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Originally published In Press as doi:10.1074/jbc.M414215200 on June 21, 2005

J. Biol. Chem., Vol. 280, Issue 33, 29708-29716, August 19, 2005
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Conserved Structural and Functional Control of N-Methyl-D-aspartate Receptor Gating by Transmembrane Domain M3*

Hongjie Yuan{ddagger}, Kevin Erreger, Shashank M. Dravid, and Stephen F. Traynelis

From the Department of Pharmacology, Emory University School of Medicine, Rollins Research Center, Atlanta, Georgia 30322-3090

Received for publication, December 17, 2004 , and in revised form, June 20, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The molecular events controlling glutamate receptor ion channel gating are complex. The movement of transmembrane domain M3 within N-methyl-D-aspartate (NMDA) receptor subunits has been suggested to be one structural determinant linking agonist binding to channel gating. Here we report that covalent modification of NR1-A652C or the analogous mutation in NR2A, -2B, -2C, or -2D by methanethiosulfonate ethylammonium (MT-SEA) occurs only in the presence of glutamate and glycine, and that modification potentiates recombinant NMDA receptor currents. The modified channels remain open even after removing glutamate and glycine from the external solution. The degree of potentiation depends on the identity of the NR2 subunit (NR2A < NR2B < NR2C,D) inversely correlating with previous measurements of channel open probability. MTSEA-induced modification of channels is associated with increased glutamate potency, increased mean single-channel open time, and slightly decreased channel conductance. Modified channels are insensitive to the competitive antagonists D-2-amino-5-phosphonovaleric acid (APV) and 7-Cl-kynurenic acid, as well as allosteric modulators of gating (extracellular protons and Zn2+). However, channels remain fully sensitive to Mg2+ blockade and partially sensitive to pore block by (+)MK-801, (-)MK-801, ketamine, memantine, amantadine, and dextrorphan. The partial sensitivity to (+)MK-801 may reflect its ability to stimulate agonist unbinding from MT-SEA-modified receptors. In summary, these data suggest that the SYTANLAAF motif within M3 is a conserved and critical determinant of channel gating in all NMDA receptors.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The N-methyl-D-aspartate (NMDA)1 subtype of ionotropic glutamate receptor plays a major role in physiological (long-term synaptic plasticity) and pathological (epilepsy, excitotoxicity in stroke) processes in the brain (1). NMDA receptors function as heteromeric assemblies composed of glycine-binding NR1 subunits in combination with at least one type of glutamate-binding NR2 subunit (2-4). NMDA receptors have three transmembrane domains (M1, M3, and M4) plus a cytoplasmic re-entrant membrane loop (M2) (5). Contained within the M3 transmembrane domain is the motif with the strictest amino acid conservation (SYTANLAAF, Fig. 1) among all the members of the ionotropic glutamate receptor family, indicating an important role in channel function (6-8). In the naturally occurring lurcher mutant mice, GluR{delta}2 receptors contain an alanine to threonine mutation in this motif (9). The lurcher mice suffer cerebellar degeneration resulting in ataxia and impaired motor learning. Introducing the analogous lurcher mutation into NR1, GluR1, or GluR6 receptors decreases agonist EC50 values and/or decreases the rate of receptor deactivation (10-12). This suggests that transmembrane domain M3 may play a critical role in the channel gating of ionotropic glutamate receptors. Jones et al. (13) reported that replacement of the alanine at position 7 of the SYTANLAAF motif of NR1 or NR2A with cysteine (hereafter A7C) produced agonist-induced accessibility changes for sulfhydryl-modifying reagents. This residue was susceptible to covalent modification by extracellular MTSEA only when it was co-applied during channel activation by glutamate and glycine, suggesting that a change in solvent accessibility of this residue is associated with channel activation. MTSEA modification dramatically slowed the deactivation of the mutated NMDA receptors, indicating that M3 functions as a transduction element whose conformational change couples ligand binding with channel opening. Here we demonstrate that this role for M3 is conserved not only between NR1 and NR2A, but also for NR2B, NR2C, and NR2D. The MTSEA modification of the mutation A7C on NR1, NR2A, NR2B, NR2C, or NR2D potentiates currents to a varying degree depending on the identity of the NR2 subunit, and the MTSEA-modified channels remain open even following removal of glutamate and glycine from the external solution. These modified channels are insensitive to competitive NMDA antagonists (APV and 7-Cl-kynurenic acid) and allosteric modulators of gating (low pH and Zn2+). MTSEA-modified channels are inhibited by channel blockers (Mg2+, (+)MK-801, (-)MK-801, ketamine, memantine, amantadine, dextrorphan), although divergent effects for some of these blockers were observed in the absence or presence of the agonists. We interpret these results as evidence that the M3 transmembrane domain plays a conserved role in channel function for all NMDA subunits.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Site-directed mutagenesis was performed using the QuikChange kit (Stratagene, La Jolla, CA) as described previously (14). cDNAs for NR1 and NR2 subunits (GenBankTM numbers: NR1, U11418 [GenBank] and U08261 [GenBank] ; NR2A, D13211 [GenBank] ; NR2B, U11419 [GenBank] ; NR2C, M91563 [GenBank] ; NR2D, L31611 [GenBank] ) were provided by Drs. S. Heinemann (Salk Institute), S. Nakanishi (Kyoto University), and P. Seeburg (University of Heidelberg). Preparation and injection of cRNA, as well as two electrode voltage-clamp recordings from Xenopus laevis oocytes were performed as previously described (15). Briefly, oocytes were injected with 5-10 ng of cRNAs synthesized in vitro from linearized template cDNA and stored at 15 °C in Barth's solution. The ratio of NR1 to NR2-injected cRNA was 1:2. Two-electrode voltage-clamp recordings were made 2-3 days postinjection at room temperature (23 °C). The recording solution contained (in mM): NaCl 90, KCl 1, HEPES 10, BaCl2 0.5, plus 2-10 µM EDTA; pH was adjusted to 7.6 with NaOH. EDTA was added to chelate contaminant divalent ions including Zn2+. Solution exchange was computer controlled through 8-modular valve positioner (Digital MVP Valve, Hamilton). Voltage and current electrodes were filled with 0.3-3.0 M KCl, and current responses were recorded at a holding potential of -40 mV. Data acquisition and voltage control were accomplished with a two-electrode voltage-clamp amplifier (OC-725, Warner Instrument, Hamilton, CT). Only currents greater than 50 nA were included in the analysis. 50 µM Glutamate, 30 µM glycine, and 0.2-2.0 mM MTSEA were used in all oocyte experiments unless otherwise stated.



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  		FIG. 1.
Mutation of a highly conserved residue in NR1 and NR2 subunits. The alanine (A) at position 7 of the SYTANLAAF motif in M3 of NR1, NR2A, NR2B, NR2C, or NR2D was substituted with cysteine (C). This region is thought to be near the extracellular end of the M3 transmembrane domain and close to the linker tethering the agonist binding domain to the transmembrane domain. The cartoon shows only 2 of 4 subunits that comprise an NMDA receptor, and for clarity does not show the amino-terminal domain.

 
Human embryonic kidney (HEK) 293 cells were maintained as described previously (16) and plated onto 12-mm glass coverslips that were coated with poly-D-lysine. cDNAs encoding NR1-1a and NR2A-A650C in pcIneo were transiently transfected into cells using the calcium phosphate precipitation method (17). The ratio of cDNAs encoding green fluorescent protein/NR1/NR2 was 1:2:4, with a final green fluorescent protein cDNA concentration of 0.1 µg/ml. Cells remained in the transfection solutions for 6-8 h, after which the media was replaced and supplemented with 2-3 mM Mg2+ and 200 µM APV. Recordings were typically made over 24 h after transfection. Glutamate was applied using a piezoelectric translator (Burleigh Instruments, Fishers, NY) to move a double-barreled flow pipe constructed from theta tubing. External recording solution for all experiments was composed of the following (in mM): 150 NaCl, 10 HEPES, 3 KCl, 0.7 CaCl2, and 0.2 EDTA, pH 7.4 (NaOH) (310-330 mosmol, 23 °C). The reduction of free extracellular Ca2+ to 0.5 mM reduced the frequency of subconductance levels (16, 18); EDTA was added to remove contaminant divalent cations. All experiments were performed at 23 °C. The internal solution was composed of the following (in mM): 110 gluconic acid, 30 CsCl, 4 NaCl, 5 HEPES, 5 BAPTA, 0.5 CaCl2, and 2 MgCl2, pH 7.3 (CsOH) (290-300 mosmol). All solutions contained a maximal concentration of glycine (30 µM or >5 x EC50). For single-channel recording, thick-walled borosilicate glass (1.5 mm outer diameter; 0.85 mm inner diameter; Warner Instruments) was fire polished to a resistance of 6-9 M{Omega}, and Sylgard (Dow Corning, Midland, MI) was applied to the pipette tip. Single-channel data were digitized at 40 kHz using Clampex version 9.0 (Axon Instruments, Union City, CA) after filtering at 5 kHz (eight-pole Bessel, -3 dB; Frequency Devices, Haverhill, MA). Single-channel records were idealized using the Viterbi algorithm (19) in QUB (www.qub.buffalo.edu). Open-duration histograms were fitted with two exponential components in ChanneLab (www.synaptosoft.com) using the maximum likelihood method (20).

All reagents were purchased from Sigma except MTSEA (Toronto Research Chemicals Inc., Toronto, Canada), APV, (+)MK-801 maleate, and (-)MK-801 maleate (Tocris Cookson, Bristol, UK). Ketamine and dextrorphan were gifts from Dr. S. Holtzman (Emory University).

Data were expressed as mean ± S.E., and analyzed statistically using paired or unpaired t test and one-way analysis of variance analysis. Significance for all tests was set at p < 0.05. Error bars in all figures are S.E.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Potentiation by MTSEA of the A7C Mutant Depends on the Identity of the NR2 Subunit—The alanine at position 7 of the conserved SYTANLAAF motif of NR1, NR2A, NR2B, NR2C, or NR2D was substituted with cysteine using site-directed mutagenesis (A7C). The mutant receptors used in this study were (numbered relative to the initiating methionine) NR1-A652C, NR2A-A650C, NR2B-A651C, NR2C-A661C, and NR2D-A675C (Fig. 1). Extracellular application of MTSEA did not induce a current response (n = 12) from oocytes injected only with the NR1-A7C mutant (Fig. 2A). MTSEA also had no significant effect (n = 8) on NR1-A7C/NR2A or NR1-A7C/NR2D currents when applied in the absence of glutamate and glycine (Fig. 2B). Similarly, MTSEA applied to NR1-A7C co-expressed with NR2B or NR2C in the absence of agonists only minimally potentiated currents compared with control by 12.5 ± 3.4 (n = 7) and 3.6 ± 1.1% (n = 9), respectively. This small effect may be because of some degree of solvent accessibility in the closed state for these subunits or trace concentration of agonist in the bath solution. MTSEA (2 mM) reversibly inhibited the agonist-evoked currents of wild-type recombinant NR1/NR2A (58.2 ± 4.3%, n = 8), NR1/NR2B (46.2 ± 3.4%, n = 10), NR1/NR2C (25.6 ± 2.9%, n = 8), and NR1/NR2D (18.3 ± 2.5%, n = 4), which may reflect channel block. Inhibition was minimal with 0.2 mM MTSEA (data not shown).

Co-application of 2 mM MTSEA with glutamate and glycine potentiated the agonist-evoked currents for the NR1-A7C cysteine mutant in a manner dependent on the identity of the NR2 subunit (NR2A, 2.5 ± 0.1-fold, n = 23; NR2B, 19.9 ± 2.0-fold, n = 37; NR2C, >100-fold, n = 10; NR2D, >100-fold, n = 9; Fig. 2, C-G). There was no significant difference between potentiation by 2 mM and 0.2 mM MTSEA (2.5 ± 0.1-fold, n = 23, versus 2.4 ± 0.1-fold, n = 29) on NR1-A7C/NR2A. Potentiation was long lasting as previously described (13), and the current was sustained even in the absence of agonist (see Fig. 5A; see also Ref. 13). Following washout of agonist, a very slow decay of current was observed in the MTSEA-modified channels. This decay may be either because of unbinding of agonist or slow washout of MTSEA, because full recovery of MTSEA-modified channels is not seen with reapplication of agonists (data not shown). The degree of the potentiation, which depends on the identity of the NR2 subunit (Fig. 2, C-G), was inversely correlated with previous measurements of channel open probability for wild-type NR2 subunits (8). The magnitude of potentiation of NR1-A7C/NR2A by MTSEA was independent of the holding potential during MTSEA application (2.5 ± 0.1-fold at -40 mV, n = 23; 2.5 ± 0.6-fold at 0 mV, n = 10; p > 0.05), suggesting that any voltage-dependent blockade of the channel by cationic MTSEA did not alter reactivity of A7C.



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  		FIG. 2.
MTSEA potentiation of NR1-A7C depends on the identity of the NR2 subunit. 2 mM MTSEA was applied in the presence (A, C-F) or absence (B) of the agonists (50 µM glutamate and 30 µM glycine). A, co-application of MTSEA and agonists had no significant effect on currents evoked from homomeric NR1-A7C. B, MTSEA application alone had no significant effect on the currents of NR1-A7C/NR2A. C-F, application of MTSEA potentiates the agonist-evoked NMDA currents in NR1-A7C co-expressed with NR2A or 2B, 2C, or 2D. G, summary of the MTSEA potentiation on the currents of NR1-A7C/NR2A and NR1-A7C/NR2B. IGLU/GLY represents the maximal currents evoked by the agonists. IMTSEA indicates the maximal currents evoked by MTSEA co-applied with the agonists. Glu/Gly and GG indicate the agonists (glutamate, 50 µM and glycine, 30 µM) through all the figures and tables.

 



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  		FIG. 3.
MTSEA potentiation on wild-type NR1 plus NR2 subunit A7C mutants depends on the identity of NR2 subunits. A-D, application of MTSEA potentiates the agonist-evoked NMDA currents in wild-type NR1 co-expressed with NR2A-A7C, NR2B-A7C, NR2C-A7C, or NR2D-A7C.

 
Wild-type NR1 was subsequently co-expressed with the A7C cysteine mutant for each of the NR2 subunits. Similar to the NR1-A7C mutant expressed with wild-type NR2 (Fig. 2B), MTSEA had no appreciable effect when applied in the absence of agonist on any of the NR2-A7C mutant subunits expressed with wild-type NR1 (n = 3-8). For all four NR2-A7C mutant subunits (NR2A-D), application of MTSEA in the presence of glutamate and glycine potentiated the current responses (Fig. 3). Also similar to the NR1-A7C mutant, the degree of potentiation by MTSEA of NR2-A7C mutants was strongly dependent on the identity of the NR2 subunit. The rank order of MTSEA-induced potentiation of wild-type NR1/NR2-A7C mutants was again inversely correlated with previous reports of channel open probability (8), and was: NR2A-A7C, 2.2 ± 0.8-fold; NR2B-A7C, 15.9 ± 1.4-fold; NR2C-A7C, >100-fold; NR2D-A7C, >100-fold (n = 12-22 per NR2 subunit; Fig. 3, A-D).

To understand more clearly the effects of MTSEA on channel function, we recorded single channel currents in outside out patches excised from HEK 293 cells transiently transfected with cDNA encoding NR1 and NR2A-A7C subunits (Fig. 4). For each patch, we first recorded in the presence of 100 nM glutamate plus 30 µM glycine, and subsequently in the presence of co-agonists plus 0.2 mM MTSEA, followed by a washout of both MTSEA and glutamate. Unmodified NR1/NR2A-A7C receptors had a significantly longer mean open time (6.9 ± 1.2 ms, n = 5) than wild-type receptors (3.7 ms (21)). Fig. 4 shows the strong increase in opening frequency following MTSEA treatment. During 0.2 mM MTSEA treatment, there was a flickery block of channel openings, which we interpret to reflect brief blockages of the conduction pathway by MTSEA. Following washout of MTSEA, flickery block subsides and there remains a significant increase in mean channel open time to 277 ± 33% of control (p < 0.05, n = 3; Fig. 4E). Interestingly, there is a modest 32% decrease in single channel chord conductance for MTSEA-modified receptors from 64.1 ± 1.2 to 43.5 ± 2.1 pS (n = 3, Fig. 4D). One interpretation of the reduction in conductance by covalent modification is that MTSEA-modified channels have a reduced pore diameter.



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  		FIG. 4.
MTSEA modification of NR1/NR2A-A7C single channel currents. A, an outside-out patch from HEK 293 cell containing at least three NR1/NR2A-A7C receptors was exposed to 100 nM glutamate and 30 µM glycine. B, the same patch is shown during exposure to 0.2 mM MTSEA in the presence of agonists. Note the flickery block, which is most likely because of MTSEA channel block. C, the same patch is shown during washout with no agonist or MTSEA. D, all point histograms from the traces depicted in A and C were generated using QUB software. Note the decrease in mean amplitude of single channel current and an increase in the relative occupancy of open state. E, open time histograms pooled from three independent patches were fitted with two exponential components using ChannelLab (non-modified channels: 292 events; MTSEA-modified channels: 354 events). The time constant, {tau}1, before MTSEA application was 0.37 ms and after MTSEA was 0.27 ms. The time constant, {tau}2, increased from 9.6 to 24.8 ms after MTSEA modification.

 
MTSEA-modified Channels Are Insensitive to Competitive Antagonists and Allosteric Modulators—Simultaneous application of MTSEA and agonists not only potentiated the current amplitude, but also markedly slowed deactivation (Fig. 5A) such that the modified channels remain open even after removing glutamate and glycine from the external solution. The sustained current response allowed us to test the sensitivity of the currents from modified channels to inhibition by competitive antagonists and allosteric modulators of gating in the absence of the modifying reagent.

Co-application of 50 µM APV (a competitive antagonist for the glutamate binding site on NR2) or 100 µM 7-Cl-kynurenic acid (a competitive antagonist for the glycine binding site on NR1) with glutamate and glycine reversibly reduced the agonist-evoked currents of unmodified NR1-A7C/NR2A by 87.6 ± 1.9 (n = 14, Fig. 5, A and B) and 92.8 ± 0.8% (n = 20, Fig. 5C), respectively. This is consistent with the predicted level of inhibition given KB values for these antagonists at wild-type NMDA receptors (22, 23). By contrast, the antagonist-induced inhibition of the MTSEA-modified channels after removal of agonists from the external solution was significantly reduced, being 1.1 ± 0.2% (p < 0.001) for APV and 26.3 ± 2.6% (p < 0.001) for 7-Cl-kynurenic acid (Fig. 5, B and C). These data suggest that the MTSEA-modified channels are insensitive to previously effective concentrations of competitive antagonists at the binding site of glutamate and glycine.

We also tested the sensitivity of the MTSEA-modified channels to allosteric modulators of gating. A reduction in the external pH from 7.6 to 6.6 or addition of 1 µM Zn2+ inhibited the agonist-evoked currents by 82.8 ± 2.0 (n = 22, Fig. 5D) and 81.8 ± 6.0% (n = 5, Fig. 5E) for unmodified NR1-A7C/NR2A, respectively. This is consistent with the effects of protons and Zn2+ on wild-type NR2A containing receptors (14, 24), suggesting that NR1-A7C does not alter proton or Zn2+ sensitivity. By comparison, the MTSEA-modified channels were inhibited by protons and Zn2+ by only 16.1 ± 1.4 and 14.5 ± 4.7%, respectively (Fig. 5, D and E), suggesting that the MTSEA-modified channels are less sensitive to allosteric modulators of gating. This is consistent with the idea that protonation reduces the activation rate by slowing or blocking a conformational change that precedes channel opening (25). Moreover, the parallel effect of MTSEA modification on proton and Zn2+ inhibition further supports the hypothesis that Zn2+ inhibition proceeds through enhancement of tonic proton inhibition (14, 26).



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  		FIG. 5.
Competitive antagonists and allosteric modulators of gating had reduced effects on the MTSEA-modified channel currents of NR1-A7C/NR2A. A and B, 50 µM APV, an antagonist of the glutamate site, inhibited the agonist-evoked control currents but had less effect on the MTSEA-modified channel currents. C, 100 µM 7-Cl-kynurenic acid, an antagonist of the glycine site, blocked the agonist-evoked currents, but caused significantly less inhibition of the MTSEA-modified channel currents. D, lowering extracellular pH from 7.6 to 6.6 inhibited the agonist-evoked currents, but had less effect on the MTSEA-modified channel currents. E, a concentration of Zn2+ (1 µM) sufficient to block the agonist-evoked currents had minimal effect on the MTSEA-modified channel currents. **, p < 0.001.

 
MTSEA-modified Channels Show Differential Sensitivity to Open Channel Blockers—We examined the effect of MTSEA modification of NMDA receptors on inhibition by use-dependent trapping and non-trapping channel blockers. Mg2+ (1 mM; Fig. 6, A and D) inhibited the agonist-evoked currents (90.5 ± 2.0%, n = 10, VHOLD -40 mV), as well as the current from the MTSEA-modified channels in the absence (89.4 ± 3.3%, n = 10) or presence (90.9 ± 2.8%, n = 10) of the agonists on NR1-A7C/NR2A. There was no significant difference (p = 0.92, one-way analysis of variance) among the Mg2+ inhibition of the agonist-evoked currents and the MTSEA-modified channel currents under any conditions, suggesting that modification of the A7C residue does not perturb the structural determinants of Mg2+ blockade deep within the pore. (+)MK-801, a channel blocker that can become trapped in the pore following agonist dissociation, similarly blocked the agonist-evoked currents in unmodified NR1-A7C/NR2A receptors (1 µM; 92.1 ± 2.0% inhibition, n = 10; Fig. 6, B and E) and MTSEA-modified receptor currents in the absence of the agonists (88.4 ± 3.1%, n = 10; Fig. 6, C and E). Surprisingly, the (+)MK-801 inhibition of MTSEA-modified NR1-A7C/NR2A currents was significantly reduced by the co-application of agonists with (+)MK-801 (13.0 ± 1.2%, n = 10; Fig. 6, C and E) compared with (+)MK-801 inhibition of the MTSEA-modified channels in the absence of agonists (p < 0.001, paired t test).



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  		FIG. 6.
NMDA channels blockers and the MTSEA-modified channel currents (A-E, NR1-A7C/NR2A; F and G, NR1/NR2A-A7C). A and D, 1 mM Mg2+ inhibited the agonist-evoked currents and the MTSEA-modified channel currents in the absence or presence of agonists. B, C, and E, 1 µM (+)MK-801 inhibited the agonist-evoked currents and the MTSEA-modified channel currents in the absence of the agonists, but had less effect on MTSEA-modified channel currents in the presence of the agonists. Mg2+ (F) or (+)MK-801 (G) showed similar effects on NR1/NR2A-A7C as on NR1-A7C/NR2A.

 
NR1 and NR2 subunits are known to make differential contributions to the intra-pore determinants of gating and block (27-29). We therefore tested whether Mg2+ and (+)MK-801 showed these distinct effects in MTSEA-modified NR1/NR2A-A7C. Similar to NR1-A7C/NR2A receptors, Mg2+ (1 mM; Fig. 6F) inhibited the agonist-evoked currents in unmodified NR1/NR2A-A7C receptors (91.6 ± 0.7%, n = 9) and MTSEA-modified NR1/NR2A-A7C channels in the absence (91.2 ± 3.0%) or presence (87.3 ± 1.6%) of the agonists. (+)MK-801 (1 µM; Fig. 6G) blocked the agonist-evoked currents in unmodified (91.1 ± 2.5%, n = 8) and MTSEA-modified NR1/NR2A-A7C channels in the absence of the agonists (87.4 ± 4.2%), but had less effect in the presence of the agonists (17.0 ± 3.1%). These data suggest that NR1-A7C/NR2A and NR1/NR2A-A7C receptors show similar effects of MTSEA on the sensitivity to the channel blockers.



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  		FIG. 7.
Effect of MK-801 on glutamate potency for NR1-A7C/NR2A and NR1/NR2A-A7C receptors. Representative two-electrode voltage-clamp recording obtained from oocytes expressing NR1/NR2A-A7C receptors in which the currents were evoked by glutamate at the concentration indicated (µM) in the presence of a maximal concentration of glycine: A, control; B, MTSEA-modified channel without (+)MK-801; and C, MTSEA-modified channels with (+)MK-801. D and E, mean normalized concentration-response curve for glutamate-evoked current in oocytes expressing NR1/NR2A-A7C (D) and NR1-A7C/NR2A (E). Note that MTSEA modification significantly decreased the glutamate EC50 (dotted line), whereas (+)MK-801 increased the EC50 after MTSEA (solid line).

 
We draw two conclusions from these data. First, MTSEA modification reduces channel block by (+)MK-801 in the presence of agonists, unlike block of modified channels by Mg2+, which is effective in the presence or absence of agonists. This likely reflects the different mechanism of (+)MK-801 and Mg2+ block. Second, it appears that (+)MK-801 may accelerate the unbinding of glutamate and/or glycine from the MTSEA-modified channels. We provide additional evidence for this hypothesis by measuring glutamate potency (quantified by EC50) in the presence or absence of (+)MK-801 following modification by MTSEA (Fig. 7). The potential effects of MK-801 on co-agonist EC50 cannot easily be measured in wild-type receptors because the channel is non-conducting in the presence of (+)MK-801. However, in MTSEA-modified channels, there is an opportunity to reduce receptor responses with (+)MK-801 in the absence of agonist, and then compare the concentration-response curve for re-activation of the current by glutamate in the absence or presence of (+)MK-801 (Fig. 7). Table I summarizes the effects of MTSEA on glutamate EC50, which is decreased 588- and 317-fold, for MTSEA modification of NR1-A7C/NR2A and NR1/NR2A-A7C receptors, respectively. In addition, the EC50 of glutamate at MTSEA-modified receptors is increased by (+)MK-801 11.6- and 7.1-fold for NR1-A7C/NR2A and NR1/NR2A-A7C receptors, respectively, compared with control. These data are consistent with our working hypothesis that (+)MK-801 can accelerate the unbinding of glutamate by reducing its potency.


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  		TABLE I
Mean EC50 values and Hill Slopes for glutamate activation of NR1-A7C and NR1/NR2A-A7C The number of oocytes recorded is shown in parentheses.

 



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  		FIG. 8.
Hypothesized model of MTSEA modification of A7C mutants and the mechanism of inhibition of modified channels by trapping blockers. A, MTSEA modification is agonist dependent and increases open probability. B, MK-801 stimulates agonist unbinding from the modified channels. Hypothesis 1: MK-801 binding at an undetermined site directly decreases the inherent agonist affinity. Hypothesis 2: MK-801 facilitates closure of channel gate, resulting in a decrease in agonist potency reflective of increased time spent in a low-affinity closed conformation. Double dotted lines indicate the plasma membrane with the extracellular side up. For clarity, the amino-terminal domain was omitted and only 2 of 4 subunits are shown.

 
Because of the unexpected dependence of (+)MK-801 block of MTSEA-modified channels on the occupancy of agonist binding sites, the sensitivity of NR1-A7C/NR2A after MTSEA treatment to a series of organic channel blockers was examined (Table II). The concentration of the channel blockers was chosen to inhibit the agonist-evoked currents in the dynamic range of the concentration-effect curve. Block of unmodified NR1-A7C/NR2A receptors was measured for (-)MK-801, ketamine, dextrorphan, memantine, amantadine, and subsequently retested for each blocker after MTSEA modification of the channel. (-)MK-801 and ketamine, two trapping blockers, had similar properties to (+)MK-801 in that the inhibition after MTSEA with the agonists was significantly less than the inhibition without the agonists in the bath solution (Table II). Dextrorphan, a channel blocker with structural determinants for binding that are unique from those of MK-801 (30), showed reduced channel block after MTSEA modification that was insensitive to the presence of agonist. Similarly, memantine and amantadine, which appear to be partial trapping blockers at hyperpolarized potentials with perhaps two intra-pore binding sites (31-33), also showed reduced channel block after MTSEA modification that was identical in the absence and presence of agonists following MTSEA modification (Table II). Thus, receptor block by dextrorphan, amantadine, and memantine following MTSEA modification of NR1-A7C/NR2A mutant receptors was significantly reduced compared with unmodified receptors (p < 0.001), yet insensitive to the absence or presence of agonist.


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  		TABLE II
Effect of organic channel blockers on the agonist-evoked currents and the MTSEA-modified channel currents of NR1-A7C/NR2A *, p < 0.001, comparing % inhibition after MTSEA without Glu/Gly and % inhibition in Glu/Gly on unmodified currents. **, p < 0.05; ***, p < 0.001, comparing % inhibition after MTSEA with and without Glu/Gly (mean ± SE; paired or unpaired t test).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Jones et al. (13) previously showed that replacement of alanine at position 7 of the highly conserved 9-amino acid motif (SYTANLAAF) of NR1 or NR2A with cysteine produced agonist-induced changes in accessibility of cysteine-modifying reagents (Fig. 8A). These authors interpreted this result to suggest that the M3 domain undergoes an important conformational change that is involved in gating. In the present study, alanine at the same position in NR1, NR2A, NR2B, NR2C, or NR2D was replaced with cysteine. Mutant NR1 was co-expressed with wild-type NR2A-D, and wild-type NR1 was co-expressed with mutant NR2A-D. Co-application of MTSEA with glutamate and glycine potentiated the currents in all subunit combinations, with the degree of the MTSEA potentiation dependent on the identity of the NR2 subunit. Potentiation inversely correlated with previous measurements of channel open probability (8), which is consistent with the idea that MTSEA modification shifts the open probability of NMDA receptor to nearly 1.0. Modification by MTSEA also increased the potency for glutamate (Fig. 8A). Our data demonstrate that the structurally conserved 9-amino acid motif of M3 plays a functionally conserved role in the channel gating across all NMDA receptor subunits.

Functional Effects of MTSEA Modification of A7C—The amplitude of the agonist-evoked currents from NR1-A7C/NR2A or NR1/NR2A-A7C was comparable with that of the wild-type receptors. However, the currents evoked by application of saturating glutamate and glycine to NR1-A7C plus NR2B, NR2C, or NR2D, and NR1 plus NR2B-A7C, NR2C-A7C, or NR2D-A7C showed consistently smaller currents than wild-type receptors, or even no resolvable response to glutamate and glycine. For these channels, MTSEA modification initiated a robust current in the presence of the agonists, indicating the mutation of subunits to cysteine influenced the maximal channel open probability or the unitary conductance. Receptors with an open probability of 0.5 (e.g. NR2A (21)) can at most have their maximal response roughly doubled following an increase in open probability, whereas receptors with a low open probability such as 0.04 for NR2D (34) can have their maximal responses potentiated at least 25-fold. If we assume that MTSEA modification increases open probability to ~1.0, we can calculate open probabilities for the mutant NR1/NR2-A7C channels prior to modification from the degree of potentiation and the measured decrease in chord conductance. We estimate that unmodified NR1/NR2A-A7C receptors have a maximal open probability of 0.31. If the MTSEA-induced change in NR2A conductance occurs in other NR2-A7C subunits, then maximal open probability for unmodified receptors would be 0.04 for NR2B-A7C and <0.01 for NR2C/D-A7C. The estimated maximal open probabilities for all NR2-A7C receptors appear lower than wild-type channel maximal open probability (8).

Saturating concentrations of competitive glycine and glutamate site antagonists were unable to block MTSEA-modified channels. One hypothesis to explain this observation is that MTSEA-modified channels have a higher affinity for agonists than unmodified channels, with glutamate locked on its binding site (in the absence of MK-801, see below). Although we cannot directly measure the affinity of glutamate for its binding site, as defined by the ratio of the microscopic dissociation rate to the association rate, we do resolve a dramatic difference in glutamate potency. Our data show a greater than 300-fold decrease in glutamate EC50 and persistent channel activation in the absence of agonist following MTSEA modification. A shift in EC50 could result from either: 1) a change in binding, the inherent association or dissociation rates of glutamate for its binding site, or 2) a change in gating, the rate of one or more conformational changes controlling the conduction of the ion channel pore. Differentiating between changes in binding versus gating for ion channels is not straightforward (35). However, the single channel data (Fig. 4) demonstrate that the change in potency involves at least in part an effect on gating. We further speculate that an additional contribution of increased agonist affinity is required to cause such a dramatic shift (>300-fold) in glutamate EC50.

Allosteric modification of gating by protons prohibits channel opening (25), and protons had greatly reduced effects on MTSEA-modified channels. This can be understood because MTSEA-modified NMDA receptors spend the most time in the open state, and therefore protons, which slow opening but do not strongly promote channel closure (25), have a reduced opportunity to modify function. The lack of effect of Zn2+ following MTSEA modification is consistent with Zn2+ inhibition of NR2A containing receptors being an enhancement of tonic proton inhibition at physiological pH (14, 26).

In contrast to competitive antagonists, we predicted that channel blockers should inhibit MTSEA-modified channels similarly to wild-type channels activated by agonists, provided that MTSEA modification does not perturb the intra-pore structure. However, the results with open channel blockers were surprisingly complicated. The normal sensitivity of MTSEA-modified channels to Mg2+ suggests that these channels possess an intra-pore configuration that is similar to the normal conformation at the Mg2+ binding site deep within the conduction path. However, the other channel blockers tested can be divided into two classes. Trapping blockers such as (+)MK-801, (-)MK-801, and ketamine were rendered less effective in the presence of glutamate and glycine in the bath following MTSEA treatment, suggesting that MTSEA modified determinants of blockade beyond those involved in Mg2+ block. One idea that emerges is that MK-801 may have a relatively low affinity for open channels of unmodified receptors, yet may greatly stabilize the closed state thereby promoting its own trapping (36). In this situation MTSEA modification increases the maximal open probability causing a decrease in the affinity of MK-801 for MTSEA-modified receptors. This idea has previously been raised as a potential mechanism for other organic channel blockers (36).

Both dextrorphan and the partial trapping blockers memantine and amantadine were less efficacious in inhibiting currents from MTSEA-modified channels than agonist-induced currents in unmodified channels. This result suggests that the structural determinants of these blockers are perturbed by MTSEA modification of M3, but perhaps less so than full trapping blockers.

In addition to the reduction in overall blocker effectiveness, we found another surprising result of MTSEA modification of the channel. Trapping channel blockers stimulate agonist unbinding following MTSEA modification. This conclusion is supported by two observations. First, trapping blockers inhibit the current through modified channels in the absence of agonists in the bath, but the current can be restored by reapplication of agonists. Second, (+)MK-801 decreases the potency (increases the EC50) for glutamate to activate the modified channels. As described above for the problem of differentiating the effects of modification on binding versus gating, the interpretation of the (+)MK-801 effect is not straightforward. The decreased potency and accelerated unbinding could reflect an outright change in agonist affinity by blocker binding (possibly at a site distinct from the channel pore), or alternatively a blocker-induced promotion of channel closure with subsequent reduction in agonist affinity (because closed channels have a lower affinity for agonist than open channels (36, 37)), or some combination of both (Fig. 8). The ability of channel blockers to facilitate agonist unbinding was clearly observable for full trapping blockers, but not for partial trapping blockers or nontrapping blockers. This result raises the question of whether the mechanism by which the trapping blockers decrease agonist potency is related to the site at which they are trapped, or perhaps a shallower site, or even a site outside the channel pore. Moreover, it is not clear whether the effects are specific for A7C mutant receptors or transfer to wild-type receptors. Nevertheless, the observation that MK-801 isomers induce a reduction in glutamate potency raises interesting questions about how trapping blockers interact with the gating machinery that translates agonist binding to channel opening.

M3 Transmembrane Region and NMDA Receptor Gating— The results of this study suggest that M3 plays a conserved role across all NMDA receptor subunits in transducing agonist binding to channel gating. Structural rearrangements in M3 or other more shallow regions may in part constitute a channel gate, consistent with studies using sequential channel blockers (38, 39) as well as with the observation here that covalent modification can increase maximal open probability. Alternatively, the channel gate may be deeper in the channel pore than the residue that we have focused on in this study (29). This hypothesis is also consistent with our results. Changes in the conformation of the agonist-binding domain may cause a rotation or translocation of M3, inducing a conformational change in the pore-lining M2 domain. Under this scenario, the activation gate directly regulating the flow of ions may be deep in the pore, with M3 acting to transduce agonist binding to channel gating.


   FOOTNOTES
 
* This work was supported by grants from the NINDS, National Institutes of Health (to S. F. T.), National Alliance for Research on Schizophrenia and Depression (to S. F. T.), and the Howard Hughes Medical Institute (to K. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Pharmacology, Emory University School of Medicine, Rollins Research Center, 1510 Clifton Rd., Atlanta, GA 30322-3090. Tel.: 404-727-1375; Fax: 404-727-0365; E-mail: hyuan{at}emory.edu.

1 The abbreviations used are: NMDA, N-methyl-D-aspartate; MTSEA, methanethiosulfonate ethylammonium; APV, D-2-amino-5-phosphonovaleric acid; HEK, human embryonic kidney. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Jon Johnson and Anders Kristensen for critical comments on the manuscript, and Dr. Philip Chen for helpful suggestions regarding experimental design. We also thank Drs. Lonnie Wollmuth, Fang Zheng, and Tue Banke for sharing mutant NMDA receptor subunits, and Dr. S. Holtzman for providing channel blockers. We thank Antoine Almonte, Polina Lyuboslavsky, and Phuong Le for excellent technical assistance.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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