Received: 12 July 2019 | Revised: 4 December 2019 | Accepted: 4 December 2019

DOI: 10.1111/jnc.14939

ORIGINAL ARTICLE

Desensitization of NMDA channels requires ligand binding to

both GluN1 and GluN2 subunits to constrict the pore beside
the activation gate

Yu-Shian Chen1 | Ya-Chi Tu1 | Yi-Chen Lai2 | Erin Liu1 | Ya-Chin Yang2,3,4 |
Chung-Chin Kuo1,5

1
Department of Physiology, National Taiwan
University College of Medicine, Taipei, Abstract
Taiwan N-methyl-D-aspartate (NMDA) receptor channels are activated by glutamate (or
2
Department of Biomedical Sciences,
NMDA) and glycine. The channels also undergo desensitization, which denotes
College of Medicine, Chang Gung University,
Tao-Yuan, Taiwan decreased channel availability, after prolonged exposure to the activating ligands.
3
Graduate Institute of Biomedical Sciences, Glycine apparently has a paradoxical negative effect on desensitization, as the in-
College of Medicine, Chang Gung University,
Tao-Yuan, Taiwan
crease in ambient glycine in concentrations required for channel activation would
4
Neuroscience Research Center, Chang increase sustained NMDA receptor currents. We hypothesized that this classi-
Gung Memorial Hospital, Linkou Medical cal “glycine-dependent desensitization” could be glycine-dependent activation in es-
Center, Tao-Yuan, Taiwan
5 sence. By performing electrophysiological recordings and biophysical analyses with
Department of Neurology, National Taiwan
University Hospital, Taipei, Taiwan rat brain NMDA receptors heterogeneously expressed in Xenopus laevis oocytes, we
characterized that the channel opened by “only” NMDA (in nominally glycine-free
Correspondence
Ya-Chin Yang, Department of Biomedical condition probably with the inevitable nanomolar glycine) would undergo a novel
Sciences, Chang Gung University, Kwei-
form of deactivation rather than desensitization, and is thus fully available for subse-
Shan, Tao-Yuan, Taiwan.
Email: ycyang@mail.cgu.edu.tw quent activation. Moreover, external tetrapentylammonium ions (TPentA), tetrabu-
Chung-Chin Kuo, Department of Physiology, tylammonium ions, and tetrapropylammonium ions (TPA, in higher concentrations)
National Taiwan University College of block the pore and prohibit channel desensitization with a simple “foot-in-the-door”
Medicine, Taipei, Taiwan
Email: chungchinkuo@ntu.edu.tw hindrance effect. TpentA and TPA have the same voltage dependence but show dif-

Funding Information
ferent flow dependence in binding affinity, revealing a common binding site at an
This work was supported by Grants electrical distance of ~0.7 from the outside yet differential involvement of the flux-
MOST107-2311-B-182-004 (to Y.-C. Yang),
MOST108-2311-B-182-001 (to Y.-C. Yang),
coupling region in the external pore mouth. The smaller tetraethylammonium ion
MOST 108-2321-B-007-003-MY2 (to Y.-C. and the larger tetrahexylammonium and tetraheptylammonium ions may block the
Yang), MOST106-2320-B-002-014-MY3 (to
C.-C. Kuo), and MOST108-2321-B-002-007
channel but could not affect desensitization. We conclude that NMDA receptor de-
(to C.-C. Kuo) from Ministry of Science sensitization requires concomitant binding of both glycine and glutamate, and thus
and Technology, Taiwan, Grant NHRI-
EX107-10503NI (to C.-C. Kuo) from
movement of both GluN1 and GluN2 subunits. Desensitization gate itself embodies
the National Health Research Institute, a highly restricted pore reduction with a physical distance of ~4 Å from the charged
Taiwan, and Grants CMRPD3E0263 and
CMRPD1H0091-2 (to Y.-C. Yang) from
nitrogen atom of bound tetraalkylammonium ions, and is located very close to the
Chang Gung Medical Foundation, Taiwan. activation gate in the bundle-crossing region in the external pore vestibule.

Abbreviations: GluN1, a N-methyl-D-aspartate receptor subunit name; GluN2, a N-methyl-D-aspartate receptor subunit name; IACUC, Institutional Animal Care and Use Committee;
NMDA, N-methyl-D-aspartate; RRID, Research Resource Identifier; SEM, standard error of mean; TAA, tetraalkylammonium; TBA, tetrabutylammonium; TEA, tetraethylammonium;
TheptylA, tetraheptylammonium; ThexylA, tetrahexylammonium; TMA, tetramethylammonium; TOA, tetraoctylammonium; TPA, tetrapropylammonium; TpentA,
tetrapentylammonium.

Yu-Shian Chen and Ya-Chi Tu contributed equally to this work.

Journal of Neurochemistry. 2020;153:549–566. wileyonlinelibrary.com/journal/jnc © 2019 International Society for Neurochemistry | 549
550 | CHEN et al.
KEYWORDS
external pore mouth, gating mechanism, glutamate, glycine-dependent desensitization,
tetraalkylammonium ions
1 | I NTRO D U C TI O N
N-methyl-D-aspartate (NMDA) receptors, a subtype of ionotropic
glutamate receptors showing significant Ca2+ permeability, play a
critical role in synaptic transmission and plasticity in the mamma-
lian central nervous system (Collingridge, Kehl, & McLennan, 1983;
Collingridge, Peineau, Howland, & Wang, 2010; Dingledine, Borges,
Bowie, & Traynelis, 1999; Lai, Shyu, & Wang, 2011; Paoletti & Neyton,
2007; Traynelis et al., 2010; Wigstrom & Gustafsson, 1984). NMDA
receptor channels are opened upon binding of the activating ligands
glutamate (or NMDA) and glycine to the GluN2 and GluN1 subunits,
respectively (Hirai, Kirsch, Laube, Betz, & Kuhse, 1996; Kleckner &
Dingledine, 1988; Laube, Kuhse, & Betz, 1998). In the continuous
presence of activating ligands, the channels may enter the non-con-
ducting desensitized state, quite analogous to inactivation in volt-
age-gated channels. The desensitization thus contributes to suitable
duration and magnitude of synaptic charge transfer and Ca2+ entry
through the NMDA receptor channels. However, the molecular sub-
strates for NMDA receptor desensitization still remain elusive.
There are reportedly three major forms of desensitization men-
2+
tioned in the literature, namely Ca -dependent, glycine-dependent,
and glycine-independent desensitization. Desensitization of NMDA
receptors could be sensitive to Ca2+ influx and intracellular Ca2+ con-
centration (Clark, Clifford, & Zorumski, 1990; Legendre, Rosenmund,
& Westbrook, 1993; Rosenmond and Westbrook, 1993; Medina et al.,
1995; Rosenmund, Feltz, & Westbrook, 1995; Krupp, Vissel, Heinemann,
& Westbrook, 1996; Krupp, Vissel, Thomas, Heinemann, & Westbrook,
1999; Ehlers et al., 1998; 1996; Zhang, Ehlers, Bernhardt, Su, & Huganir,
1998; Dingledine et al., 1999; Vissel, Krupp, Heinemann, & Westbrook,
2002), although the conductance change induced by extracellular Ca2+
binding to the DRPEER motif in GluN1 has been recently depicted
(Tu, Yang, & Kuo, 2016). The development of the traditional type of
Ca2+-dependent desensitization involves second messenger systems
(Rosenmond and Westbrook 1993; Krupp et al., 1999; Lieberman &
Mody, 1994; Raman, Tong, & Jahr, 1996; Tong & Jahr, 1994; Zhang et al.,
1998) and, therefore, could be too slow (in the order of seconds) to play
2+
a major role in fast synaptic transmission. In addition, Ca -dependent
desensitization reflects more a gating modification secondary to Ca2+
binding or influxes rather than a protein conformational change directly
induced by the prolonged presence of the activating drive, a classical
view of channel desensitization or inactivation. Activation of NMDA
receptors requires not only NMDA or glutamate, but also the co-ago-
nist glycine in low micromolar or even submicromolar concentrations.
The same concentration range of glycine, however, seems to prohibit
desensitization (Johnson & Ascher, 1992; Kleckner & Dingledine, 1988;
Mayer, Vyklicky, & Clements, 1989). Apparently at odds with the con-
ventional definition of channel desensitization that is promoted by the
continuous presence of activating ligands (e.g., glycine), NMDA receptor
desensitization is manifest with submicromolar glycine, and decreases
with higher concentrations of glycine before saturation at 10–30 μM
glycine. This is referred to as glycine-dependent desensitization, tenta-
tively viewed as a time-dependent negative cooperativity between the
glutamate and glycine-binding sites (Benveniste, Clements, Vyklicky, &
Mayer, 1990; Cummings & Popescu, 2015; Lerma, Zukin, & Bennett,
1990; McBain & Mayer, 1994; Vyklicky, Benveniste, & Mayer, 1990).
However, this model is challenged by the findings that alterations
of glycine affinity by mutation or allosteric modulators do not affect
glutamate binding concomitantly (Hansen, Ogden, & Traynelis, 2012;
Hirai et al., 1996). On the other hand, there is still a form of desensiti-
zation considered as glycine-independent desensitization in the pres-
ence of more than 10–30 μM glycine (Benveniste et al., 1990; Chen,
Li, Murphy, & Rayond, 2004; Hu & Zheng, 2005a; Lerma et al., 1990;
Lester, Tong, & Jahr, 1993; Mayer et al., 1989; McBain & Mayer, 1994;
Nahum-Levy, Lipinski, Shavit, & Benveniste, 2001; Regalado, Villarroel,
& Lerma, 2001; Sessoms-Sikes, Honse, Lovinger, & Colbran, 2005). This
form of desensitization could be the most influential for fast synaptic
transmission because the concentration of glycine at the glutamater-
gic synaptic cleft may be higher than 30 μM during neuronal activation
(Vandenberg & Aubrey, 2001). From a structural perspective, the amino
terminal or LIVBP-like domain and the four amino acids immediately be-
fore the first transmembrane segment (M1) have been presumed as the
structural determinants of glycine-independent desensitization (Krupp,
Vissel, Heinemann, & Westbrook, 1998; Rumbaugh, Prybylowski,
Wang, & Vicini, 2000; Villarroel, Regalado, & Lerma, 1998), implicating
a decrease in the "tension" exerted by the pre-M1 region on M1 and
thus closure of the pore at the external entrance (Krupp et al., 1998;
Rumbaugh et al., 2000; Villarroel et al., 1998). This proposal, however,
lacks a direct evidence for “closure” of the pore (the molecular mech-
anism underlying desensitization). Also, it does not address the site
of closure (the location of the desensitization gate) or its relationship
with the activation gate, which is also located at the external vestibule
(Chang & Kuo, 2008a; Karakas & Furukawa, 2014; Lee et al., 2014). In
this regard, it is worthy of note that the binding site of anticonvulsant
felbamate, which binds preferentially to the desensitized state of the
NMDA receptor channel, is located in the SYTANLAAF motif contain-
ing the activation gate (Chang & Kuo, 2007, 2008b). Consistently, the
same motif has been inferred to play a role in glycine-independent de-
sensitization (Hu & Zheng, 2005b ; Klein & Howe, 2004; Kohda, Wang,
& Yuzaki, 2000). It is thus desirable to investigate the molecular entities
of glycine-independent as well as glycine-dependent desensitization in
more detail, and to explore its relevance to channel activation.
Tetraalkylammonium (TAA) ions have a common tetrahedral
structure but different molecular size and local configuration with
increasing number of the methylene groups in the alkyl chain. TAA
ions have been applied to probe the dimension of pore entrances
of different channels, such as the Kir2.1 inward rectifier K+ channel
CHEN et al.
(Guo & Lu, 2001; Guo, Ramu, Klem, & Lu, 2003). TAA ions could also
block the NMDA receptor channel with a one-to-one binding stoi-
chiometry (Sobolevsky, 2000; Sobolevsky, Koshelev, & Khodorov,
1999; Zarei & Dani, 1995). Notably, there is a hook tail current
following wash-off of the activating ligands and tetrapentylam-
monium (TpentA) ions, apparently signaling a deterring effect of
TpentA ions on NMDA receptor desensitization (Sobolevsky, 2000;
Sobolevsky et al., 1999). In this study, we took a large series of dif-
ferent TAA ions as a set of probes to dissect the biophysical and
structural mechanisms underlying NMDA receptor desensitization.
We found that desensitization requires the concomitant presence
of glycine and NMDA, and that TAA ions preclude desensitization
by a simple and direct “foot-in-the-door” effect. Moreover, NMDA
receptor desensitization most likely physically embodies a highly
restricted pore reduction adjacent to the activation gate in the
bundle-crossing region in the external vestibule.
2 | M ATE R I A L S A N D M E TH O DS
2.1 | Study approval
The care and use of animals was carried out in accordance with
the ethical information guidelines of and approved by the National
Taiwan University College of Medicine and College of Public Health
Institutional Animal Care and Use Committee, Taiwan (IACUC
Approval No. 20150061). All efforts were made to minimize animal
suffering. All experiments were conducted in compliance with the
ARRIVE guidelines. The study was not pre-registered.
2.2 | Expression of NMDA receptors
The rat cDNA clones of NMDA receptors used in this study are the
GluN1a variant and the GluN2B clone (gifts from Dr. Keith Williams,
Department of Physiology and Pharmacology, SUNY Downstate
Health Sciences University, U.S.A.). The sequence of amino acid
residues in the GluN1 and GluN2B subunits is numbered from the
initiator methionine as in the original papers of NR1 (Moriyoshi
et al., 1991) and NR2B (Monyer et al., 1992), respectively. The
capped cRNA transcripts of GluN1 and GluN2B were synthesized
using T7 and T3 mMESSAGE mMACHINE transcription kits (Cat.
No. AM1344 and AM1348, Ambion), respectively. Adult female
Xenopus laevis (RRID:XEP_Xla100; The African Xenopus Facility,
Western Cape, South Africa) were housed in non-toxic plastic con-
tainers equipped with a filtered recirculation system in an aquatic
animal room having temperature and lighting controls. The stock-
ing density was one frog for each 3 liters of water. Frogs were fed
two times a week, and the containers were cleaned one hour after
feeding. For harvesting of oocytes, one frog at a time was used,
and the frog was anesthetized in cold water containing 0.1 g/L of
tricaine (ethyl 3-aminobenzoate methanesulphonic acid, Cat.No.
A5054, Sigma), a commonly used anesthetic for Xenopus laevis,
| 551
before the ovarian lobes were removed to obtain stage V or VI
oocytes. The surgery was performed during the daytime. After
the surgery, the frog was carefully maintained in clean water for
recovering from tricaine at room temperature. To minimize animal
suffering, the surgery was done rapidly (normally less than 10 min)
and with care to avoid damage of the other visceral organs. A Frog
had oocytes collected up to four times, with at least one month
between collections. Following the final collection of oocytes, the
animals were killed by decapitation after anaesthetization with
cold water and tricaine. Totally about 20 frogs were used in this
study. A mixture of GluN1 and GluN2B cRNAs in a ratio of 1:5
(i.e. 0.1–4 ng of GluN1 and 0.5–20 ng of GluN2) was injected into
oocytes (Kashiwagi et al., 2002), which were then maintained in
the culture medium (96 mM NaCl, 2 mMKCl, 1.8 mMMgCl2 , 1.8
mMCaCl2 , 5 mMHEPES, and 50 μg/ml gentamycin, pH 7.6) at
18°C for 2–3 days before electrophysiological recordings. NaCl:
Cat.No. 3624–05, J.T.Baker, Avantor, Radnor, PA, U.S.A.; KCl: Cat.
No. 3040–01, J.T.Baker; MgCl2: Cat.No. M8266, Sigma; CaCl2:
Cat.No. 131,232, PanReac AppliChem ITW Reagents; HEPES: Cat.
No. HB0264, Bio Basic, Toronto, Canada; gentamycin: Cat.No.
G1272, Sigma. All reagents and compounds were purchased on
various occasions from 2015 to 2018.
2.3 | Outside-out patch recordings
Oocytes were placed in a recording chamber containing Mg2+-free,
modified Tyrode's solution (150 mM NaMeSO3, Cat.No. 471348
from Sigma, 5 mMNaCl, and 10 mMHEPES, pH 7.4). Outside-out
patch recordings were obtained using fire-polished pipettes pulled
from borosilicate capillaries (outer diameter, 1.55–1.60 mm; Cat.
No. 1407458, Hilgenberg, Malsfeld, Germany). Recording pipettes
(0.1 ~ 0.2 MΩ) were filled with 150 mMNaMeSO3, 5 mMNaCl,
5 mMEGTA, 10 mMHEPES (pH 7.4). EGTA: Cat.No. 03777, Fluka,
Honeywell, Mexico City, Mexico. A seal was formed, and the out-
side-out patch configuration was obtained in the modified Tyrode's
solution. The patch was then lifted from the bottom of the chamber
and moved in front of a set of “theta glass” tubes (2.0 mm outer di-
ameter pulled to an opening of ~300 μm in width; Cat.No. 804446,
Warner Instrument) emitting either control or drug-containing ex-
ternal recording solutions. The standard external solution was Mg 2+-
free, modified Tyrode's solution (pH 7.4), except for Figure 7 where a
Na+-free Tyrode's solution (a solution containing 150 mM N-methyl-
D-glucamine ions instead of 150 mM Na+) was also used. The glass-
tube holder was connected to a stepper motor (SF-77B perfusion
system, Warner Instrument) to achieve fast switch of the theta
glass tube and get rapid solution change. The rate of solution ex-
change was quantified by the method described previously (Kuo,
Lin, Chang, & Hsieh, 2004), namely the rate of change in current
amplitude between two different external solutions (the Mg2+-free,
modified and the Na+-free Tyrode's solutions mentioned above). For
the theta-glass tube, 10%–90% solution exchange took ~1.8 ms (the
time constant was ≤1 ms when we fitted the fastest phase with a
552 | CHEN et al.
monoexponential function). Macroscopic currents were recorded at
−70 mV (or different voltages if indicated in the figures) using an
Axopatch 200A amplifier, digitized at 20 kHz, filtered at 1 kHz with
a four-pole Bessel filter and stored using a Digidata-1322A analog/
digital interface along with the pCLAMP software (all from Axon
Instruments). All average data are expressed as mean ± SEM. NMDA
(Research Biochemicals International, Cat.No. 508-651-8151), gly-
cine (Sigma, Cat.No. G5417-100G), tetramethylammonium chlo-
ride (TMA; Fluka, Cat.No. 87718), tetraethylammonium bromide
(TEA; Sigma, Cat.No. T-7012) tetrapropylammonium chloride (TPA;
Fluka, Cat.No. 88108), tetrabutylammonium chloride (TBA; Arcos
Organics, Cat.No. 174120250), and tetrapentylammonium chloride
(TpentA; Sigma, Cat.No. 258962-1G) were dissolved in water, and
tetrahexylammonium chloride (ThexylA; Arcos Organics, Cat.No.
420410050), tetraheptylammonium chloride (ThepylA; Fluka, Cat.
No. 87292), and tetraoctylammonium chloride (TOA; Fluka, Cat.
No. 87991) were dissolved in dimethyl sulfoxide (J.T.Baker, Cat.No.
9224–01) to make 100 mMstock solutions. The stock solutions were
then diluted into Mg2+-free modified Tyrode's solution to make the
final concentrations. The final concentration of dimethyl sulfoxide
(~0.1%) was found to have no detectable effect on NMDA currents.
All reagents and compounds were purchased on various occasions
from 2015 to 2018.
2.4 | Concentration–response curves
To study the apparent affinity of TpentA in different processes
(Figures 3 and 5), the concentration–response curves are fitted with
the Hill equation with the Hill coefficient set at 1 (i.e. a one-to-one
binding equation),
Relative sustained current (or Relative initial speed in Figure 5) = I∕Imax
(1)
( ([ ] ))
= 1∕ 1 + TpentA ∕Kd
or
Relative hook current = I∕Imax
([ ] ) ( ([ ] )) (2)
= m TpentA ∕Kd ∕ 1 + TpentA ∕Kd
Where I is the sustained or peak hook NMDA current in the pres-
ence of TpentA, Imax is the maximum NMDA current of the same kind,
[TpentA] is the TpentA concentration, Kd is the apparent affinity of
TpentA to the channel, and m is the maximum for the relative current.
2.5 | Single-channel analysis
Microscopic currents were recorded using an Axopatch 200A am-
plifier with Clampex software and were initially filtered at 2 kHz
using a low-pass Bessel filter and digitized at 10 kHz. All recordings
were performed at −70 mV. Pipettes were pulled from thick-walled
borosilicate glass and fire polished immediately before use. Pipette
resistances were 5–25 MΩ when filled with the pipette solution and
measured in the bathing solution. The level of the bath solution was
kept as low as possible. Records were idealized and analyzed using
the Clampfit software (Version 10.6). Sweeps were rejected when
openings of more than one channel overlapped. Current amplitude
for the two major conductance levels was derived from all-point his-
tograms and defined as the open and closed states. Openings and
closings were determined by the 50% (half of the unitary current
amplitude) threshold method (Colquhoun & Sigworth, 1983). The
durations of the open and closed events were not corrected for the
missed events but rounded up to the closest 0.5 ms precision for the
construction of histograms with a semi-log scale and equally spaced
bins in the X-axis as well as cumulative events (for the bin and be-
yond) in the Y-axis. The histograms were fitted by single or two
exponential functions to give the mean open and (short and long)
closed times. There may be subconductances in the open state, but
they were treated without further discrimination in the analysis.
2.6 | Homology modeling
The NMDA receptor channel homology modeling was performed
according to the X-ray crystal structural data built for the Xenopus
NMDA receptor channel (Lee et al., 2014, PDB ID: 4TlL). Because
the DRPEER motif in rat GluN1 subunit corresponds to the RRPEER
in the Xenopus GluN1 subunit, we replaced the arginine with aspar-
tate before the actual modeling process (Tu et al., 2016). Glycine
and NMDA were also always introduced into the modeling, so that
the simulations were based on a presumable activated/desensi-
tized channel. Molecular dynamics simulation was performed using
Discovery Studio v4.0 programs (Accelrys Inc., San Diego, CA USA)
and the chemistry at Harvard Molecular Mechanics (CHARMm)
force field, and provides a “steepest descent” energy minimization
with positional restraints. Tetraalkylammonium ions in the space-
filling model were also obtained with the same programs, and intro-
duced to the external pore mouth outside the SYTANLAAF motifs.
A second conjugated gradient energy minimization was then per-
formed until no significant energy change could be detected. A con-
stant temperature of 300 K and a constant pressure of 1 bar were
maintained while Berendsen coupling was applied for the simulation
parameters. The van der Waal's force was modeled with a cut-off
value of 10 Å. To integrate the equation of motion, the Leapfrog
Verlet procedure was used. Up to ~25 candidate models were ob-
tained and the one with the lowest potential energy was selected.
2.7 | Statistics
All data are presented as mean ± standard error of mean (SEM).
Sample sizes (n) are number of oocyte patches (without sample calcu-
lation). The experiments were done with heterogeneously expressed
channels in oocytes, and thus no randomization was performed to
allocate subjects in the study. No blinding was performed. The study
was exploratory, and all healthy oocytes successfully expressing the
CHEN et al. | 553

F I G U R E 1 The N-methyl-D-aspartate (NMDA) receptor channel could be transiently opened by NMDA with very low (< 0.05 µM)
ambient glycine. (a) A prominent transient NMDA receptor current is elicited by the application of 300 μM NMDA and decays with a time
constant of ~270 ms. In contrast, NMDA receptor currents could not be elicited by the application of 30 μM glycine. (b) Similar currents are
elicited by 300 μM NMDA plus 0–0.05 μM concomitantly added glycine, all showing fast initial activation but little sustained currents. The
current with 0.1 μM added glycine shows a significant late sustained phase of the currents with similar peak amplitude to those with lower
concentrations of added glycine (left). The sustained currents are more manifest with higher concentrations (e.g., 0.3–30 μM) of added
glycine (right). (c) The currents are normalized to that in 300 μM NMDA and 30 μM glycine to give the relative currents (n = 3–7). Note the
very similar relative peak current and the small sustained current in 0–0.05 μM glycine. Also, although the initial peak currents in 0–0.3 μM
glycine are quite similar, the sustained currents are markedly larger in 0.1–0.3 μM glycine. This could signal the slow macroscopic-binding
rates of glycine at such low concentrations (Tu & Kuo, 2015). n indicates number of oocyte patches

NMDA channels were included in the analysis. However, electro-
physiological data were excluded from analyzes if the amplitude of
the leak currents at −70 mV exceeds 150 pA. Individual data are given
in Figures S6 and S7. Those associated with bar graphs (in Figures 1,
3, 4, 5, and 7) are also given in the bars. For statistical comparison,
non-parametric Wilcoxon signed-rank tests or Friedman tests for
multiple comparisons were used (PASW statistics 18.0, IBM, USA).
Methods for statistical comparison are described in the figure leg-
ends, with a value of p < .05 indicating significant difference.
3 | R E S U LT S
3.1 | A NMDA receptor-mediated current with rapid
decay is elicited by application of NMDA alone
Figure 1a shows that application of only NMDA (in a saturating
concentration of 300 μM, Tu & Kuo, 2015) without addition of
glycine produces a small transient current through the NMDA re-
ceptor. Application of only glycine (in a saturating concentration of
30 μM, Tu & Kuo, 2015), on the other hand, never elicits discerna-
ble NMDA receptor currents. Up to a few tens of nanomolar glycine
is a common contamination even with extreme caution (Kleckner &
Dingledine, 1988; Lerma et al., 1990). Therefore, we added 0.01,
0.03, or 0.05 μM of glycine with 300 μM NMDA, and found that es-
sentially the same transient current is elicited (Figure 1b and c). This
finding suggests that the presence of up to 0.05 μM (plus the back-
ground or “contaminating”) glycine would not affect the gating pro-
cesses that lead to the tiny transient current. In contrast, the NMDA
receptor current peak is significantly increased and a marked late
sustained current develops with co-application of higher concen-
tration of glycine (0.1–30 μM). Both peak and sustained currents
get larger with higher glycine concentrations, consistent with the
conventional feature of “glycine-dependent desensitization” as if
0.1 μM or higher glycine antagonizes desensitization (Johnson &
Ascher, 1992; Kleckner & Dingledine, 1988; Mayer et al., 1989).
554 | CHEN et al.

The very low levels (presumably at least up to 0.05 μM) of glycine,
however, could not have the antagonizing effect so that the elicited
currents show a manifest phase of rapid decay (see below).
3.2 | NMDA receptor desensitization requires co-
activation of GluN1 and GluN2 subunits
In the following experiments, saturating concentrations of
NMDA (300 μM) or glycine (30 μM) will be used unless otherwise
specified. For clarity, we use the term “NMDA alone” to describe
a condition of no added glycine what could be just nominally
glycine-free. We then examined the nature of the rapid decay of
NMDA receptor current elicited by NMDA alone in more detail.
In general, a process of inactivation (for the voltage-gated chan-
nels) or desensitization (for the ligand-gated channels) is defined
as a decrease in opening probability upon prolonged exposure to
activating drives (e.g., voltages or ligands), and signals decreased
responsiveness or availability of the channel to the persistent
drive. After complete decay of the currents with the continuous

F I G U R E 2 The N-methyl-D-aspartate (NMDA) channel could not be desensitized by one single ligand glycine or NMDA. (a) The patches
is consecutively exposed to a pulse of 300 μM NMDA and a pulse of 300 μM NMDA plus 30 μM glycine without any gap in between.
Immediately after a short gap in Tyrode's solution (free of any activating ligands) for 200 ms (top trace) or 550 ms (bottom trace), a second
300 μM NMDA plus 30 μM glycine pulse is applied to elicit current. The peak current in this second pulse is evidently smaller than that
in the first pulse with NMDA plus glycine, and is larger with a longer gap in Tyrode's solution, during which more NMDA channels are
recovered from desensitization (compare the bottom trace to the top trace). The recovery time course from desensitization in Tyrode's
solution is consistent with previous reports (e.g., a recovery time constant of ~540 ms, Tu et al., 2016). A long gap in Tyrode's solution
for 5 s is subsequently applied to recover essentially all desensitized channels, as evidenced by a same-size peak current elicited again by
the following 300 μM NMDA plus 30 μM glycine pulse. (b) After pre-exposure to 300 μM NMDA, the current elicited by an immediate
subsequent pulse of 300 μM NMDA plus 30 μM glycine are essentially the same as that without pre-exposure to NMDA (the relative peak
and sustained currents of 1.0 ± 0.022 and 1.0 ± 0.0050, respectively, n = 4). Refer to Figure 1a for the initial current generated by exposure
to 300 μM NMDA. (c) After pre-exposure to 30 μM glycine, the current elicited by a subsequent pulse of 300 μM NMDA plus 30 μM glycine
are essentially the same as that without pre-exposure to glycine (the relative peak and sustained currents of 1.0 ± 0.0032 and 1.0 ± 0.0024,
respectively, n = 3). Refer to Figure 1a for the lack of any currents generated by exposure to 30 μM glycine. n indicates number of oocyte
patches
CHEN et al.
presence of NMDA alone, co-application of glycine and NMDA
in saturating concentrations always elicits currents with essen-
tially the same amplitude and decay kinetics as that without
pre-exposure to NMDA in the same patch (Figure 2a and b). This
finding strongly argues against that the decay of the transient
currents in the presence of NMDA alone (Figures 1a and 2a) is a
form of desensitization. Moreover, desensitization would ensue
only with a pulse of both NMDA and glycine, and necessitates
a long-enough ligand-free gap to be recovered before the chan-
nel could be activated by NMDA plus glycine again (Figure 2a). In
contrast, deactivation rather than desensitization happens in the
presence of only NMDA, and thus a pulse of NMDA plus glycine
readily elicits full currents even if there is no ligand-free gap in
between (Figure 2b). Similar cases were found with continuous
pre-exposure to glycine alone (Figure 2c). These findings suggest
that substantial co-activation of GluN1 and GluN2 subunits (by
binding of glycine and NMDA, respectively) may be a prerequisite
of channel desensitization. The channel opened by “only” NMDA
actually rapidly undergoes a novel form of deactivation rather
than desensitization because it is fully available for subsequent
activation. In this regard, the “glycine-dependent desensitiza-
tion” is then in fact glycine-dependent activation, which increases
peak and sustained currents simultaneously with 0.1 to 30 μM of
glycine (Johnson & Ascher, 1992; Kleckner & Dingledine, 1988;
Mayer et al., 1989). The apparent different kinetics in the deac-
tivation phase in Figure 2b and c are because of the much slower
unbinding of glycine than NMDA from the channel upon washing
off the ligands. This is further demonstrated in Figure S1.
3.3 | TPentA precludes nmda receptor
desensitization and keeps the same open state as that
in control
We then designed a series of biophysical approaches taking tetrap-
entylammonium ions (TpentA) (Sobolevsky, 2000; Sobolevsky et
al., 1999) as probes to dissect the molecular nature of NMDA
receptor desensitization. 30–1000 μM TpentA applied extracel-
lularly with NMDA plus glycine inhibit the elicited NMDA recep-
tor currents in a dose-dependent fashion (Figure 3a and b). The
inhibition develops faster with higher concentrations of TpentA,
consistent with a pore-blocking effect. Notably, upon washing-off
of both the activating ligands (i.e. NMDA and glycine) and TpentA,
a prominent “hook” current is produced with its peak roughly the
same size of the sustained current in control (i.e., in the absence
of TpentA, Figure 3c). TpentA thus should not significantly facili-
tate the open-to-desensitized state transition, in which case the
hook current should be significantly smaller than the sustained
current in control. The concentration–response curves for the sus-
tained currents and for the increase in the hook currents from the
sustained levels (Figure 3b and c) are almost mirror images with
very similar IC 50 or EC 50 values at ~50 μM, suggesting that inhi-
bition of sustained currents and generation of hook currents are
| 555
consequences of the same molecular event. Moreover, the kinetics
of the decay phase of hook currents are independent of TpentA
concentrations, and are similar to the channel deactivation kinet-
ics in control (Figure 3d). These findings imply that TpentA unbinds
first and thus generates the hook currents without significantly
changing the open state conformation. Moreover, the existence or
binding of TpentA in the pore seems to preclude channel desensiti-
zation, considering the relatively large peak amplitude (comparable
to the sustained current in control) and the relatively slow rising
time (signaling the unbinding rate of TpentA from the open chan-
nel, also see Figure 4c) of the hook current. These ideas were fur-
ther verified by washing off TpentA in the continuous presence of
NMDA plus glycine (Figure 3, e and f). Unblock of TpentA produces
transient hook currents larger than the sustained current (the cur-
rent after desensitization by NMDA plus glycine), indicating that
TpentA not only blocks the open channels but also keeps the chan-
nel in the open conformation (by precluding the development of
desensitization). Moreover, the hook currents decay (desensitize
in the presence of NMDA plus glycine) with a rate independent
of TpentA concentrations and essentially the same as the macro-
scopic desensitization rate of the NMDA receptor currents in the
absence of TpentA (Figure 3a, e, and f). Thus, the same open state
is very likely reached after washing-off of TpentA and then relaxes
toward the same desensitized state.
3.4 | TPentA does not alter the mean open and
(short) closed times of the NMDA receptor channel
The same unitary current amplitude is obtained in the early
decay and late sustained phases of the NMDA currents in con-
trol (TpentA-free condition), as well as immediately after wash-
off of TpentA in the presence of NMDA plus glycine (Figure 4a).
Also, the mean open time and the mean closed time immediately
after wash-off of TpentA are similar to that upon exposure to the
activating ligands (glycine plus NMDA) in the absence of TpentA
(Figure 4b). Note that the latency to the first open event after
wash-off of TpentA is consistent with the time to the macroscopic
hook current peak (Figure 4c). In the long sustained currents in the
continuous presence of NMDA and glycine (but not TpentA), the
open and the short closed times, presumably the dwelling time in
the open and closed state in the steady-state condition, respec-
tively (Figure S2, where the long closed events probably signal the
dwelling time in the desensitized state), are also very similar to
that in Figure 4b. These findings argue against that the genesis of
hook currents are consequences of permeation changes such as
augmented unitary conductance of the channel. Instead, TpentA
apparently prohibits desensitization and “keeps” the channel in
the same open state as that in control. Mechanistically, prohibi-
tion of desensitization by TpentA could be based on either sta-
bilization of the open state or destabilization of the desensitized
state. The experimental data, especially the unchanged open
time, clearly indicate a molecular mechanism of the latter than the
556 | CHEN et al.
CHEN et al. | 557

F I G U R E 3 TpentA dose-dependently inhibits N-methyl-D-aspartate (NMDA) currents and gives rise to a hook phase after its wash-
off. (a) NMDA currents are elicited by application of 300 μM NMDA plus 30 μM glycine with different concentrations of TpentA. Note the
currents abruptly increase upon wash-off of TpentA, NMDA, and glycine, and then return to the zero level (i.e., the “hook” currents). The
“hook” currents are replotted in the right panel to better demonstrate the current amplitude. (b) The relative sustained current, defined by
the ratio between the amplitude of the late sustained currents (the average current amplitude between 2 and 2.5 s of the pulse) in TpentA
and that in control (TpentA-free condition) is plotted against the TpentA concentration (n = 3–6). The solid line is the best fit to the data
points using Equation 1 (see Materials and Methods) with a Kd of 50.0 μM. (c) The relative hook current (measured from the sustained level)
to that in 100 μM of TpentA is plotted against the TpentA concentration (n = 3–6). The solid line is the best fits to the data points using
Equation 2 with a Kd of 61.1 μM. (d) Monoexponential fits to the decay phase of the hook currents in part a give similar time constants with
30–300 μM TpentA and slightly faster (deactivation) time constant in control (each n = 3). (e) A hook current is elicited by wash-off of TpentA
in the continuous presence of 300 μM NMDA plus 30 μM glycine. The dashed line indicates the current level of the continuous presence of
300 μM NMDA plus 30 μM glycine without TpentA. (f) The time constants of the decay phase of the hook currents in part e and the time
constant of macroscopic desensitization of the NMDA currents in control from part a (n = 6). n indicates number of oocyte patches

former. Moreover, the destabilization of the desensitized state by
TpentA is very likely a simple hindrance of closure of the desensi-
tization gate (e.g., a “foot-in-the-door” effect) rather than a more
complicated form of conformational modification (e.g., generation
of new gating states).
3.5 | TpentA binds to the closed and open NMDA
receptor channels equally well
If external TpentA binds to the pore to preclude desensitization with-
out altering the open conformation, then it would be desirable to ex-
amine the possibility of differential affinity of TpentA toward the open
and the closed states considering that the activation gate is located at
the external pore vestibule (Chang & Kuo, 2008a). After pre-treatment
of TpentA, the initial rising phase of the NMDA receptor currents elic-
ited by a pulse of NMDA plus glycine is slowed in a TpentA concentra-
tion-dependent fashion (Figure 5a, note that TpentA is present only
before but not during the NMDA plus glycine pulse). This finding dem-
onstrates that TpentA also blocks the closed channel pore, and ion per-
meation could ensue only when TpentA leaves the channel. Moreover,
the slower onset of the currents is always associated with smaller peak
current (and thus apparently decreased fraction of desensitization as
the steady-state level of the currents remains the same, Figure 5b),
consistent with an idea that the macroscopic peak amplitude is deter-
mined by kinetically-“coupled” sequential conformational changes in
channel activation and desensitization. The concentration–response
curve in Figure 5c shows an apparent binding affinity very similar to
that of TpentA binding to the open channel in Figure 3. TpentA there-
fore binds to the closed and the open channels with quite the same
affinity. Again, these data support the foregoing view in Figures 3 and
4 that TpentA binds to the channel pore to preclude ion permeation
as well as desensitization without induction of novel conformational
changes in the channel protein itself.
would then be interesting to explore whether TpentA acts on GluN1
or GluN2 to prohibit desensitization. We have found that movement
of only GluN2 by application of NMDA alone produces transient cur-
rents that intriguingly decay to a new non-conducting state but do not
desensitize (Figure 1). Similar NMDA (alone)-elicited transient currents
can be obtained with pre-treatment of TpentA, TpentA plus NMDA, or
TpentA plus NMDA plus glycine (Figure S4a). The new non-conducting
state generated with the continuous presence of NMDA alone is also
prohibited by TpentA, so that a hook current is produced by wash-off
of TpentA (middle trace of Figure S4a). The unblocked open channel
then enters the same non-conducting state in the presence of NMDA
alone with the same decay kinetics as that without TpentA (Figure S4b).
TpentA thus very likely interacts with GluN2 movement and keeps
GluN2 in the same open conformation. In the continuous presence of
NMDA and glycine, which move both GluN1 and GluN2 to a activated/
desensitized state, wash-off of only glycine leads to a slow decay of
the current presumably because of unbinding of glycine and deactiva-
tion of GluN1 (Figure 2b). If TpentA is applied to the activated/desen-
sitized channel driven by NMDA and glycine (bottom trace of Figure
S4a), wash-off of glycine and TpentA leads to a decay of the current
faster than the case without TpentA (Figure 2a) and slower than the
case without glycine (middle trace of Figure S4a, and see also Figure
S4b). This finding suggests that TpentA also alters the movement of
GluN1. TpentA therefore seems to have an effect on the movement
of both GluN1 and GluN2. Moreover, a hook current is produced with
unaltered decay kinetics by wash-off of TpentA either from the acti-
vated GluN2 only (Figure S4a and b) or from the co-activated GluN1
and GluN2 (Figure 3e and f). The new non-conducting state generated
by activated GluN2 alone, although not functionally desensitized, may
share part of the conformation of the desensitized state of the channel
(see Discussion).
3.7 | ThexylA and TheptylA could not, and tea could
only partially, preclude channel desensitization

3.6 | TpentA has an effect on the movement of both
GluN1 and GluN2
We have argued that NMDA receptor desensitization requires ac-
tivation (movement) of both GluN1 and GluN2 (Figures 1 and 2). It
We then probed the gating conformational changes in the external
pore vestibule mouth with other tetraalkylammonium (TAA) ions
(Figure 6). The diameters of tetramethylammonium (TMA), tetraethyl-
ammonium (TEA), tetrapropylammonium (TPA), tetrabutylammonium
(TBA), and tetrapentylammonium (TpentA) are roughly ~7, 8, 9, 10,
558 | CHEN et al.

F I G U R E 4 The N-methyl-D-aspartate (NMDA) receptor channel desensitizes after wash-off of TpentA with the same unitary currents
as that in control. (a) Single channel sweeps for the first 400 ms after exposure to 300 μM NMDA plus 30 μM glycine (without TpentA,
left traces, the “control” group), and for the first 400 ms after wash-off of only TpentA but not 300 μM NMDA plus 30 μM glycine (middle
traces, the “TpentA” group). This observation time window is made as short as 400 ms considering that the effect of TpentA on channel
conformation may fade more with longer period after TpentA wash-off. Note the short openings before wash-off of TpentA in the latter
condition, indicative of transient binding configuration change or a relatively “loose” binding of TpentA. These transient openings would
never exceed ~5 ms in duration. We therefore start the 400 ms period for gating analysis from the first open event longer than 5 ms after
wash-off of TpentA to avoid contamination of the closed time by the off time of TpentA. (c) and (o) denote the closed and open states of
the channel, respectively. The unitary currents are very similar among these conditions (n = 5–6, right panel). (b) The mean open time and
the mean closed time in the first 400 ms of exposure to 300 μM NMDA plus 30 μM glycine (without exposure to TpentA, left two panels,
respectively) are very similar to that in the first 400 ms after wash-off of only TpentA (right two panels, respectively) (14 and 13 samples
from 4 oocyte patches in each conditions). (c) The time to peak after wash-off of only TpentA in the continuous presence of 300 μM NMDA
plus 30 μM glycine in Figure 3e (n = 6) is rather similar to the first latency (from wash-off to the first open event longer than 5 ms, n = 6) in
part a, demonstrating that it takes ~100 ms for the bound TpentA to unbind from the channel after wash-off. n indicates number of oocyte
patches

and 11 Å, respectively (Robinson & Stokes, 1968), but the alkyl chain
tends to be more flexible (adaptable to the microenvironment) with
the increase in length. 100 µM TBA has a comparable effect on the
sustained currents and the generation of hook currents to 100 µM
TpentA (Figure 6a and c). Consistent with the idea that the two TAA
ions effectively preclude channel desensitization (e.g. the closure of
the desensitization gate), the recovery from desensitization (e.g. open-
ing of the desensitization gate) tends to be faster in the presence of
TBA and TpentA (Figure S3). 100 µM TPA and TEA (not shown), on
the other hand, have only a negligible effect on the sustained currents
with no discernible hook currents upon wash-off. However, if the con-
centration is increased to 1 and 10 mM, respectively, TPA and TEA
(but not 10 mM TMA) both could also substantially block the sustained
currents (Figure 6b and d). Notably, despite of the similar blocking ef-
fect on the sustained currents, the hook currents are markedly smaller
in 10 mM TEA than in 1 mM TPA. Binding of TAA ions to the exter-
nal pore vestibule of the NMDA receptor channel therefore is likely
dependent on hydrophobic interactions between the alkyl chains and
CHEN et al. | 559

F I G U R E 5 TpentA binds to the closed and open N-methyl-D-aspartate (NMDA) receptor channel with similar affinity. (a) The patch is
moved from the NMDA- and glycine-free external solution containing 0 (control) to 1,000 μM TpentA to the external solution containing
300 μM NMDA plus 30 μM glycine without TpentA. (b) Pre-treatment of TpentA dose-dependently decreases apparent desensitization in
macroscopic NMDA currents (n = 3– 7). (c) The initial speed is defined as the slope of current increase between 20 and 50 ms after switch
of the external solution (from TpentA to NMDA plus glycine) in part a, and is normalized to the initial speed in control in the same patch to
give the relative initial speed (n = 3). The solid line is the best fit to the data points using Equation 1 with a Kd value of 62.0 μM. n indicates
number of oocyte patches

the channel wall, so that the binding affinity is positively correlated
with the length of the alkyl chain (and thus TMA in a concentration
up to 10 mMcould not have any significant interaction with the pore).
The same pore-blocking but differential effect on hook currents be-
tween 1 mM TPA and 10 mM TEA would further implicate that the
alkyl chains also play a critical role in the prohibition of desensitization.
Also, channel desensitization could in essence be a rather restricted
change in the pore caliber so that a difference in the alkyl chain by
just one methylene group matters. 100 µM ThexylA and TheptylA
larger than TpentA readily block essentially all of the sustained cur-
rents (Figure 6a and c), well consistent with the view that the length of
the alkyl chain is positively correlated with the affinity of external TAA
ions to the NMDA receptor channel pore. However, there is barely any
discernible hook current after wash-off of ThexylA or TheptylA, which
is just slightly larger than TpentA. Even larger TOA ion could neither
block the sustained currents nor generate hook currents. It is plausi-
ble that ThexylA and TheptylA bind to the external vestibule at a site
located too externally to interact effectively with the desensitization
gate. TOA may take an even more external position where the exter-
nal pore vestibule has a wide-enough caliber, and thus fail to block
ion permeation. These findings are also consistent with the view that
NMDA receptor desensitization involves a highly restricted pore re-
duction external to the activation gate.
3.8 | The desensitization gate is located ~2Å internal
to a weak flux-coupling external pore region and with
an electrical distance close to ~0.7 from the outside
The delicately size-dependent effect of different TAA ions on NMDA
receptor desensitization strongly indicates a fairly localized or focal in-
teraction. We therefore endeavored to locate the binding site of TAA
ions and thus the desensitization gate in more detail. Figure 7a shows
that the blocking effect of TPentA is voltage-dependent, consistent
with a blocking site in the pore. The apparent Kd at different mem-
brane voltages is deduced based on a one-to-one binding process, and
560 | CHEN et al.
CHEN et al. | 561

F I G U R E 6 The inhibitory effect on the sustained currents could be dissociated from the generation of hook currents for different
tetraalkylammonium (TAA) ions. (a) N-methyl-D-aspartate (NMDA) currents are elicited by application of 300 μM NMDA plus 30 μM glycine
in the absence (control) and presence of 100 μM TAA ions. (b) The same experiments in part a were repeated in higher concentrations of
tetrapropylammonium (TPA), tetraethylammonium (TEA), and tetramethylammonium (TMA). (c) and (d) The relative sustained current (blue
symbols) is defined as the ratio between the amplitude of the late sustained currents (the average amplitude between 1 and 1.5 s of the
pulse, blue dots) with 100 μM (part c) or 1–10 mM(part d) TAA ions and that in the control (TAA-free) condition. The relative hook current
(black symbols) is defined by the ratio between the peak hook current (after wash-off of TAA ions and activating ligands) and the sustained
current in the control TAA-free condition in the same patch (n = 3–4). n indicates number of oocyte patches

is found to change e-fold per ~33 mV (Figure 7b and c). This finding lo-
cates the charged nitrogen (N) atom of TpentA at an electrical distance
of ~0.7 from the outside. If the interaction between the TAA ions and
the NMDA receptor channel pore is chiefly hydrophobic (Figure 6), 0.7
is just a rough estimate given the possibilities of changes in binding con-
figuration of the charged N atom as well as the alkyl chains at different
membrane voltages. In any case, the nearly identical voltage depend-
ence between 1 mM TPA and 100 µM TPentA may indicate a common
binding position of the N atoms in these two blockers. It is of note that
this binding position of the N atom may be very close to or even colo-
calized with the blocking position of external Mg2+ (electrical distance
~0.7 from the outside, Yang, Lee, & Kuo, 2010). Moreover, the inhibitory
effect of TPA is significantly decreased if the external Na+ ions are re-
placed with N-methyl-D-glucamine (NMG) ions to turn the current flow
from inward to outward at −40 mV. The inhibitory effect of TpentA is
also decreased with the outward currents, but to a smaller extent than
TPA (Figure 7d and e). The differential inhibitory effects of TPA and
TpentA with preponderant outward and inward ionic flow at the same
membrane voltage would strongly implicates that the blocking site of
the charged N atom are located at or near, and functionally coupled to,
a flux-coupling region. There has been only one flux-coupling region re-
ported for the external vestibule of the NMDA receptor channel so far,
a region which is coupled to the movement of the external Mg2+ (Yang
et al., 2010). The flow-dependent inhibitory effect would therefore lend
a further support not only for a blocking site of the TAA ions in the pore
but also for a colocalization of the sites for the positively charged N
atoms and external Mg2+. The flux-coupling feature is more apparent
with TPA as if the ~2Å longer alkyl chains in TpentA may have a more
complete “coverage” of the flux-coupling region and more disruption
of the flux-coupling feature (see Figs. S5 and 8). Taken together, these
findings suggest that the single positive charge in the N atom of TPA
and TpentA may reach the same site in the external pore vestibule (of
an electrical distance of as much as ~0.7 from outside), with the alkyl
chains splayed externally to have hydrophobic interactions with the
channel wall. The hydrophobic interactions may in turn have a delicate
local effect on not only channel gating but also ion permeation.
4 | D I S CU S S I O N
4.1 | TpentA prohibits channel desensitization by a
direct “foot-in-the-door” rather than an allosteric effect
We have shown that TpentA renders more NMDA channels to stay
in the open (although blocked) rather than the desensitized state in
the continuous presence of glycine plus NMDA (e.g. the hook currents
even larger than the sustained current in control in Figure 3e). We have
also demonstrated similar Kd values of TpentA for the production of
hook currents and for the inhibition of sustained currents (Figure 3b
and c). Moreover, there are very similar kinetics between the hook cur-
rent decay (upon wash-off of both TpentA and glycine/NMDA) and
macroscopic deactivation (Figure 3d), and between the hook current
decay (upon wash-off of only TpentA) in the continuous presence of
glycine/NMDA and macroscopic desensitization (Figure 3e and f). In
addition, the unitary current amplitude as well as the single-channel
open and closed times immediately following wash-off of TpentA are
all very similar to that in control condition (Figure 4a–c). The open state
immediately after wash-off of TpentA thus is the same as the open
state directly activated by glycine plus NMDA. Also, it is unlikely that
the major closed conformation is significantly altered by TpentA bind-
ing. TpentA thus most likely prohibits desensitization with a simplistic
action such as “foot-in-the-door” (Armstrong, 1971; López-Barneo,
Hoshi, Heinemann, & Aldrich, 1993), rather than stabilization of a new
open state. The immediate resumption of the original open conforma-
tion and gating kinetics after unbinding of TpentA would also impli-
cate that desensitization is chiefly a conformational change directly
continual (coupled) to channel activation or opening. Consistently,
it is of note that TpentA binds to the closed and open channels with
very similar affinity (Figure 5), indicating an open desensitization gate
in the presence of a closed activation gate. This is reminiscent of the
molecular mechanisms underlying inactivation of voltage-gated chan-
nels (Bezanilla & Armstrong, 1977; Armstrong and Bezanilla, 1997), and
well illustrates an intriguing evolutionary kinship in the rationales of ion
channel gating.
4.2 | Desensitization denotes a highly restricted
pore reduction in the bundle-crossing region deep
in the external pore vestibule
Different TAA ions tend to have similar chemical properties, with
increasing molecular size and decreasing charge density as the alkyl
chains get longer. From TMA to TpentA, the diameter of TAA mole-
cules roughly increases by ~1 Å for each added methylene group (from
~7 to ~11 Å, Robinson & Stokes, 1968). TAA ions thus were applied to
probe the dimension of pore entrances of different channels (Guo & Lu,
2001; Guo et al., 2003; Sobolevsky, 2000; Sobolevsky et al., 1999). For
example, the blocking effect on Kir2.1 channels is the highest for TEA
and gradually decreases toward TpentA, implicating an internal pore
mouth no smaller than ~12 Å (Guo & Lu, 2001). We have estimated the
562 | CHEN et al.
CHEN et al. | 563

F I G U R E 7 The blocking effects of TpentA and tetrapropylammonium (TPA) show the same voltage dependence but distinct flow
dependence. (a) N-methyl-D-aspartate (NMDA) currents are elicited by application of 300 μM NMDA plus 30 μM glycine in the absence
(control) and presence of 100 μM TpentA or 1 mM TPA ions at different membrane voltages (−70 to + 20 mV). (b) The cumulative results of
the relative current, defined by the ratio between the amplitude of the late sustained currents (the average current amplitude between 2
and 2.5 s of the pulse) in 100 μM TpentA or 1 mM TPA and that in control in part a. (c) The apparent dissociation constants (Kd) derived from
the mean relative currents in part b based on a one-to-one binding reaction and the concentration of the blocker are plotted against the
membrane voltages (n = 3). The solid lines are the best fits to the data points with a slope of an e-fold change per ~ 33 mV for both cases.
(d) and (e) The same experiments as those in part a at −40 mV but with the Na+-free Tyrode's solution (designated as NMG, see Materials
and Methods) as the external solution so that the currents are now outward. The cumulative results of the relative currents show a larger
difference between the inhibitory effect on inward and outward currents (at the same voltage of −40 mV) in the case of TPA than TpentA
(**p = .0023 and ***p = .00037 for the cases of TPA and TpentA, respectively, Wilcoxon signed-rank test, each n = 6). n indicates number of
oocyte patches

diameter of the open activation gate at the bundle-crossing point, pre-
sumably deep in the external vestibule at A652(GluN1)/A651(GluN2),
as ~17 Å (side chain tip to tip, Chang & Kuo, 2008a). Consistently, the
external vestibule of the NMDA channel pore is wide enough even for
TOA (roughly of a diameter of 14 Å). Molecular dynamic simulations
also support that the external vestibule of the NMDA channel could
accommodate TMA to TOA (Figure S5), although the binding configu-
ration may be different according to the optimal hydrophobic interac-
tions (Figures S5 and 8). Among TAA ions, ThexylA and TheptylA have
the strongest pore-blocking but negligible desensitization-prohibiting

F I G U R E 8 Schematic drawings illustrate the interaction between the desensitization gate and different tetraalkylammonium (TAA) ions
in the external pore mouth of the N-methyl-D-aspartate (NMDA) receptor channel. Tetramethylammonium (TMA) to tetraoctylammonium
(TOA) (blue) could be accommodated by the external vestibule of the NMDA receptor channel (see also the molecular dynamic simulations
in Figure S5). Tetraethylammonium (TEA) to TheptylA block the pore with increasing potency positively correlated with the length of the
(alkyl) side chain. The binding affinity between TAA ions and the channel pore thus is most likely contributed by the hydrophobic interaction
between the alkyl side chain and the peptide chain. The electrostatic force probably plays only a minor role, although the positively
charged N atom (indicated by a cross) does go deep into the pore in terms of the electrical distance (Figure 7). The smallest TMA could
not effectively bind to the pore to block ionic passage. The largest TOA could not block the pore either, presumably because the most
favorable hydrophobic interaction between the alkyl chain of TOA and the channel pore do not allow a deep-enough binding position to
have a pore-blocking effect. Similar cases are noted for TpentA and ThexylA (Figure 6c), although the N atom in ThexylA is located slightly
more externally for an optimal hydrophobic interaction. TEA to TpentylA could block the pore with the positively charged N atom reaching
roughly the same deep position (electrical distance ~0.7 from the outside, probably right external to the SYTANLAFF motif or the activation
gate, see Figure S5 and Discussion). 10 mM TEA and 1 mM tetrapropylammonium (TPA) show the same blocking effect on the sustained
currents but distinct hook-generating effects, whereas 1 mM tetrapropylammonium (TPA) shows comparable hook currents to 100 μM
TpentA (Figure 6d). These findings suggest that channel desensitization is prohibited by the binding of the alkyl chains rather than the N
atom, and is a highly restricted conformational change within ~4 Å from the N atom (see Discussion)
564 | CHEN et al.
effect (Figure 6). TOA abruptly turns ineffective, as if TAA ions must
secure a deep-enough position to have a pore-blocking effect. With
just a small difference in the length of the alkyl chain (~1 Å) between
TpentA and ThexylA, however, the marked difference in the hook
currents after wash-off of TpentA and ThexylA (as well as TheptylA,
Figure 6c) are unlikely because of an extraordinarily slower unbind-
ing rate of ThexylA than TpentA. The small difference (presumably no
more than a few angstroms) of the blocking positions between TpentA
and ThexylA very likely determines the direct “foot-in–the-door” pro-
hibiting effect on desensitization. Consistently, the molecular mode-
ling data show that only TpentA or smaller TAA ions but not ThexylA or
larger TAA ions could touch the activation gate at the bundle-crossing
region demarcated by the SYTANLAAF motif (Figure S5). The desensi-
tization gate thus should be located external to but within a few ang-
stroms of the activation gate (i.e. a very small area covered by the
bound TpentA but not ThexylA, Figure 8). Moreover, 1 mM TPA and
10 mM TEA have roughly the same pore-blocking effect, but only the
former has a comparable hook-generating effect to TpentA, indicating
that the desensitization gating conformational changes involve only up
to a range of ~4 Å from the N atom (in TEA to TpentA) in the pore.
NMDA channel desensitization therefore is likely a highly restricted
conformational change co-localized with the activation gate in the
bundle-crossing region.
4.3 | NMDA channel desensitization could
proceed only with co-activation of GluN1 and GluN2
subunits and is thus both glycine- and NMDA-
dependent
Glycine was considered to have an antagonistic effect on NMDA
channel desensitization (“glycine-dependent desensitization”,
Benveniste et al., 1990; Cummings & Popescu, 2015; Lerma et al.,
1990; Mayer et al., 1989; McBain & Mayer, 1994; Vyklicky et al.,
1990). The desensitization that remains manifest in saturating con-
centrations of glycine is therefore defined as glycine-independent
desensitization (Benveniste et al., 1990; Chen et al., 2004; Hu &
Zheng, 2005a; Lerma et al., 1990; Lester et al., 1993; Mayer et al.,
1989; McBain & Mayer, 1994; Nahum-Levy et al., 2001; Regalado
et al., 2001; Sessoms-Sikes et al., 2005). We have seen that NMDA
“alone” (with 0.05 μM or lower ambient glycine) could elicit small
transient currents through the NMDA channel (Figures 1 and 2a).
The currents decay almost completely in the continuous presence
of NMDA, seemingly a form of desensitization but in fact a novel
form of deactivation, as a subsequent NMDA plus glycine pulse al-
ways evokes full currents (Figure 2). This novel form of deactiva-
tion could be envisaged with a gating scheme with the gate proper
directly served by only GluN2, which tends to move more rapidly
than GluN1 (see Tu and Kuo, 2015). If GluN1 is not activated, the
activated GluN2 with binding of NMDA will be distributed to a
new closed conformation with a time constant of ~200 ms via an
open conformation (Figure 1). The open conformation is thus a rela-
tively high-energy conformer in this situation and only transiently
trespassed during the process of distribution to the new closed
state (and thus no sustained current in this case). If enough glycine
is added to activate GluN1 at this moment, GluN2 would be quickly
moved “back” to the open conformation (Figure 2), which is one of
the lowest-energy conformers, but not the only one, in this new
situation. A portion of GluN2 would then relax to a new non-con-
ducting conformation (the desensitized state) with a time constant
of ~400 ms (Figure 1). NMDA currents do not decay to a level of
zero with desensitization. The free energy levels of the open and
the desensitized states with fully activated GluN1 and GluN2 thus
should not be very different, although the intervening energy bar-
rier between the two states seems to be significant, making the
relatively slow transition rates between the two states (i.e. the
slower macroscopic desensitization than the activation rates). It is
thus plausible that the open and desensitized conformations may
be fundamentally similar and are both made by restricted as well as
small-scale peptide movement at the bundle-crossing region, but
with energetically unstable transitional intermediates in between.
This could illustrate a basic molecular picture of formation, disrup-
tion, and formation again of multiple different bonds at the contact
points between bundles at the crossing region, and is also compat-
ible with the new closed state generated by fully activated GluN2
in the presence of a relatively deactivated GluN1. In any case, the
desensitized conformation of GluN2 is feasible only with an acti-
vated GluN1. NMDA channel desensitization thus is dependent on
co-activation of both GluN1 and GluN2 subunits, or the presence of
both glycine and NMDA, just like activation.
AC K N OW L E D G E M E N T S
The authors are grateful to the Neuroscience Research Center of
Chang Gung Memorial Hospital, Linkou, Taiwan.
COMPETING INTERESTS
The authors declare no financial conflicts of interest.
ORCID
Ya-Chin Yang https://orcid.org/0000-0002-9983-0607
Chung-Chin Kuo https://orcid.org/0000-0001-8208-6829
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S U P P O R T I N G I N FO R M AT I O N
Additional supporting information may be found online in the
Supporting Information section.
How to cite this article: Chen Y-S, Tu Y-C, Lai Y-C, Liu E, Yang
Y-C, Kuo C-C. Desensitization of NMDA channels requires
ligand binding to both GluN1 and GluN2 subunits to constrict
the pore beside the activation gate. J. Neurochem.
2020;153:549–566. https​://doi.org/10.1111/jnc.14939​