in NMDA receptors
Glycine-dependent NMDA receptor desensitization is pres- containing GluN1 splice variants that lack calmodulin-binding
ent only in subsaturating glycine concentrations (Mayer et al., sites (Ehlers et al., 1996, 1998) or harbor mutations in calm-
1989) and occurs as a result of a negative allosteric interaction odulin-binding sites in the GluN1 CTD (Zhang et al., 1998;
between the glutamate- and glycine-binding sites, such that the Krupp et al., 1999).

Conclusion Al-Hallaq, R.A., T.P. Conrads, T.D. Veenstra, and R.J. Wenthold. 2007. NMDA
di-heteromeric receptor populations and associated proteins in rat hip-
Recent discoveries from genetic analyses that link NMDA recep- pocampus. J. Neurosci. 27:8334–8343. https://doi.org/10.1523/JNEURO
tors to specific disease conditions, the emerging evidence of the SCI.2155-07.2007
antidepressant effects of NMDA receptor antagonists, and accel- Alsaloum, M., R. Kazi, Q. Gan, J. Amin, and L.P. Wollmuth. 2016. A Molecular
Determinant of Subtype-Specific Desensitization in Ionotropic Gluta-
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This work was supported by grants from National Institutes of https://doi.org/10.1074/jbc.271.26.15527
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S.F. Traynelis is a consultant for Janssen Pharmaceuticals, Inc., Atlason, P.T., M.L. Garside, E. Meddows, P. Whiting, and R.A. McIlhinney.
the principal investigator on a research grant awarded to Emory 2007. N-Methyl-D-aspartate (NMDA) receptor subunit NR1 forms the
University from Janssen Pharmaceuticals, a member of the Scien- substrate for oligomeric assembly of the NMDA receptor. J. Biol. Chem.
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tific Advisory Board for Sage Therapeutics, and co-founder of Neu- Auerbach, A., and Y. Zhou. 2005. Gating reaction mechanisms for NMDA re-
rOp, Inc. K.B. Hansen is the principal investigator on a research ceptor channels. J. Neurosci. 25:7914–7923. https://doi.org/10.1523/JNE
grant awarded to University of Montana from Janssen Pharmaceu- UROSCI.1471-05.2005
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tors. Nat. Neurosci. 6:144–152. https://doi.org/10.1038/nn1000
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Lesley C. Anson served as editor. NMDA receptors in a nonconducting state. J. Neurosci. 25:42–51. https://
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Submitted: 10 May 2018 Barygin, O.I., V.E. Gmiro, K.Kh. Kim, L.G. Magazanik, and D.B. Tikhonov. 2009.
Blockade of NMDA receptor channels by 9-aminoacridine and its deriva-
Accepted: 3 July 2018 tives. Neurosci. Lett. 451:29–33. https://doi.org/10.1016/j. neulet.2008.12.036
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HHS Public Access
Author manuscript
Epilepsy Behav. Author manuscript; available in PMC 2017 November 01.
Author Manuscript

Epilepsy Behav. 2016 November ; 64(Pt A): 166–170. doi:10.1016/j.yebeh.2016.09.034.
Serotonergic agents act on 5-HT3 receptors in the brain to block

seizure-induced respiratory arrest in the DBA/1 mouse model of
SUDEP
Carl L. Faingolda,*, Marcus Randalla, Chang Zengb,c, Shifang Pengc, Xiaoyan Longd, and
Hua-Jun Fengb,*
aDepartment of Pharmacology and Neurology, Southern Illinois University, School of Medicine,
Author Manuscript
Springfield, IL, USA

bDepartment of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital
and Harvard Medical School, Boston, MA, USA
cHealth Management Center, Xiangya Hospital, Central South University, Changsha, China
dDepartment of Neurology, Xiangya Hospital, Central South University, Changsha, China
Abstract
Drugs that enhance the action of serotonin (5-hydroxytrypamine, 5-HT), including several
selective serotonin reuptake inhibitors (SSRIs), reduce susceptibility to seizureinduced respiratory
arrest (S-IRA) that leads to death in the DBA/1 mouse model of sudden unexpected death in
Author Manuscript
epilepsy (SUDEP). However, it is not clear if specific 5-HT receptors are important in the action of
these drugs and whether the brain is the major site of action of these agents in this SUDEP model.
The current study examined the actions of agents that affect the 5-HT3 receptor subtype on S-IRA
and whether intracerebroventricular (ICV) microinjection of an SSRI would reduce S-IRA
susceptibility in DBA/1 mice. The data indicate that systemic administration of SR 57227, a 5-
HT3 agonist, was effective in blocking S-IRA in doses that did not block seizures, and the S-IRA
blocking effect of the SSRI, fluoxetine, was abolished by co-administration of a 5-HT3 antagonist,
ondansetron. Intracerebroventricular administration of fluoxetine in the present study was also
able to block S-IRA without blocking seizures. These findings suggest that 5-HT3 receptors play
an important role in the block of S-IRA by serotonergic agents, such as SSRIs, which is consistent
with the abnormal expression of 5-HT3 receptors in the brainstem of DBA mice observed
previously. Taken together, these data indicate that systemically administered serotonergic agents
Author Manuscript
act, at least in part in the brain, to reduce S-IRA susceptibility in DBA/1 mice and that 5-HT3
receptors may be important to this effect.
*
Corresponding authors: cfaingold@siumed.edu and feng.huajun@mgh.harvard.edu.
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Conflict of interest
None of the authors has any conflict of interest to disclose.
Faingold et al. Page 2
Keywords
Author Manuscript
5-HT; SSRIs; antagonist; audiogenic seizures; intracerebroventricular
1. Introduction
Sudden unexpected death in epilepsy (SUDEP) is a shattering consequence of epilepsy in
adult and pediatric patients [1–5] and results in a major cause of lost patient years second
only to stroke among neurological disorders [6]. In the majority of witnessed cases of
SUDEP, a generalized convulsive seizure and severe respiratory dysfunction were observed
prior to death [3]. DBA/1 and DBA/2 mice have been shown to be relevant models of
SUDEP, because they exhibit generalized convulsions that lead directly to seizure-induced
respiratory arrest (S-IRA), resulting in death if resuscitation is not rapidly instituted [7–9].
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Several drugs that enhance the activation of serotonin (5-hydroxytryptamine, 5-HT)

receptors, including selective serotonin reuptake inhibitors (SSRIs), prevent S-IRA without
blocking seizures in DBA mice, and 5-HT antagonists increase S-IRA susceptibility in non-
susceptible DBA mice [10–12]. Seizures induced by maximal electroshock or pilocarpine in
Lmx1b(f/f) mice resulted in elevated seizure-induced mortality because of breathing
cessation, which was also reduced by SSRI administration [13]. Since seizure susceptibility
is not blocked by SSRIs in DBA mice at doses that block S-IRA, this suggests that these
agents exert selective effects on respiratory arrest rather than a general anticonvulsant effect.
Respiratory dysfunction after seizures is common in patients with epilepsy [14, 15], and
patients who exhibited partial seizures and who had been taking SSRIs exhibited less
respiratory dysfunction than untreated patients in a retrospective study [16].
Author Manuscript
Serotonin is known to play a role in the normal regulation of respiration, in part, by acting
on neurons in the medullary respiratory centers to enhance respiration in response to
elevated CO2 levels [17, 18]. Although our previous studies demonstrated that the SSRI,
fluoxetine, at a dose that suppresses S-IRA, did not enhance respiratory ventilation in the
absence of seizures in DBA/1 mice, SSRIs may prevent respiratory arrest via maintaining
respiratory function during generalized tonic-clonic seizures [12]. Serotonin is also involved
in the arousal response via ascending projections from raphe nuclei [19, 20]. Thus, deficits
of serotonergic neurotransmission in both respiratory and arousal systems may contribute to
S-IRA in DBA/1 mice.
Specific subtypes of 5-HT receptors are thought to be selectively relevant to control of

respiration, arousal and epilepsy [21–23]. At least seven subtypes of 5-HT receptors are
Author Manuscript
expressed in the brainstem [24, 25], and the absence of 5-HT2C receptors in transgenic mice
is associated with seizure susceptibility that can result in seizure-induced death [26, 27],
similar to that seen in DBA mice. The 5-HT2A receptors are reported to contribute
importantly to S-IRA induced by electroshock [13]. Our previous studies indicate that levels
of several 5-HT receptor subtype proteins, including 5-HT2B, 5-HT2C and 5-HT3 receptors,
were significantly reduced in DBA/1 mice compared with those in a seizure-resistant mouse
strain, although a 5-HT2B/2C receptor agonist (mCPP) was ineffective in reducing S-IRA in
DBA/1 mice [28]. Therefore, the present study examined the involvement of 5-HT3

receptors, the only ionotropic receptor among all of the 5-HT receptor subtypes identified
Author Manuscript
[29], in S-IRA in DBA/1 mice. The 5-HT receptors are also known to exist peripherally and
can potentially be an important target for these S-IRA suppressing SSRIs, particularly by
exerting effects in the lung [30]. Therefore, the present study also examined the effect of
administration of an SSRI directly into the brain using intracerebroventricular (ICV) route.
2. Materials and methods

2.1. Animals
The male and female DBA/1 mice were obtained from Harlan Laboratories (Indianapolis,
IN) and were housed and or bred in the animal facility with food pellets and water available
ad libitum. Multiple groups of DBA/1 mice were screened for susceptibility to S-IRA
induced by audiogenic seizures (AGSz), beginning on postnatal day (PND) 24–30, and
acoustic stimulation was presented daily for 3–4 days at which time 90 to 100% of DBA/1
Author Manuscript
mice in each group developed S-IRA susceptibility, as previously described [8]. This
“priming” process at a young age is important to developing the chronic consistent S-IRA
susceptibility that allows the animals to model SUDEP and the testing of SUDEP prevention
treatments [8, 31]. A total of 97 mice were used in the current study. The operational
definition of S-IRA is described below. Only those mice that consistently exhibited S-IRA
were used in these experiments. The experimental protocols used in this study were
approved by the Institutional Animal Care and Use Committee (IACUC) of Southern Illinois
University School of Medicine and Massachusetts General Hospital, which are in
accordance with the National Institute of Health guidelines for care and use of laboratory
animals. Measures were included in the protocols to minimize the pain and discomfort of the
animals and to minimize animal usage.
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2.2. Seizure induction and resuscitation

All DBA/1 mice were subjected to an acoustic stimulation paradigm, consisting of a broad-
band acoustic stimulus generated by an electrical bell (Heath Zenith Model #172C-A or FOS
4771L, Tecumseh, MI) at an intensity of 96–110 dB SPL. Mice were individually placed in a
plastic cylinder within a sound-isolation room. The stimulus was given for a maximum
duration of 60 s or until the mouse exhibited a tonic seizure, which ended in tonic hindlimb
extension convulsions and consistently resulted in S-IRA. Behaviors were recorded on
videotape, and seizure-related behaviors were quantified visually off-line. After S-IRA was
evoked, all DBA/1 mice received respiratory support to assist in recovery of respiration, as
described below. The operational criteria for S-IRA were defined by the appearance of a
deep respiratory gasp and relaxation of the pinnae determined visually, which were invariant
indicators in previous studies that S-IRA had begun and death was imminent [8]. Although
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S-IRA was behaviorally determined by qualitative (visual) breathing assessment, our

previous quantitative studies using plethysmography indicated that S-IRA leads to terminal
asystole and death in DBA/1 mice [32]. Resuscitation was accomplished by placing the
outflow polyethylene tube (4.4 mm external diam.) of a rodent respirator (Harvard
Apparatus 680, Holliston, MA) over the nostrils of the supine mice. The respirator was
previously in operation pumping room air (180 strokes/min), and when the outflow tube was
placed over the nostrils, the one cc volume induced visible displacement of the chest.

Initiation of resuscitation began within 5 s after the final deep respiratory gasp to effectively
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revive the mice [7, 8]. The mice were re-subjected to the acoustic stimulation paradigm 24 h
and 48 h after drug administration and at 24 h intervals thereafter, if necessary, to determine
if the susceptibility to S-IRA had returned.
2.3. Intracerebroventricular (ICV) injection

The ICV cannulation was carried out as described in earlier studies [33]. In brief, mice were
anesthetized with ketamine/xylazine (100/10 mg/kg, i.p.). A guide cannula (26G, Plastics
One, Roanoke, VA) was stereotaxically implanted based on coordinates from Paxinos and
Franklin (1997) [34] (AP − 0.4 mm; ML − 1.0 mm; and V −2.0 mm). The guide cannula was
fixed to the skull using mounting screws (BASi, West Lafayette, IN) and dental cement (A–
M Systems, Sequim, WA). A stainless steel stylet was used to occlude the guide cannula
when not in use. The animals were then allowed to recover for a week during which period
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tetracycline (1 g/l) was administered in the drinking water to reduce infection. One week
after implant surgery, each mouse was tested to verify that it remained susceptible to
seizures and S-IRA and then, resuscitated. The following day microinjections were made
using a Hamilton syringe and a pump (11 Elite Nanomite, Harvard Apparatus), which was
connected to the internal cannula (33G, Plastics One) by a polyethylene tubing, and 1.0 µl of
fluoxetine or the vehicle, dimethyl sulfoxide (DMSO), was administered at a rate of 0.5
µl/min into the left lateral ventricle. The injection cannula was left in place for a further one
min before being slowly withdrawn to avoid back flow.
2.4. Histology
Verification of the placement of the ICV guide cannula was done at the end of experiments.
Fast green was injected ICV to mark the ventricular space. Each mouse was euthanized with
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an overdose of ketamine/xylazine and transcardially perfused with 30 ml PBS (pH 7.4),

followed by 30 ml 4% paraformaldehyde. The brains were removed and stored in 4%
paraformaldehyde at 4°C. Each brain was sectioned into 50-µm thickness of coronal slices
using a freezing microtome (CM 1850 UV, Leica, Buffalo Grove, IL). The placement of the
guide cannula was observed using a light microscope. Only data from animals with correct
cannula placement were used for statistical analysis.
2.5. Drugs
Because of the very small volume used for ICV injection, the dose of fluoxetine (120 nmol)
was not soluble in saline, and dimethyl sulfoxide (DMSO) was used as the vehicle for these
experiments. The DBA/1 mice received the following drugs acutely: the SSRI, fluoxetine,
administered i.p. (40 mg/kg in saline) 30 min prior to AGSz induction or ICV (60, 90, or
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120 nmol in DMSO) 15 min prior to AGSz. The selective 5-HT3 agonist SR 57227 (20–40
mg/kg in saline) or the 5-HT3 antagonist ondansetron (0.5–1 mg/kg in distilled water) was
administered 30 and 35 min prior to AGSz, respectively. All drugs were obtained from
Sigma-Aldrich (St. Louis, MO).

2.6. Statistics
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The incidence of S-IRA between drug and vehicle groups was compared using Mann-
Whitney U test. Statistical significance was inferred if p<0.05.
3. Results
3.1. A 5-HT3 receptor agonist reduces S-IRA
The effects of systemic administration of the selective agonist for the 5-HT3 receptor (SR
57227) in primed DBA/1 mice that consistently exhibited S-IRA are shown in Fig. 1. SR
57227 significantly reduced S-IRA at doses that did not affect the severity or susceptibility
to AGSz. Thus, at doses of 35 and 40 mg/kg, the 5-HT3 agonist was effective in inhibiting S-
IRA without changing AGSz susceptibility. The saline vehicle and lower doses of SR 57227
(20 and 30 mg/kg) were ineffective in reducing S-IRA. The suppressive effect of this 5-HT3
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agonist on S-IRA was reversible, and most mice returned to S-IRA susceptibility 24 h after
drug administration. Susceptibility to S-IRA did not return in one DBA/1 mouse likely
because of delayed sequelae of the S-IRA, including nasal exudates and labored breathing,
suggestive of pulmonary edema. This mouse was euthanized at 72 hr after drug
administration because of its compromised health status. Another mouse was not
successfully resuscitated.
3.2. Fluoxetine effect on S-IRA is blocked by a 5-HT3 receptor antagonist

The role of 5-HT3 receptors in the occurrence of S-IRA in primed DBA/1 mice was further
evaluated by examining the ability of a selective 5-HT3 antagonist, ondansetron (OND), to
alter the ability of the SSRI, fluoxetine, to suppress S-IRA. When given sequentially, this 5-
HT3 antagonist blocked the ability of fluoxetine to reduce S-IRA (Fig. 2). Thus, OND (0.5,
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0.75, 1.0 mg/kg or vehicle i.p.) was administered 5 min prior to fluoxetine (40 mg/kg, i.p.),
and 30 min later, AGSz were induced (Note, OND in doses up to 2.0 mg/kg had no effect on
AGSz or S-IRA in DBA/1 mice in preliminary studies). Thirty min after fluoxetine
administration, a complete suppression of S-IRA was induced in mice pre-treated with
vehicle or the lower doses of OND, but AGSz susceptibility was not affected. A dose-
dependent reduction in the suppressive effect of fluoxetine was induced by OND, which
reached statistical significance at 1.0 mg/kg (p < 0.05). The incidence of AGSz was not
significantly affected by any of the pre-injections.
3.3. Fluoxetine ICV blocks S-IRA

Administration of fluoxetine directly into the brain reduced the incidence of S-IRA in
primed DBA/1, and this effect was dose-related (Fig. 3A). In the 60 nmol ICV group, only
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one of the 7 mice did not develop S-IRA after AGSz, which was not significantly different
from the DMSO control (S-IRA occurrence 85.7% vs. 100%). In the 90 nmol ICV group,
the S-IRA rate was reduced to 70% (n = 10) but still did not reach statistical significance. In
the 120 nmol ICV group, fluoxetine blocked only S-IRA but not AGSz activity in 2/7 of
DBA/1 mice tested. While fluoxetine blocked both S-IRA and tonic seizures in the rest 5/7
of mice, these mice still exhibited AGSz (wild running and/or clonic seizures). Therefore,
fluoxetine at this dose blocked S-IRA in all mice tested, which is significantly different from

control (p < 0.01). Two of the animals that received the 120-nmol dose exhibited tremor,
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hypothermia, hunched back and bradypnea, which are consistent with the serotonin
syndrome that is seen in rodents [35]. These two mice were found dead 24–72 h after
fluoxetine microinjection. The rest of the mice returned S-IRA susceptibility by 96 h after
fluoxetine microinjection. In 32 of 40 implanted DBA/1 mice, the cannula hit the ventricle
(Fig. 3B). Intracranial injections of fluoxetine in those mice in which the cannula did not hit
the ventricle (8 mice) exerted no effect on S-IRA.
4. Discussion
The present study indicates that a selective 5-HT3 agonist can suppress S-IRA without
affecting seizure susceptibility, which is the first selective 5-HT agonist that was effective in
DBA/1 mice, since neither a 5-HT2B/2C agonist nor a 5-HT7 agonist was effective in this
model of SUDEP [28, 31]. The current findings with agents that act selectively on the 5-HT3
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receptor, which is the only ionotropic receptor among the 7 classes of 5-HT receptor
subtypes [29], are consistent with previous studies that showed abnormal levels of 5-HT3
receptor protein in both DBA/1 and DBA/2 mouse models of SUDEP [28, 36]. The ability of
a selective 5-HT3 antagonist, ondansetron, to block the effect of fluoxetine in the present
study is further evidence of the importance of 5-HT3 receptors in the ability of SSRIs to
suppress S-IRA that was also seen previously [8, 11, 12].
The ability of a selective 5-HT3 receptor agonist to block S-IRA in DBA/1 mice seen in the
current study is consistent with the effectiveness of other selective 5-HT agonists to block S-
IRA in DBA/2 mice (5-HT2B/2C) [28] as well as in seizure-induced mortality in Lmx1b(f/f)
mice treated with pilocarpine or by maximal electroshock (5-HT2A) [13]. The 5-HT2C
receptors may also play a role in seizure-related respiratory dysfunction, since 5-HT2C
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knockout mice exhibit AGSz that can lead to death [26]. These findings suggest that the
specific 5-HT receptor that modulates S-IRA susceptibility may vary among the different
strains of animals. Further studies are needed to examine if other 5-HT receptor subtypes are
involved in S-IRA in DBA/1 mice.
The present finding that a selective 5-HT3 antagonist can block the effects of fluoxetine on
S-IRA supports the idea that the ability of SSRIs to suppress S-IRA [8, 11–13] is due to
inhibition of 5-HT re-uptake rather than other actions exerted by SSRIs, such as effects on
cholinergic, noradrenergic and sigma receptors, and transient receptor potential vanilloid
type 1 channels as well as TREK-2 [tandem of pore domains in a weak inwardly rectifying
K+ channel (TWIK)-related K+ channel (TREK)-2] potassium channels [9, 37–40]. Further
support of an important role of 5-HT in the susceptibility to S-IRA is the ability of non-
selective 5-HT antagonists to increase the susceptibility to S-IRA of both DBA/1 and DBA/2
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mouse models of SUDEP [7, 31]. Selective serotonin reuptake inhibitors can also reduce
electroshock or pilocarpine seizure-induced death, but not in mice with genetic deletion of 5-
HT neurons [13]. 5-HT3 receptors are highly expressed in the interneurons of the forebrain
structures [41]. It is not known how 5-HT3 receptor activation maintains breathing after
seizures. However, it was reported that activation of 5-HT3 receptors increases the levels of
norepinephrine in the locus coeruleus, a structure that is involved in arousal response [42]. It
is possible that stimulation of 5-HT3 receptors reduces S-IRA in DBA/1 mice via enhancing

arousal response. Future studies are needed to examine this possibility. It should be noted
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that the expression of 5-HT3 receptors is also significantly reduced in the brainstem of
DBA/2 mice compared with that of audiogenic seizure-resistant C57BL/6J mice [36]. It is
possible that activation of 5-HT3 receptors may suppress S-IRA in DBA/2 mice. Further
studies are needed to confirm this.
It has been well established that SSRIs act in humans primarily via actions in the brain even
in single doses [43], as used in the present and most of the previous studies. The present
study examined this issue in DBA/1 mice by administering fluoxetine directly into the brain.
These experiments indicated that ICV administration of fluoxetine could block S-IRA
without affecting seizure susceptibility, supporting the hypotheses that this effect of SSRIs is
largely mediated via actions in the brain, since the amount of fluoxetine administered ICV is
considerably below that required to block S-IRA when administered systemically [10].
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As noted earlier, 5-HT release from the neurons in the brainstem raphe nuclei mediates the
response to elevated CO2 by stimulation of respiration [22] and by inducing arousal under
normal conditions [19]. Mice with a genetic deletion of 5-HT neurons are not aroused from
sleep by elevated CO2 levels but can be aroused by other stimuli [19]. Selective 5-HT2A but
not 5-HT2C receptor antagonists blocked CO2-induced arousal during sleep in normal mice
[23]. Thus, defects in serotonergic mechanisms in the raphe nuclei may be important in
hypercarbic conditions, which commonly occur peri-ictally in patients and animals
undergoing epileptic seizures [7, 14, 15, 44]. The ability of an SSRI to significantly alter
neural activity in certain raphe nuclei post-ictally in DBA/1 mice seen in our recent
preliminary data suggests a role of these nuclei in the prevention of S-IRA by this agent
[45]. Other deficits in serotonergic neurotransmission in DBA mice may also contribute to
S-IRA [9]. Although the doses of SSRIs that prevent S-IRA in the DBA mouse models of
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SUDEP exceed the doses used in the treatment of depression clinically, the antidepressant
effects of SSRIs were originally discovered in rodents, and the doses used in those
experiments and in recent rodent depression studies (5–20 mg/kg) [39, 46] are in a similar
dosage range to that used to block S-IRA in these DBA mouse models of SUDEP (15–70
mg/kg) [7, 10–12]. Although it remains to be evaluated whether these agents that enhance
the activation of 5-HT receptors are relevant to a potential use in the prevention of SUDEP
in patients [47], one retrospective study observed that patients who experienced partial onset
seizures and had been under treatment with SSRIs exhibited a significantly lesser degree of
respiratory dysfunction than an age-matched group who were not under SSRI treatment [16].
This potentially exciting finding has not yet been evaluated in a prospective study.
Interpretations of these human results are made more complex because the specific SSRIs
used in each of the patient groups were not specified and subsequent animal studies in
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DBA/1 mice showed that not all SSRIs are effective in suppressing S-IRA [31]. Further
epidemiological studies are needed to investigate if the SSRIs that are effective in
suppressing S-IRA in animal models also reduce the incidence of SUDEP in patients [9].
The existentially vital function of control of respiration is a multifaceted process, involving

multiple neurotransmitters, neuromodulators and ion channels, including adenosine [5, 44,
48, 49]. Dravet syndrome is a form of human epilepsy that exhibits an extremely high
incidence of SUDEP, and this syndrome is thought to be mainly due to a loss of function

mutation of sodium channels [50, 51]. Two drugs that alter the availability of 5-HT have
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been shown to have potential usefulness in treating Dravet syndrome in patients, including
an SSRI and d-fenfluramine, which enhances 5-HT release from nerve endings in the brain
[52]. Seizures in an animal model of Dravet syndrome are also inhibited by D-fenfluramine
[53]. Interestingly, 5-HT3 receptors are known to flux sodium ions, which have the potential
to mediate the positive effects of serotonergic drugs seen in Dravet syndrome [54].
In summary, the present findings suggest that agents that enhance the activation of 5-HT
receptors, including agents that are agonists at 5-HT3 receptors, appear to act largely within
the brain, providing potentially useful information in the quest to prevent SUDEP in
patients.
Acknowledgments
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This study was supported by Epilepsy Foundation (CF) and R03NS078591, CURE (Citizens United for Research in
Epilepsy) foundation grant and fund from the Department of Anesthesia, Critical Care and Pain Medicine at
Massachusetts General Hospital (HJF).
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Highlights
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• 5-HT3 receptor agonist reduces S-IRA in DBA/1 mice
• 5-HT3 receptor antagonist reduces the suppressing effect of fluoxetine

on S-IRA
• Fluoxetine administered into the brain reduces S-IRA

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Fig. 1.
Effect of a 5-HT3 agonist (SR 57227) on the incidence of audiogenic seizures (AGSz) and
seizure-induced respiratory arrest (S-IRA) in DBA/1 mice.
The effects of systemic (i.p.) administration of SR 57227 [20, 30, 35 or 40 mg/kg in saline
(vehicle) or saline shown as zero dose] were examined in S-IRA susceptible DBA/1 mice.
Thirty min after SR 57227 injection, the 35 and 40 mg/kg dose significantly reduced the
incidence of S-IRA evoked by acoustic stimulation as compared with vehicle control. The
incidence of AGSz was not significantly affected by any of the other injections. White bars
indicate the incidence of S-IRA; black bars indicate the incidence of AGSz.
*Significantly different from vehicle control, p < 0.05. N is the number of animals for each
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dose.

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Fig. 2.
Effect of a 5-HT3 antagonist (ondansetron) on S-IRA suppressing action by fluoxetine in
DBA/1 mice.
Systemic (i.p.) administration of ondansetron (OND), 0.5, 0.75 or 1.0 mg/kg or vehicle
shown as zero dose, 35 min prior to audiogenic seizures (AGSz) on the ability of fluoxetine
(40 mg/kg, i.p.), 30 min prior to AGSz, to reduce the incidence of AGSz and seizure-induced
respiratory arrest (S-IRA) in primed DBA/1 mice that consistently exhibited S-IRA before
treatment. Fluoxetine plus vehicle induced a complete suppression of S-IRA without
blocking AGSz. A dose-dependent reduction in the suppressive effect of fluoxetine was
induced by OND, which reached statistical significance at 1.0 mg/kg. The incidence of
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AGSz was not significantly affected by any of the injections. White bars indicate the
incidence of S-IRA; black bars indicate the incidence of AGSz.
*Significantly different from fluoxetine alone, p < 0.05. N is the number of animals for each
dose.

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Fig. 3.
Intracerebroventricular (ICV) injection of fluoxetine reduces S-IRA in DBA/1 mice.
A: The effects of fluoxetine [1.0 µl, 60, 90 or 120 nmol in DMSO or the DMSO (vehicle)
shown as zero dose] administered ICV on the incidence of audiogenic seizures (AGSz) and
seizure-induced respiratory arrest (S-IRA) in DBA/1 mice. Fifteen min after fluoxetine
injection, the 120-nmol dose significantly reduced the incidence of S-IRA evoked by
acoustic stimulation as compared with vehicle control. The incidence of AGSz was
unaffected by any of the other fluoxetine doses. White bars indicate the incidence of S-IRA;
black bars indicate the incidence of AGSz. B: a representative example of histology showing
that the drug and vehicle were successfully delivered into the ventricle.
** Significantly different from vehicle control at p<0.01. N is the number of animals for
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each dose.
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Crystal Structures of the M1 and M4 Muscarinic Acetylcholine

Sciences, Monash University, Parkville, 3052, Victoria, Australia

Reprints and permissions information is available at www.nature.com/reprints.

The M1 – M5 muscarinic acetylcholine receptors constitute an important family of Class A

Crystallization of the M1 and M4 receptors

Nature. Author manuscript; available in PMC 2016 September 17.

Comparison of muscarinic receptor structures

Nature. Author manuscript; available in PMC 2016 September 17.

residues in stabilizing different inactive-state conformations with QNB potentially making

Allosteric binding and cooperativity

Nature. Author manuscript; available in PMC 2016 September 17.

We investigated the interaction between the well-characterized PAM, LY203329813-15,20,21,

Nature. Author manuscript; available in PMC 2016 September 17.

structure (Fig. 4, Extended Data Fig. 6).

Nature. Author manuscript; available in PMC 2016 September 17.

Another noteworthy feature of LY2033298 is that it is selective towards the M4 receptor

could arise either through differential binding affinities of LY2033298 or through a

M1 and M4 receptor expression and purification

M4 receptor was removed by cleavage with HRV 3C protease at a concentration of 2%

Nature. Author manuscript; available in PMC 2016 September 17.

Pharmacology of crystallization constructs

and stored in liquid nitrogen before use.

Data collection, processing, and structure determination

Nature. Author manuscript; available in PMC 2016 September 17.

reported in Extended Data Table 1.

Induced fit docking of pirenzepine

Molecular modelling of active M4 receptor

Molecular modelling of active M1 receptor

Nature. Author manuscript; available in PMC 2016 September 17.

Receptor mutagenesis and generation of cell lines

Radioligand binding assays

Radioligand inhibition binding assays were performed by co-incubating 10–100 μg of

Nature. Author manuscript; available in PMC 2016 September 17.

Nature. Author manuscript; available in PMC 2016 September 17.

Extended Data Figure 1. Crystallization construct design, purification and crystallization

Nature. Author manuscript; available in PMC 2016 September 17.

observed under circularly polarized light.

Nature. Author manuscript; available in PMC 2016 September 17.

Nature. Author manuscript; available in PMC 2016 September 17.

Nature. Author manuscript; available in PMC 2016 September 17.

Nature. Author manuscript; available in PMC 2016 September 17.

Extended Data Figure 6. Ligand interaction diagrams for the M4 receptor

Nature. Author manuscript; available in PMC 2016 September 17.

Nature. Author manuscript; available in PMC 2016 September 17.

Nature. Author manuscript; available in PMC 2016 September 17.

Extended Data Figure 9. LY2033298 binding to active-state M1 and M4 models

Nature. Author manuscript; available in PMC 2016 September 17.

Extended Data Table 1

Data collection* M1-T4L•tiotropium M4-mT4L•tiotropium

Rmerge (%) 15.5 (83.6) 21.3 (94.7)

Average B-factors (Chain A / Chain B; Å2) 73.6 74.0 / 70.5

Allowed regions (%) 3.6 0.8

Nature. Author manuscript; available in PMC 2016 September 17.

Refer to Web version on PubMed Central for supplementary material.

for schizophrenia. Am J Psych. 2008; 165:1033–1039.

vivo validation of allosteric modulation at the M4 muscarinic acetylcholine receptor. British

Nature. Author manuscript; available in PMC 2016 September 17.

learning. J Neurosci. 2009; 29:14271–14286. [PubMed: 19906975]

285:19012–19021. DOI: 10.1074/jbc.M110.125096 [PubMed: 20406819]

subtype selectivity of the allosteric interactions of gallamine at muscarinic acetylcholine receptors.

Nature. Author manuscript; available in PMC 2016 September 17.