A Site in the Fourth Membrane-associated Domain of the
N-Methyl-D-aspartate Receptor Regulates
Desensitization and Ion Channel Gating*
Hong
Ren,
Yumiko
Honse,
Brian J.
Karp,
Robert H.
Lipsky, and
Robert
W.
Peoples
From the Unit on Cellular Neuropharmacology, Laboratories of
Molecular and Cellular Neurobiology and Neurogenetics, National
Institute on Alcohol Abuse and Alcoholism,
Bethesda, Maryland 20892-8115
Received for publication, September 16, 2002, and in revised form, October 28, 2002
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ABSTRACT |
The N-methyl-D-aspartate
(NMDA) receptor has four membrane-associated domains, three of which
are membrane-spanning (M1, M3, and M4) and one of which is a re-entrant
pore loop (M2). The M1-M3 domains have been demonstrated to influence
the function of the ion channel, but a similar role for the M4 domain
has not been reported. We have identified a methionine residue
(Met823) in the M4 domain of the NR2A subunit that
regulates desensitization and ion channel gating. A tryptophan
substitution at this site did not alter the EC50 for
glycine or the peak NMDA EC50 but decreased the
steady-state NMDA EC50 and markedly increased apparent
desensitization, mean open time, and peak current density. Results of
rapid solution exchange experiments revealed that changes in
microscopic desensitization rates and closing rates could account for
the changes in macroscopic desensitization, steady-state NMDA
EC50, and current density. Other amino acid substitutions
at this site could increase or decrease the rate of desensitization and
mean open time of the ion channel. Both mean open time and
desensitization were dependent primarily upon the hydrophobic character
of the amino acid at the position. These results demonstrate an
important role for hydrophobic interactions at Met823 in
regulation of NMDA receptor function.
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INTRODUCTION |
The N-methyl-D-aspartate
(NMDA)1 receptor is a subtype
of ionotropic glutamate receptor that plays essential roles in
neuronal development, synaptic plasticity, and several types of
neurological disorders (1). NMDA receptors are heteromers containing
NR1 subunits, which bind the co-agonist glycine (2, 3), and NR2
subunits, which bind the agonist glutamate (4, 5). The agonist-binding
domains of all ionotropic glutamate receptor subunits consist of two
lobes (S1 and S2) that are formed by the region of the extracellular
N-terminal domain preceding the first membrane-associated (M) domain
and the loop between the M3 and M4 domain, respectively (5-8), and
that together form a clamshell structure that undergoes a
conformational change to enclose the ligand upon binding (6, 9, 10).
Observations from x-ray crystallographic studies on non-NMDA glutamate
receptor constructs suggest that the degree of binding domain closure
induced by a particular agonist appears to determine both the degree of
receptor activation and the extent of desensitization produced by that
agonist (10) and that desensitization in these receptors results from
the dissociation of dimers of the ligand-binding domains of adjacent
subunits (11). NMDA receptors exhibit apparent desensitization
sensitive to glycine or intracellular Ca2+ (1, 12) in
addition to true glycine-insensitive desensitization (subsequently
referred to simply as desensitization). NMDA receptor desensitization
is physiologically relevant, as it can influence the amplitude,
duration, and following frequency of NMDA receptor-mediated synaptic
events (13-16). Determinants of NMDA receptor desensitization have
been localized to two regions in or near the S1 ligand-binding site in
the N-terminal domain, one that shows homology to
leucine/isoleucine/valine-binding proteins and one located in the
region immediately preceding the M1 domain (17, 18), as well as to a
highly conserved motif (YTANLAAF) in the C-terminal portion of the M3
domain preceding the S2 ligand-binding lobe (19). The pre-M1 and M3
domains have been suggested to be involved in transducing the
conformational changes induced by agonist binding into those
responsible for ion channel gating (17, 19), but the nature of the
conformational changes and molecular determinants underlying NMDA
receptor ion channel gating remain unclear. The YTANLAAF motif in M3
has been shown to play an important role in the regulation of ion
channel gating, as a point mutation in this region of the NR1 subunit increases mean open time in receptors formed from coexpression with
NR2A subunits (19). Mutations at a tryptophan residue in the M2 domain
of the NR1 or NR2A subunit have also been reported to subtly affect
mean open time and opening frequency (20). We report here that a
residue in the M4 domain of the NR2A subunit exerts a powerful
regulatory influence on the desensitization and gating of the NMDA
receptor ion channel.
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EXPERIMENTAL PROCEDURES |
Mutagenesis, Transfection, and Cell Culture--
Site-directed
mutagenesis in plasmids containing NR1-1a and NR2A subunit cDNA
(Drs. D. R. Lynch, University of Pennsylvania; and D. M. Lovinger, Vanderbilt University) was performed using the QuikChange kit
(Stratagene, La Jolla, CA), and all mutations were verified by DNA
sequencing. Human embryonic kidney (HEK) 293 cells were seeded in 35-mm
dishes, allowed to grow to 70-95% confluence, and transfected with
cDNA for the wild-type or mutant NR1 and NR2A subunits and green
fluorescent protein at a 2:2:1 ratio, respectively, using LipofectAMINE
2000 or calcium phosphate (both from Invitrogen). The culture medium
during and after the transfection step contained 100 µM
ketamine and 200 µM
DL-2-amino-5-phosphonovaleric acid to minimize cell death
due to excitotoxicity. Cells were used in experiments 18-72 h after transfection.
Western Blot Analysis--
Biotinylation and membrane
solubilization were performed as described by Chen et al.
(21) and Huh and Wenthold (22), respectively. Transfected HEK 293 cells
were washed with phosphate-buffered saline and incubated with the
membrane-impermeable reagent sulfosuccinimidyl 2-(biotinamido)ethyl-1,3'-dithiopropionate (1.5 mg/ml; Pierce) in
Buffer A (0.5 mM MgCl2 and 1 mM
CaCl2 in phosphate-buffered saline) for 30 min at 4 °C.
Cells were washed and incubated with Buffer A containing 100 mM glycine for 30 min at 37 °C and then harvested in
Buffer B (50 mM Tris (pH 7.5), 150 mM NaCl,
0.02% NaN3, 1 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, and 2 µg/ml aprotinin). After
centrifugation (2000 × g for 15 min), pellets were
solubilized with 2% SDS in Buffer B for 3 min at 90 °C. The solubilized membrane fraction was centrifuged at 49,000 × g for 30 min at 15 °C, and then the supernatant was
diluted six times with 2% Triton X-100 in Buffer B. Aliquots were
subjected to 7% SDS-PAGE (lysate) and protein assay (bicinchoninic
acid protein assay kit, Pierce), and the remainder was incubated with
NeutrAvidin-linked beads (Pierce) overnight at 4 °C.
Following washing and centrifugation, the samples were subjected to 7%
SDS-PAGE. Proteins were transferred to nitrocellulose membranes and
blocked for 1 h in TBST (Tris-buffered saline with 0.1% Tween 20)
containing 5% nonfat milk. Nitrocellulose membranes were incubated
with rabbit polyclonal anti-NR2A antibody (1:1000; Chemicon
International, Inc., Temecula, CA) in TBST with 1% nonfat milk
overnight at 4 °C. After washing with TBST, the membranes were
incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG
as the secondary antibody (1:1000; Santa Cruz Biotechnology, Santa
Cruz, CA) for 1 h. Proteins were visualized by enhanced
chemiluminescence (PerkinElmer Life Sciences) and quantitated using
Typhoon and ImageQuant Version 5.1 (Amersham Biosciences).
Electrophysiological Recording--
Patch-clamp recording was
performed at room temperature using an Axopatch 1D or 200B amplifier
(Axon Instruments, Inc., Union City, CA). For whole-cell
recording, gigaohm seals were formed using patch pipettes with tip
resistances of 1-7 megaohms, and series resistances of 4-10 megaohms
were compensated by 80%. In some experiments, cells were lifted off
the surface of the dish after obtaining a gigaohm seal to increase the
speed of the solution exchange; in these experiments, patch pipettes
were pulled from thin-walled glass capillaries and had series
resistances of 1-2.5 megaohms. For single-channel recordings, patch
pipettes were coated with R6101 elastomer (Dow-Corning), and had
tip resistances of 7-15 megaohms following fire polishing. Cells were
voltage-clamped at
50 mV, unless noted otherwise. Data were filtered
(2-kHz low-pass 8-pole Bessel for whole-cell recording and 1-kHz
low-pass 8-pole Butterworth for fluctuation analysis) and acquired at
5-20 kHz on a computer using a DigiData interface and pClamp software
(Axon Instruments, Inc.). Cells were superfused at 1-2 ml/min in
extracellular medium containing 150 mM NaCl, 5 mM KCl, 0.2 mM CaCl2 , 10 mM HEPES, and 10 mM glucose; the pH was
adjusted to 7.4 using NaOH, and the osmolality was adjusted to 340 mmol/kg using sucrose. The patch pipette solution in whole-cell
recordings contained 140 mM CsCl, 2 mM
Mg4ATP, 10 mM BAPTA, and 10 mM HEPES; the pH was adjusted to 7.4 using CsOH, and the
osmolality was adjusted to 310 mmol/kg using sucrose. In recordings
from cell-attached patches, the patch pipette solution contained 1 µM NMDA, 50 µM glycine, and 10 µM EDTA in extracellular medium. Solutions of agonists
and drugs were prepared fresh daily in extracellular medium and applied
to cells using a stepper motor-driven rapid solution exchange apparatus
used without the metallic manifolds supplied by the manufacturer
(Fast-Step, Warner Instrument Corp.) and 600-µm inner diameter square
three-barrel glass tubing. The 10-90% rise time for solution exchange
in lifted cells was ~1.5 ms, which was determined using steps from 0 to 150 mM Na+ in the continuous presence of agonist.
Calculation of Physicochemical Properties of Amino
Acids--
Molecular (van der Waals) volumes of amino acids were
calculated using Spartan Pro (Wavefunction, Inc., Irvine, CA) following structural optimization using the AM1 semi-empirical parameters. Values
used for amino acid hydropathy, hydrophilicity, and polarity were
reported previously (23-25).
Data Analysis--
Concentration-response data were analyzed
using the nonlinear curve-fitting program ALLFIT (26), which allows
statistical comparison of parameters from multiple curves by measuring
the degradation in goodness of fit (using an F test for the sums of squares of the residuals) that results from constraining these parameters to be equal. Values reported for half-maximal concentration (EC50) and slope factor (n) are those obtained
by fitting the data to Equation 1,
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(Eq. 1)
|
where x and y are concentration and
response, respectively, and Emax is the maximal
response. In fluctuation analysis experiments, fast Fourier
transformations of 25-60 data sweeps of 600-800 ms were averaged;
background spectra were subtracted; and the data were fitted with a
lorentzian function of the form shown in Equation 2,
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(Eq. 2)
|
where Sf is the spectral density at frequency
f (in hertz), S0 is the zero
frequency asymptote, and fc is the corner frequency.
Time constants (
) were obtained from the relationship
= 1/2
fc. Mean current amplitude and variance of the
data were obtained using Clampfit Version 8.0 (Axon Instruments, Inc.);
single-channel conductances (
, in picosiemens) were determined
from the relationship
= i/Vm,
where Vm is the membrane holding potential (in
volts) and i is the slope (in picoamperes) of the
least-squares linear fit to the initial region of a current variance
versus amplitude plot. Data from single-channel recordings
were idealized using the program SKM (QUB suite)
(27),2 which utilizes a
hidden Markov event detection algorithm. These data were filtered at 10 kHz (8-pole Bessel) to ensure optimal detection of brief
(submillisecond) openings and closings using SKM; similar results were
obtained using 2-kHz filtering and a 50% threshold event detection
criterion. Maximal likelihood multiple exponentials were fitted to
dwell time histograms using Clampfit, and gaussian functions were
fitted to all-points histograms using TAC (Bruxton Corp., Seattle, WA).
No attempt was made to correct for missed events, as no appropriate
correction procedure is currently available for NMDA receptors (28).
Data obtained in rapid solution exchange experiments were fitted to a
simple five-state kinetic model (Scheme
1) in which A represents agonist; R
represents receptor; and the subscripts C, O, and D represent closed,
open, and desensitized states of the channel, respectively. In this
model, k1 and k
1 are the respective agonist binding and unbinding rates;
and
are the
respective channel opening and closing rates; and
d1 and d
1 are the rates
for entry into and recovery from the desensitized state, respectively.
Data from rapid solution exchange experiments were fitted to the model
using the program SCoPFit (Simulation Resources, Redlands, CA), with
k1 constrained to the value determined in
cultured hippocampal neurons (under conditions in which NMDA receptor
subunit expression should have been predominantly NR1/NR2A) (29), 0.005 µM
1 ms
1 (30); with
constrained to the inverse of the mean open time determined from noise
analysis (0.311 and 0.0926 ms
1 for receptors containing
NR2A and NR2A(M823W) subunits, respectively); and with all other rates
allowed to vary. Simulated current values were generated using the
program SCoP, with all parameters in the model set to their respective
average values determined from fits to the data. Analysis of variance
(ANOVA), correlation analysis, and linear regression analysis were
performed using the program StatView (SAS Institute, Inc., Cary, NC).
Fisher's protected least significant difference test was used to
determine differences among means following ANOVA. All values are
reported as the means ± S.E.
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RESULTS |
Effect of the NR2A(M823W) Mutation on the NMDA Concentration
Response--
The results of a previous study have demonstrated that
Val816-Ala821 in the M4 domain of the NMDA
receptor NR1 subunit are not exposed to the lumen of the ion channel
(31). Mutation of these residues to tryptophan, which, as the largest
and most hydrophobic amino acid, should be the substitution most likely
to disrupt protein-protein interactions (32), produced little or no
change in receptor function (as measured by apparent desensitization)
(data not shown), with the exception of the NR1(M818W) mutant, which
was nonfunctional. This methionine residue is conserved in NMDA
receptor NR1 and NR2 subunits (Fig. 1).
However, tryptophan substitution of the corresponding residue in the
NR2A subunit had a pronounced effect on receptor function. Fig.
2A illustrates current
activated by various concentrations of NMDA in receptors composed of
wild-type NR1/NR2A subunits (upper traces) and
NR1/NR2A(M823W) subunits (lower traces). As is evident, the
NR2A(M823W) mutant exhibited a greater degree of apparent
desensitization at all NMDA concentrations tested. The NR2A(M823W)
mutation did not alter the EC50 of NMDA for activation of
peak current (35.1 ± 4.39 versus 40.2 ± 6.39 µM; ALLFIT analysis; p > 0.05) (Fig.
2B), but significantly lowered the EC50 of NMDA
for activation of steady-state current (4.98 ± 1.08 versus 23.4 ± 3.76 µM; ALLFIT analysis;
p < 0.05). In contrast, the concentration-response
curve for glycine was not altered by the NR2A(M823W) mutant (Fig.
2C). The NR2A(M823W) mutation did not alter the
EC50 of glycine for activation of peak current (4.03 ± 0.322 versus 3.76 ± 0.442 µM; ALLFIT
analysis; p > 0.05) or steady-state current (2.97 ± 0.424 versus 3.41 ± 0.274 µM; ALLFIT analysis; p > 0.05).

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Fig. 1.
A methionine residue in the M4 domain is
conserved in all NMDA receptor subunits. A, sequences
of the M4 domains (underlined) of the NR1 and NR2 subunits.
The arrow shows the position of the conserved methionine
(boldface). B, topological model of the NR2A
subunit showing membrane-associated domains M1-M4, ligand-binding
domains S1 and S2 (thick lines), and the presumed position
of Met823 (shaded band).
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Fig. 2.
The NR2A(M823W) mutation alters the
EC50 for steady-state (but not peak) NMDA-activated
current. A, currents activated by various micromolar
concentrations of NMDA and 10 µM glycine in HEK 293 cells
expressing NR1/NR2A (upper traces) or NR1/NR2A(M823W)
(lower traces) subunits. The time and current scales for the
upper traces apply to all traces. B,
concentration-response curves for NMDA (in the presence of 10 µM glycine) in HEK 293 cells expressing wild-type
(WT) NR1/NR2A or mutant NR1/NR2A(M823W) subunits. Data
points are the means ± S.E. of four to nine cells, and the curves
are least-squares fits to Equation 1 given under "Experimental
Procedures." C, concentration-response curves for glycine
(in the presence of 25 µM NMDA) in HEK 293 cells
expressing NR1/NR2A or NR1/NR2A(M823W) subunits. Data points are the
means ± S.E. of three to eight cells, and the curves are
least-squares fits to Equation 1 given under "Experimental
Procedures."
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Effect of the NR2A(M823W) Mutation on Desensitization--
As
noted above, the NR2A(M823W) mutation dramatically altered the apparent
desensitization of the NMDA-activated current. We accordingly tested
the effect of the mutation on the desensitization kinetics of current
activated by different concentrations of NMDA in lifted cells using a
fast solution exchange apparatus. In the majority of cells expressing
wild-type NR2A or mutant NR2A(M823W) subunits, desensitization of
NMDA-activated current following prolonged application of NMDA
exhibited two exponential components (Fig.
3A). The time constant for the
faster component was not altered by NMDA concentration in wild-type or
mutant receptors (ANOVA; p > 0.05) (Fig.
3B) and did not differ between wild-type and mutant
receptors (ANOVA; p > 0.05). This fast component of desensitization was most probably attributable to the presence of
ambient Zn2+ (33, 34), as it was abolished in the presence
of 10 µM EDTA (data not shown). In contrast, the time
constant for the slower component was significantly dependent upon NMDA
concentration in receptors containing both wild-type and mutant
NR2A(M823W) subunits (ANOVA; p < 0.001) and was
~3-7-fold lower in NR1/NR2A(M823W) receptors compared with wild-type
receptors (ANOVA; p < 0.0001).

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Fig. 3.
Effect of the NR2A(M823W) mutation on
desensitization kinetics. A, traces illustrating
desensitization of current activated by various micromolar
concentrations of NMDA in lifted cells expressing wild-type NR1/NR2A or
mutant NR1/NR2A(M823W) subunits. The curves are double-exponential fits
to the data. B, average time constants for desensitization
of NMDA-activated current. At 30 µM NMDA, three of seven
cells expressing wild-type (WT) receptors exhibited the fast
component of desensitization; for all other treatment conditions,
n = 5-13 cells. Asterisks denote
significant differences from the same component of desensitization in
cells expressing wild-type receptors (ANOVA and Fisher's protected
least significant difference test; p < 0.0001). The
fast and slow components at each NMDA concentration in each subunit
combination differed significantly (ANOVA and Fisher's protected least
significant difference test; p < 0.001).
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Effect of the NR2A(M823W) Mutation on Ion Channel Gating--
To
determine whether the NR2A(M823W) mutation could alter other ion
channel gating parameters in addition to desensitization, we used
single-channel recording in patches from cells expressing wild-type or
mutant receptors (Fig. 4). Single-channel
conductance measured at
50 mV was not altered by the NR2A(M823W)
mutation (55.0 ± 1.26 versus 52.7 ± 1.16 picosiemens for wild-type versus NR2A(M823W) receptors,
respectively; t test; p > 0.05).
Single-channel currents in cell-attached patches from cells expressing
wild-type NR1/NR2A receptors yielded open time distributions that could be fitted with three exponential components with average time constants
of 51.7 ± 6.39 µs (59.6%), 0.831 ± 0.0616 ms (20.6%), and 3.66 ± 0.547 ms (28.4%) (n = five patches)
(Fig. 4B). Open time distributions of single-channel
currents in cells expressing NR1/NR2A(M823W) receptors exhibited four
exponential components with average time constants of 49.9 ± 6.47 µs (50.9%), 0.627 ± 0.145 ms (14.9%), 4.28 ± 1.22 ms
(22.0%), and 22.4 ± 5.79 ms (12.2%). The first three time
constants observed in patches containing NR1/NR2A(M823W) receptors did
not differ significantly from the corresponding values observed in
cells expressing wild-type NR1/NR2A receptors (ANOVA; p > 0.05); however, the presence of the additional component resulted in
a mean open time of 3.55 ± 0.836 ms, which was significantly
increased compared with the value of 1.14 ± 0.405 ms obtained in
wild-type channels (ANOVA; p < 0.05; n = five patches).

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Fig. 4.
The NR2A(M823W) mutation increases the mean
open time of the NMDA receptor. A, currents activated
by 1 µM NMDA in the presence of 50 µM
glycine and 10 µM EDTA in cell-attached patches from
cells expressing wild-type NR1/NR2A (left panel) or mutant
NR1/NR2A(M823W) (right panel) subunits. The membrane holding
potential was +50 mV; channel openings are upward. Lower
traces are segments of the upper traces shown with an
expanded time scale. B, open time histograms from
cell-attached patches containing wild-type NR1/NR2A (left
panel) or mutant NR1/NR2A(M823W) (right panel)
subunits. Dashed curves indicate maximal likelihood multiple
exponential fits to the data, and dotted curves indicate the
individual exponential components. The traces in A and the
corresponding distributions in B are from the same
individual patches; similar results were obtained in four other
patches. C, power density spectra of NMDA-activated current
in cells expressing wild-type NR1/NR2A (left panel) or
mutant NR1/NR2A(M823W) (right panel) subunits. Spectra from
individual data sweeps were averaged following fast Fourier
transformation. The curves are the best fits of the data to the
lorentzian function (Equation 2) under "Experimental Procedures."
The corner frequencies of 47.8 and 14.6 Hz obtained in cells expressing
wild-type NR1/NR2A and mutant NR1/NR2A(M823W) subunits, respectively,
correspond to time constants of 3.33 and 10.9 ms.
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Similar observations were obtained in experiments using fluctuation
analysis. Variance analysis of current activated by NMDA at
concentrations of 1-10 µM revealed that the unitary
conductance of the ion channel was not changed by the NR2A(M823W)
mutation (48.0 ± 1.87 versus 48.4 ± 2.11 picosiemens for mutant and wild-type channels, respectively; ANOVA;
p > 0.05). Fitting of lorentzian functions to power
density spectra of NMDA-activated current yielded mean open times of
3.33 ms for wild-type NR1/NR2A receptors and 10.9 ms for mutant
NR1/NR2A(M823W) receptors in the data shown from two typical cells in
Fig. 4C. On average, the mean open time of NMDA receptors
containing the NR2A(M823W) mutant was significantly increased compared
with that of wild-type receptors (10.8 ± 0.768 versus
3.22 ± 0.218 ms, respectively; ANOVA; p < 0.0001). Because the filtering used in noise analysis would essentially
eliminate the shortest open time constant observed in single-channel
recording and reduce the contribution of the second open time constant, under these conditions, the values obtained in noise analysis appear to
represent primarily the contribution of the two longest time constants.
These values agreed well with the proportionally weighted means of the
third and fourth open time constants obtained in single-channel
analysis (11.5 ± 1.24 and 3.66 ± 0.550 ms for mutant and
wild-type receptors, respectively).
Kinetic Modeling of the Effect of the NR2A(M823W)
Mutation--
The data obtained for either wild-type NR1/NR2A or
mutant NR1/NR2A(M823W) receptors in fast exchange experiments could be adequately fitted to a simple five-state kinetic model (Fig.
5A). Average values for rate
constants obtained from the fits of the data to the model indicated
that the NR2A(M823W) mutation increased the rate of entry into the slow
desensitized state (9.09 versus 0.696 s
1 for
mutant and wild-type receptors, respectively; ANOVA; p < 0.0001) and decreased the rate of exit from this state (0.0582 versus 0.798 s
1 for mutant and wild-type
receptors, respectively; ANOVA; p < 0.001), but did
not significantly alter the rates of ion channel opening (0.137 versus 0.102 ms
1 for mutant and wild-type
receptors, respectively; ANOVA; p > 0.05) or agonist
unbinding (0.217 versus 0.183 ms
1 for mutant
and wild-type receptors, respectively; ANOVA; p > 0.05). Comparison of simulated traces generated from the model (Fig.
5B) using the average values obtained from fits to the data showed that the model can account not only for the marked increase in
macroscopic desensitization produced by the NR2A(M823W) mutation, but
also for the >2-fold increase in peak current density and decrease in
steady-state current density observed in cells expressing the mutant
subunit (Fig. 5C). This increase in peak current density was
not attributable to an increase in receptor expression, as the
NR2A(M823W) mutation did not alter cell-surface receptor expression as
determined in Western blot experiments (Fig. 5, D and
E).

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Fig. 5.
Effect of the NR2A(M823W) mutation on NMDA
receptor kinetics and surface membrane expression. A,
current activated by 300 µM NMDA and 10 µM
glycine in lifted cells expressing wild-type NR1/NR2A (left
panel) or mutant NR1/NR2A(M823W) (right panel)
subunits. The superimposed dashed curves are fits of the
model in Scheme 1 to the data. B, simulated current using
average values obtained from fits of the model in Scheme 1 to current
in lifted cells expressing wild-type (WT) NR1/NR2A subunits
(solid curve; n = 13) or mutant
NR1/NR2A(M823W) subunits (dashed curve; n = 7), with the total receptor number set to 1000 for both subunit
combinations. C, current density for peak and steady-state
currents activated by 300 µM NMDA and 10 µM
glycine. Results are the means ± S.E. of 12-37 cells. **,
significantly different from peak current density in cells expressing
wild-type receptors (ANOVA and Fisher's protected least significant
difference test; p < 0.001). Average values of peak
current were 1216 ± 227 pA in wild-type subunits and 2593 ± 518 pA in NR1/NR2A(M823W) subunits. pF, picofarad.
D, typical Western blots of NMDA receptors expressing
wild-type NR1/NR2A (upper panel) or mutant NR1/NR2A(M823W)
(lower panel) subunits probed with anti-NR2A antibody. Both
blots contain (from the left) biotinylated cell-surface membrane
receptor (SUR; amount equivalent to 20 µg of lysate), 1-3
µg of lysate (3, 2, and 1), and
molecular mass markers. E, surface expression of NR2A
subunits. Surface expression was calculated by comparing the intensity
of the biotinylated surface receptor with that of lysate. Results are
the means ± S.E. of four experiments.
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Effect of Other NR2A Met823 Mutations on Ion Channel
Gating--
In an attempt to identify the physicochemical parameters
of the substituent at the NR2A Met823 site responsible for
regulating the gating of the ion channel, we constructed and tested a
panel of substitution mutants at this site. Substitution of highly
polar residues (Asp, His, and Lys) or glycine at this site did not
yield functional receptors. As was observed for the NR2A(M823W) mutant,
a number of the remaining mutant subunits showed marked differences in
the EC50 for NMDA activation of steady-state current, the
maximal steady-state current/peak current ratio, and the mean open time
(Table I), whereas the EC50
for NMDA activation of peak current and the unitary conductance were
unchanged. The single exception to this was the NR2A(M823I) mutant,
which exhibited a unitary conductance of 40 picosiemens and an
increased EC50 for NMDA activation of peak current compared with wild-type receptors. The mean open time of NR1/NR2A(M823I) receptors was, however, dramatically attenuated compared with that of
wild-type receptors (0.77 ms), which could account for an apparent
reduction in unitary conductance due to the short duration of many
channel opening events relative to the rise time of the recording
system. Linear regression analysis of the results obtained with the
series of NR2A Met823 mutants revealed a significant linear
relation between the steady-state current/peak current ratio and the
substituent amino acid hydropathy (R2 = 0.516;
linear regression ANOVA; p < 0.05) (Fig.
6A), but not molecular volume,
hydrophilicity, or polarity (R2 = 0.001, 1.01 × 10
6, and 0.288, respectively; linear
regression ANOVA; p > 0.05). Similarly, the mean open
time of the series of mutants was linearly related to hydropathy
(R2 = 0.424; linear regression ANOVA;
p < 0.01) (Fig. 6B) as well as to polarity
(R2 = 0.427; linear regression ANOVA;
p < 0.05), but not to molecular volume or
hydrophilicity (R2 = 0.262 and 0.217, respectively; linear regression ANOVA; p > 0.05).
Although there was an apparent trend toward an inverse relationship
between steady-state current/peak current ratio and mean open time in
the various NR2A Met823 mutants, these parameters were not
significantly correlated (R2 = 0.3036; Fisher's
z test; p > 0.05) (Fig.
7A). The values for the
steady-state NMDA EC50 were, however, highly correlated
with steady-state current/peak current ratios
(R2 = 0.9332; Fisher's z test;
p < 0.0001), whereas there was no correlation between
the values for the peak NMDA EC50 and steady-state current/peak current ratios (R2 = 0.0144;
Fisher's z test; p > 0.05) (Fig.
7B).
View this table:
[in this window]
[in a new window]
|
Table I
Characteristics of NMDA receptors containing NR2A Met823
substitution mutants
Iss and Ip denote steady-state
and peak current amplitudes, respectively. Values are the means ± S.E. Mean open times were determined for steady-state current activated
by 5 or 10 µM NMDA in the presence of 10 µM
glycine. Substitution of Asp, His, Lys, or Gly at this position yielded
nonfunctional receptors. WT, wild-type; pS, picosiemens.
|
|

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 6.
Regression analysis of NMDA receptor ion
channel gating properties with amino acid physicochemical parameters of
substituents at NR2A Met823. The graphs plot
steady-state/peak ratios
(Iss:Ip) of current
activated by 1000 µM NMDA and 10 µM glycine
(A) or the mean open time of channels activated by 5 µM NMDA and 10 µM glycine (B)
versus molecular volume (in cubic angstroms), hydropathy
(24), hydrophilicity (23), and polarity (25).
Iss:Ip values were
significantly linearly related to hydropathy (p < 0.05); mean open time was significantly linearly related to both
hydropathy (p < 0.01) and polarity (p < 0.05).
|
|

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 7.
Relation between NMDA receptor
desensitization and mean open time or NMDA EC50. The
graphs plot steady-state/peak ratios
(Iss:Ip) of current
activated by 1000 µM NMDA and 10 µM glycine
against the mean open time of channels activated by 5 µM
NMDA and 10 µM glycine (A) or the
EC50 for NMDA activation of peak or steady-state current
(B).
|
|
 |
DISCUSSION |
In this study, mutation of a highly conserved methionine residue
(Met823) in the NR2A subunit of the NMDA receptor to
tryptophan dramatically increased the rate of macroscopic
desensitization as well as the mean open time of the ion channel. In
wild-type NR1/NR2A receptors, the extent of desensitization
(Iss:Ip = 0.58 at 1 mM NMDA) and the slow time constant of desensitization
(
ds = 2.0 s at 300 µM NMDA) were
similar to those previously observed under similar conditions
(Iss:Ip = 0.47-0.65 and
ds = 1.1-1.9 s) (17, 34, 35). The faster of the two
components of apparent desensitization was presumably attributable
primarily to the presence of Zn2+ in the extracellular
solution, as its value was in the range (200-300 ms) reported for
apparent desensitization due to high-affinity Zn2+
inhibition (34), and as it was not observed in the presence of the
chelator EDTA. This fast component of desensitization was also not
significantly altered by the NR2A(M823W) mutation. In contrast, the
slower component of desensitization was markedly altered by the
NR2A(M823W) mutation. A marked alteration in the rates of entry into
and exit from the desensitized state in receptors containing the
NR2A(M823W) mutant was also obtained when the data were fitted to a
simple kinetic model. Although it is possible that the mutation may
have introduced a new desensitized state, this should have been evident
as an additional component of desensitization rather than as a change
in the rate of an existing component. This residue in the M4 domain
thus appears to be critical in regulating the transition of the ion
channel into and out of the slow desensitized state. In addition, NR2A
Met823 regulates the rate of ion channel closing, as was
observed in noise analysis experiments in which the NR2A(M823W)
mutation increased the mean open time of the ion channel. Results from
single-channel recordings indicated that this increase in mean open
time in receptors containing the NR2A(M823W) mutant was attributable to
the introduction of an additional long open time not present in
wild-type channels.
Results of previous studies support a physiological role for mean open
time and desensitization of NMDA receptors in the regulation of
synaptic transmission. Alterations in the phosphorylation state of NMDA
receptors can produce changes in mean open time that significantly affect the amplitude of NMDA receptor-mediated synaptic events (36,
37), and drugs such as ethanol that decrease NMDA receptor mean open
time (38, 39) depress the amplitude of the NMDA receptor component of
postsynaptic potentials (40). NMDA receptor desensitization can
influence the duration and following frequency as well as the amplitude
of synaptic events (13-16). Although the time constant of
desensitization affected by the NR2A(M823W) mutation in this study is
slow relative to that of the glutamate concentration transient in the
synaptic cleft (~1 ms) (52), this component of desensitization
may nevertheless participate in the regulation of NMDA receptor
function. NMDA receptor channel gating kinetics slow the dissociation
of glutamate (15, 41), with the result that the time constant for decay
of NMDA receptor-mediated synaptic currents can be in the range
150-300 ms (41, 42). Furthermore, time constants for recovery from
desensitization may exceed 500 ms (43). The contribution of the slow
component of NMDA receptor desensitization to regulation of synaptic
activity would thus be predicted to increase with prolonged receptor
stimulation, such as would occur during repetitive firing, which could
cause a significant proportion of the receptors to accumulate in the desensitized state.
The trend toward a relation between desensitization and open time in
the various NR2A Met823 mutants observed in this study may
indicate that, in general, the longer the channel is open, the more
likely it is to enter the desensitized state. This would seem to agree
with the molecular mechanism of desensitization recently proposed for
non-NMDA glutamate receptors (11). The lack of a significant
correlation between steady-state current/peak current ratio and mean
open time in the various mutants may indicate that other factors such
as structural differences differentially regulate desensitization and
open time at this site. Mutations at NR2A Met823 did not
influence the NMDA EC50 for peak current, with the
exception of the NR2A(M823I) mutant, in which case it is likely that
the very brief duration of ion channel opening seen in this mutant was
responsible for the increase in the NMDA EC50 for
activation of peak current due to a reciprocal influence of ion channel
gating on agonist binding (44). In contrast to the results obtained for
the NMDA EC50 for peak current, six of nine NR2A
Met823 mutations significantly decreased the NMDA
EC50 for steady-state current activation. The decreased
steady-state EC50 values were most probably attributable to
the increased desensitization observed in these mutant receptors, as
they were highly correlated with steady-state current/peak current
ratios and were also predicted by the kinetic model (data not shown).
If this interpretation is correct, the increase in apparent affinity
for mutants exhibiting a high degree of desensitization
would result from an increase in the number of agonist molecules
trapped on desensitized receptors (45), as has been observed in
-aminobutyric acid type A receptors (46), rather than from an
increase in the affinity of the binding site for agonist. This
interpretation is also consistent with the observed lack of
correspondence between peak and steady-state NMDA EC50
values in the various mutants, the lack of correlation between NMDA
EC50 values for peak current and steady-state current/peak current ratios, and the absence of a change in the agonist unbinding rate determined using kinetic modeling.
The results of linear regression analyses using a number of
physicochemical measures of amino acids were consistent with an important influence of the hydrophobicity of the substituent at NR2A
Met823 upon both mean open time and desensitization. Thus,
hydrophobic interactions between NR2A Met823 and a closely
apposed residue or region of the protein may stabilize the ion channel
in the closed and desensitized states. The observation that the
molecular volume of the substituent did not influence either parameter
in a systematic manner suggests that the regulation exerted by the
residue at this site on ion channel gating behavior does not involve a
simple volume occupation, as appears to be the case for similar
residues in
-aminobutyric acid type A and glycine receptors
(47-50). The inverted V shape of the
Iss:Ip versus molecular volume plot may indicate, however, the existence of an
optimal value for molecular volume at this position at which desensitization is minimal. The lack of an absolute relationship between ion channel open time and desensitization in the various mutants tested is consistent with a differential influence of this site
on the transition rates for the closed and desensitized states of the
ion channel. It is likely that these differences may be attributable to
subtle structural characteristics of the amino acid at the site, which
are not well represented by relatively crude measures such as overall
molecular volume and hydrophobicity of the side chain.
A previous study investigating the aqueous accessibility of residues in
the proximal region of M4 of the NR1 subunit (31) did not report any
alteration in ion channel function following cysteine substitution at
NR1 Met818, the cognate site to NR2A Met823. In
the present study, substitution of a tryptophan at this position resulted in nonfunctional receptors. Because tryptophan is the largest
and most hydrophobic amino acid, the lack of function of this mutant
NR1 subunit is most likely due to an intolerance of this position to
the increase in molecular volume or hydrophobicity. This observation,
coupled with the observation that tryptophan substitutions at the
adjacent residues in the NR1 subunit did not alter receptor function,
suggests that this residue may influence ion channel function, albeit
not in a manner identical to that of its cognate site in the NR2A subunit.
Results of studies on the NR1 subunit (31, 51) suggest that part of the
N-terminal end of the region originally identified as M4 is located
outside of the membrane. Thus, NR2A Met823 may be located
much closer to the extracellular face of the membrane than would be
originally inferred based upon its position in the sequence of the M4
domain. Assuming that the segment of the M4 domain that actually spans
the membrane is
-helical in structure, NR2A Met823 is
most likely oriented toward one or more of the other
membrane-associated domains, rather than toward the lipid membrane,
because small changes in the residue at this position (e.g.
leucine versus isoleucine) could dramatically influence ion
channel function in a manner that is consistent with a protein-protein
interaction, but difficult to envision for a protein-lipid interaction.
In addition, the results of this study clearly demonstrate that this
site influences the behavior of the ion channel gating region. Sites in
the pre-M1 and M3 domains also influence ion channel gating and
desensitization and may be involved in transducing agonist binding into
ion channel gating (17, 19). In non-NMDA glutamate receptors, agonist binding appears to induce a movement of the S2 ligand-binding domain
relative to S1 (11), which places a strain on the M3 domain that
results in ion channel opening. Desensitization then results from
separation of dimers of the S1 domains, relieving the strain on the M3
domain and allowing the channel to close (11). If similar
conformational changes underlie ion channel gating and desensitization
in NMDA receptors, the results of the present study may indicate that
NR2A Met823 influences ion channel gating via hydrophobic
interactions with an adjacent site in M3 or another membrane-associated domain.
 |
ACKNOWLEDGEMENTS |
We thank Julia Healey, Fang Li Lu, Amber Luo,
and Jerry Wright for technical advice and assistance and David
Lovinger and Randall Stewart for helpful comments on the manuscript.
 |
FOOTNOTES |
*
This work was supported by the intramural program of the
National Institute on Alcohol Abuse and Alcoholism.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Unit on Cellular
Neuropharmacology, LMCN, NIAAA, Park 5 Bldg., Rm. 150, 12420 Parklawn
Dr., MSC 8115, Bethesda, MD 20892-8115. Tel.: 301-443-1236; Fax:
301-480-6882; E-mail: bpeoples@helix.nih.gov.
Published, JBC Papers in Press, October 31, 2002, DOI 10.1074/jbc.M209486200
2
Available at www.qub.buffalo.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
NMDA, N-methyl-D-aspartate;
HEK, human embryonic
kidney;
BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid;
ANOVA, analysis of variance.
 |
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