Tuesday, February 23, 2021
highly penetrant point mutations located within the cTnT N-terminus (R92L/W
and I79N) which are associated with distinct phenotypes and severities. We
employed stopped-flow to measure the rates of calcium association (kon) to
and dissociation (koff) from IAANS-labeled cardiac troponin C in CTF systems
of increasing complexity to explain similar changes in steady-state calcium
sensitivity often associated with these mutations. We previously characterized
mutation-specific differences in koff for these mutations in CTFs. Kon to WT,
R92L, R92W, and I79N CTFs was measured over a range of calcium concen-
trations and calculated to be 9.8852.051x106M-1s-1, 9.14151.94x106M-1s-
1, 31.3752.948x106M-1s-1, and 28.252.736x106M-1s-1, respectively. Both
I79N and R92W, but not R92L, significantly increased kon compared to WT
CTFs. We then explored the effects of these mutations on koff in the presence
of heavy meromyosin (HMM). Koff from WT, R92L, R92W, and I79N
CTFsþHMM was found to be 12.9850.170s-1, 10.0850.147s-1,
7.8250.267s-1, and 9.6950.354s-1, respectively. After adding HMM to the
system, all three mutations significantly decreased koff to a similar extent
(1.3-fold). Lastly, we added C0C2, a cardiac myosin binding protein-C frag-
ment, to the WT CTF system. Adding C0C2 decreased koff in a concentration-
dependent manner that was abolished upon C0C2 phosphorylation. These
results highlight the utility of stopped-flow for probing myofilament calcium
kinetics in vitro and how mutation-specific alterations in calcium kinetics
can be used to distinguish mutations which similarly affect calcium sensitivity.
Posters: Ligand-gated Channels I
266-Pos
Heritable Mutations to the b3 Subunit of GABA-A Receptor Alter the
Receptor Function and Synaptic Localisation
Nela I. Durisic1, Joseph W. Lynch2, Ned Cotter1, Dejan Gagoski1,
Parnayan Syed1, Pankaj Sah1, Duka Skalamera1.
1
The University of Queensland, Brisbane, Australia, 2Queensland Brain Inst,
Univ Queensland, Brisbane, Australia.
Human genetic epilepsies are frequently caused by mutations in the GABA
type-A receptor (GABAAR), an essential mediator of inhibitory neurotransmis-
sion in the brain. The largest number of mutations causing severe forms of ep-
ilepsy is discovered in the GABAAR b3 subunit, which plays an important role
in the early stages of brain development. Yet it is not clear how these mutations
perturb neuronal activity. Here, we compared the expression and functional
impact of b3G32R, associated with mild epilepsy, to b3N110D, b3D120N
and b3E180G which are implicated in more severe forms of the disease. We
used a neuron-HEK293 cell hetero-synapse preparation to record inhibitory
postsynaptic currents (IPSCs) mediated by mutant-containing GABAARs in
isolation from other GABAAR isoforms. We then expressed the mutant sub-
units in cortical neurons to investigate changes in neuronal morphology, syn-
apse formation and GABAAR mobility. To simulate neuronal activity during
epileptic seizures, 4-Aminopyridine was applied. Using single-molecule local-
ization microscopy (SMLM) and electrophysiology, we show that these muta-
tions do not affect membrane trafficking and surface expression, but they
accelerate the decay of IPSCs to a different extent. Diazepam increases the
peak amplitude and the decay time constant of IPSCs mediated by all mutant
receptors. However, it is able to fully recover only the function of GABAARs
with a mutation linked with mild epilepsy, b3G32R. In cortical neurons,
b3D120N and b3E180G decrease synaptic clustering. Universal point accumu-
lation for imaging in nanoscale topography (uPAINT) revealed changes in the
mobility of mutant GABAARs as well as in the rate of exocytosis of GABAARs
during 4AP-ivoked neuronal hyperexcitability. These results provide new in-
sights into the mechanisms of epileptogenesis and suggest possible leads for
improving treatments for patients harbouring mutations in GABAAR subunits.
267-Pos
Two-Photon Photoactivation of Rubi-Gaba for Studying the Role of the
Antisecretory Factor in the Modulation of the GABAA Receptor in Rat
Cerebellar Granule Cells In Vitro
Virginia Bazzurro1, Elena Gatta1, Elena Angeli1, Aroldo Cupello1,
Stefan Lange2, Mauro Robello1, Alberto Diaspro1,3.
1
Department of Physics, University of Genoa, Genova, Italy, 2Department of
Infectious Diseases, Institute of Biomedicine, University of Gothenburg,
Gothenburg, Sweden, 3Department of Nanoscopy, Istituto Italiano di
Tecnologia, Genoa, Italy.
Caged compounds, widely diffused in neurophysiology, are molecules biolog-
ically or functionally inert until a short pulse of light enables them. Illumination
releases the caged effector allowing the activation, manipulation, and control of
selective biological functions and variations. in particular, 3D two-photon exci-
tation microscopy, coupled with the electrophysiological technique of the
patch-clamp in whole-cell configuration, provides a useful tool for studying
55a
several neurobiological processes such as the localized, accurate, and specific
receptor response of a well-defined neuronal area. Here, we report this method
to figure out the role of the protein Antisecretory Factor (AF) in the modulation
of the GABAA receptor by using the caged neurotransmitter RuBi-GABA,
rapidly photoreleased, in femtoliter volumes, in a precise and confined region
of a neuron at a defined concentration. AF acts in vivo by inhibiting intestinal
hypersecretion and various inflammation forms, although its mechanism of ac-
tion is mostly unknown. We tested the response produced by AF-16, a peptide
obtained from the N-terminal of AF, on rat cerebellar granule cellsthanks to the
uncaging of caged GABA on the soma, neurites, and the growth cone for un-
derstanding the effect on different GABAA receptor populations.
268-Pos
Influence of GABA-A Receptor Intracellular Domain on Channel
Activation and Desensitization
Anton J. Tung, Lucas M. Blecker, Cynthia Czajkowski.
Dept Neuroscience, Univ Wisconsin Madison, Madison, WI, USA.
Rapid opening and closing of GABA-A receptor (GABAR) channels regulate
signaling in the brain. Mechanisms underlying channel open, closed, desensi-
tized transitions remain unclear. GABAR cryo-EM structures provide details
about its extracellular and transmembrane domains, but little information exists
about its intracellular domain (ICD) and its role in regulating channel gating.
We constructed a GABAR ICD homology model based on a cryo-EM
5HT3AR structure, which revealed a helix (MX) at the C-terminal end of
M3 and a helix (MA) at the N-terminal end of M4 projecting intracellularly.
Four residues with potential interactions between these helices were mutated
to alanine (a1K327A in MX, a1K390A in MA, and g2K409A and g2Y413A
in MA). Mutations effects on GABA-EC50 and current desensitization were
measured using two-electrode voltage clamping of oocytes expressing wild-
type (WT) and mutant GABARs. a1K327A increased GABA EC50 6-fold
compared to a1b2g2 WT (35.98 5 1.03 mM, n=9) and significantly reduced
GABAR desensitization. Extent of current desensitization after 60 seconds in
saturating GABA (10mM) was 30.16 5 2.24 % (n=9) versus WT (62.96 5
4.15 %, n=13). a1K390A had no effect on GABA EC50 or desensitization,
and mutation of the aligned residue in g2 subunit, g2K409A, decreased
GABA EC50 2 fold without affecting desensitization. g2Y413A significantly
increased desensitization and increased GABA EC50 by 2-fold. Desensitiza-
tion extent after 10 seconds was 76.57 5 2.09 % (n=18) compared to WT
(23.71 5 2.30 %, n=10). These data indicate that structural perturbations of
the a1MX and g2MA regions in the GABAR ICD can influence macroscopic
GABAR current desensitization. Interestingly, GABAR MX regions are lysine
rich. We speculate that mechanisms underlying GABAR gating transitions may
involve charged interactions between MX and other regions of the GABAR
ICD (e.g. MA). Experiments are underway to test this idea.
269-Pos
A Conserved Arginine with Non-Conserved Function is a Key Determinant
of Agonist Selectivity in Alpha7 Nicotinic Acetylcholine Receptors
Teresa Minguez Vinas1,2, A. Sofia F. Oliveira3, B. Elizabeth Nielsen4,
Cecilia Gotti5, Adrian J. Mulholland3, Timothy Gallagher3,
Isabel Bermudez1.
1
Biological and Medical Sciences, Oxford Brookes University, Oxford,
United Kingdom, 2Biomedical and Clinical Sciences, Linköping University,
Linköping, Sweden, 3School of Chemistry, University of Bristol, Bristol,
United Kingdom, 4CONICET, Instituto de Investigaciones Bioquı́micas de
Bahı́a Blanca, Bahı́a Blanca, Argentina, 5Institute of Neuroscience, Biometra
Dept, University of Milan, Milan, Italy.
The a7 and a4b2 subtypes are the most abundant nicotinic acetylcholine recep-
tors (nAChR) in the mammalian brain, and also the most commonly targeted
nAChR subtypes in drug discovery programs for brain disorders. Activation
of a4b2 nAChR by agonists, particularly partial agonists, is a valid strategy
to intervene therapeutically in nicotine addiction. However, the development
of subtype specific agonists remains challenging, mainly due to the high degree
of sequence homology coupled to the conservation of function in the nAChR
family. Smoking cessation drugs such as varenicline or cytisine partially acti-
vate a4b2 nAChR but also behave as full agonists at other nAChR such as the
a7 subtype, which may underlie some of the off-target effects of these com-
pounds. Here, by combining molecular dynamics simulations with mutagenesis
and whole-cell and single-channel current recordings, we determined the struc-
tural underpinning of the selectivity of 10-methylcytisine, a compound that
shows high-affinity for a4b2 nAChR but has negligible selectivity for the a7
subtype. We identify a conserved arginine residue (a7R101) in the b3-strand
that impairs agonist binding in a7 nAChR. in a4b2, the equivalent arginine res-
idue establishes an inter-subunit electrostatic interaction with an aspartate res-
idue in loop B that is necessary for functional expression, whereas in the a7
56a Tuesday, February 23, 2021
subtype, the aspartate residue is exchanged for glycine residue, making the side
chain of arginine highly mobile. This enables a7R101 to interact with loop C
residues, which influences agonist binding and channel function. We conclude
that the high mobility of the arginine residue in the a7 nAChR subtype affects
agonist function by influencing agonist binding and the pathway communi-
cating agonist binding to the ion channel. The findings have implications for
rational design of subtype-selective cholinergic agents.
270-Pos
Structural Investigation into Gating and Modulation of Alpha1 Glycine
Receptor in Lipid Nanodiscs
Arvind Kumar1, Sandip Basak1, Shanlin Rao2, Mark S. Sansom2,
Sudha Chakrapani1.
1
Department of Physiology and Biophysics, Case Western Reserve
University, Cleveland, OH, USA, 2Department of Biochemistry, University
of Oxford, Oxford, United Kingdom.
Glycine receptors (GlyR) mediate fast chemical transmission of synaptic sig-
nals in the central and peripheral nervous system. GlyR are anionic members
of pentameric ligand gated ion channels and are central players in motor co-
ordination and pain perception. Potentiation of GlyR activity results in damp-
ening pain signals in the spinal cord making these receptors an attractive
target for pain therapy. Here, we present Cryo-EM structures of the full-
length alpha1 GlyR reconstituted into lipid nanodiscs that are captured in
multiple functional states that include, the unliganded state (closed) and
glycine-bound conformations (open and desensitized). in addition, we present
conformations of GlyR in various ligand-bound forms that provide insights
into the mechanisms underlying allosteric modulation. The functional state as-
signments were validated by molecular dynamics simulations. A comparison
of these states reveals global conformational changes underlying GlyR chan-
nel gating and modulation.
271-Pos
When a Gain Becomes a Loss: Gain-of-Function Glycine Receptors and
Hyperekplexia
James P. Dilger1, Mohammed A. Shanawaz2.
1
Dept Anesthesiology, Stony Brook Univ, Stony Brook, NY, USA,
2
Department of Public Health, Applied Medical Sciences College, Al-Baha
University, Al-Baha, Saudi Arabia.
Glycine receptors (GlyRs) mediate rapid synaptic inhibition in mammalian spi-
nal cord and brainstem. GlyRs mutations often produce abnormalities that
result in hyperekplexia. Most mutations cause a loss of GlyR function such
as decreased expression, conductance or ligand sensitivity. It is easily seen
how a reduction in inhibition could result in the increased excitatory symptoms
of hyperekplexia. Paradoxically, the a1V280M mutation triggers hyperekplexia
but exhibits gain of GlyR function. The a1V280M mutation increases GlyR
sensitivity to glycine and produces spontaneous channel activity but does not
alter GlyR expression. Because CSF contains resting levels of 10mM glycine
and because GlyRs undergo desensitization, we sought to determine the degree
of desensitization induced by resting glycine levels for wild-type (a1WT) and
a1V280M GlyRs. HEK-293 cells transfected with cDNA for either human
a1WT or a1V280M GlyR were prepared for outside-out patch clamp recording.
Currents were activated by five, 300ms applications of 3mM glycine every 5s
(–50mV). Between applications (background), patches were perfused with
glycine-free solution. Subsequently, the background was switched to a solution
containing between 3nM and 60mM glycine; and currents recorded. Finally, re-
covery currents were obtained. Desensitization was calculated as the peak cur-
rent in the presence of glycine background relative to that in glycine-free
background. Background glycine decreased peak currents through both
a1WT and a1V280M GlyR with a sigmoidal concentration dependence.
IC50s for current inhibition were 14mM for a1WT GlyR and 0.3mM for
a1V280M GlyR. The 10mM glycine in CSF would desensitize about half of
a1WT GlyRs. In contrast, 98% of a1V280M GlyR would be desensitized.
Thus, even though the V280M mutation produces a gain-of-function at the re-
ceptor level, it results in a loss-of-function at the synaptic level. This may
explain why patients with this mutation exhibit the symptoms of hyperekplexia.
272-Pos
Conformational Landscape of an Agonist-Binding Domain of a
Full-Length Heteromeric Kainate Receptor Gluk2/K5 Displaying Partial
Agonism
Nabina Paudyal, Elisa Carrillo, Vladmir Berka, Vasanthi Jayaraman.
Biochemistry and Molecular Biology, UT Health Science Center at Houston,
Houston, TX, USA.
Kainate receptors are the family of glutamate-gated ion channels (iGluRs)
involved in both excitatory and inhibitory synaptic transmission. Based on their
affinity for kainate, they are sub-grouped as low affinity (GluK1-3) and high
affinity (GluK4-5). Homomeric GluK2 and heteromeric GluK2/K5 are the
most abundant subtypes of kainate receptors found in human brain. Structural
data available for homomeric GluK2 have shown differential degree of cleft
closure upon binding to different agonists. On contrary, the structural under-
standing of an agonist binding to the cleft of agonist binding domain of hetero-
meric GluK2/K5 is limited. In particular, we are interested in understanding the
conformational changes induced in heteromer GluK2/K5 in presence of agonist
AMPA and iodowillardiine. Functional studies have shown that both AMPA
and iodowillardiine serve as partial agonist for heteromer GluK2/K5 and elicit
no response in homomeric GluK2. Herein, we present to you our structural
investigation for understanding the conformational changes induced in a full-
length heteromer GluK2/K5 in presence of its endogenous ligand glutamate
and its partial agonist AMPA and iodowillardiine.
273-Pos
Fatty Acids Inhibit a Pentameric Ligand-Gated Ion Channel through One
of Two Binding Sites
Noah Dietzen1, John T. Petroff1, Douglas F. Covey2, Wayland W. Cheng1.
1
Anesthesiology, Washington University in Saint Louis, St. Louis, MO, USA,
2
Developmental Biology, Washington University in Saint Louis, St. Louis,
MO, USA.
Pentameric ligand-gated ion channels (pLGICs) mediate synaptic transmission
and are inhibited by fatty acids. To investigate the structural determinants of
fatty acid inhibition of pLGICs, we developed a new photolabeling reagent,
KK-242, for the identification of fatty acid binding sites. by giant liposome
patch-clamping, fatty acids inhibit the model pLGIC, Erwinia ligand-gated
ion channel (ELIC), decreasing peak response to agonist and increasing the
rate and extent of current decay. Polyunsaturated fatty acids (PUFAs) are
more efficacious than saturated fatty acids. This modulatory effect is similar
to previous reports of fatty acid inhibition of the GABA(A) receptor and nico-
tinic acetylcholine receptor, and is consistent with a model where fatty acids
stabilize a pre-activated state. KK-242 also inhibits ELIC. KK-242 photolabels
two fatty acid binding sites per subunit, which are also binding sites for doco-
sahexaenoic acid and palmitic acid. To probe the functional significance of
these sites, a cysteine was introduced to each site and modified with
hexadecyl-methanethiosulfonate (MTS). Modification of one site with
hexadecyl-MTS recapitulates the inhibitory effect of fatty acids on ELIC func-
tion. The results show that fatty acids bind to two sites, and inhibit ELIC by
binding to just one of these sites. KK-242 may be a useful tool for identification
of fatty acid binding sites in other membrane proteins.
274-Pos
Solution Structure of the Gloeobacter Violaceus Ligand-Gated Ion
Channel Probed by Small-Angle Neutron Scattering
Marie Lycksell1, Urska Rovsnik1, Cathrine C. Bergh2, Nicolai T. Johansen3,
Anne Martel4, Lionel Porcar4, Lise Arleth3, Rebecca J. Howard1,
Erik Lindahl1,2.
1
Dept Biochem & Biophysics, Stockholm Univ, Solna, Sweden, 2Royal Inst
of Tech, Solna, Sweden, 3Niels Bohr Inst, Univ of Copenhagen, Copenhagen,
Denmark, 4Inst Laue-Langevin, Grenoble, France.
Pentameric ligand-gated ion channels transform chemical signals into electrical
ones, a process during which they undergo subtle conformational cycling re-
sulting in rapid, reversible gating of an intrinsic transmembrane pore. The
pH-gated bacterial channel GLIC has proved a valuable model system for
this receptor family, in part due to its accessibility to X-ray crystallography -
which has provided structures of closed, open, and intermediate conformations.
However, such models raise critical questions as to whether the X-ray struc-
tures represent actual functional states, and if the cycle includes additional con-
formations such as highly contracted or expanded states. Small-angle neutron
scattering (SANS) offers an approach to characterizing solution-phase struc-
ture, increasingly applicable to biomolecular problems on this scale. Here we
characterized GLIC by SANS, in combination with inline size-exclusion chro-
matography (SEC), match-out deuterated detergents, and computational
modeling of multiple putative structural models. SEC-SANS under resting con-
ditions (pH 7) enabled the characterization of non-aggegated species which fit
predictions from resting-state models, particularly following unrestrained mo-
lecular dynamics simulation of X-ray structures. Curves collected under acti-
vating conditions (pH 3) best fit a mixture of resting- and open-state models,
whereas they diverged from expanded-pore models. in addition to supporting
less-expanded models of the open state, this work demonstrates that SEC-
SANS, in combination with molecular modeling and simulations, can distin-
guish solution-phase functional states of an ion channel. As neutron source
brilliance increases and inline SEC-SANS set-ups become increasingly acces-
sible, such methods will offer valuable tools for the elucidation of receptor
conformational cycling and pharmaceutical development.