A Site of Alcohol Action in the Fourth Membrane-associated Domain of the N-Methyl-D-aspartate Receptor

Abstract

The N-methyl-d-aspartate (NMDA) subtype of ionotropic glutamate receptor is an important mediator of the behavioral effects of ethanol in the central nervous system. Although ethanol is known to inhibit NMDA receptors by influencing ion-channel gating, its molecular site of action and the mechanism underlying this effect have not been established. We have previously identified a conserved methionine residue in the fourth membrane-associated domain of the NMDA receptor NR2A subunit (Met823) that influences desensitization and gating of the ion channel. Here we report that this residue plays an important role in mediating the effect of ethanol on the NMDA receptor. Ethanol IC50 values among functional substitution mutants at this position varied over the range ∼130–225 mm. There was a weak correlation between ethanol IC50 and mean open time of NR2A(Met823) mutants that was dependent on inclusion of the value for the tryptophan mutant. In the absence of this value, there was no trend toward a correlation among the remaining mutants. Desensitization appeared to influence the action of ethanol, because ethanol IC50 of the mutants was correlated with the steadystate to peak current ratio. With the exception of tryptophan, ethanol sensitivity was significantly related to the molecular volume and hydrophobicity of the substituent. The relation between ethanol sensitivity and the molecular volume and hydrophobicity at this position suggests that this residue interacts with or forms part of a site of ethanol action and that the presence of a tryptophan residue in this site disrupts its ability to interact with ethanol. The N-methyl-d-aspartate (NMDA) subtype of ionotropic glutamate receptor is an important mediator of the behavioral effects of ethanol in the central nervous system. Although ethanol is known to inhibit NMDA receptors by influencing ion-channel gating, its molecular site of action and the mechanism underlying this effect have not been established. We have previously identified a conserved methionine residue in the fourth membrane-associated domain of the NMDA receptor NR2A subunit (Met823) that influences desensitization and gating of the ion channel. Here we report that this residue plays an important role in mediating the effect of ethanol on the NMDA receptor. Ethanol IC50 values among functional substitution mutants at this position varied over the range ∼130–225 mm. There was a weak correlation between ethanol IC50 and mean open time of NR2A(Met823) mutants that was dependent on inclusion of the value for the tryptophan mutant. In the absence of this value, there was no trend toward a correlation among the remaining mutants. Desensitization appeared to influence the action of ethanol, because ethanol IC50 of the mutants was correlated with the steadystate to peak current ratio. With the exception of tryptophan, ethanol sensitivity was significantly related to the molecular volume and hydrophobicity of the substituent. The relation between ethanol sensitivity and the molecular volume and hydrophobicity at this position suggests that this residue interacts with or forms part of a site of ethanol action and that the presence of a tryptophan residue in this site disrupts its ability to interact with ethanol. Ethyl alcohol or ethanol, one of the oldest and most widely abused drugs, produces a well known spectrum of behavioral effects primarily through actions on ion channels in the nervous system. The N-methyl-d-aspartate (NMDA) 1The abbreviations used are: NMDAN-methyl-d-aspartateBAPTA1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acidHEKhuman embryonic kidneyGABAAγ-aminobutyric acid, type AANOVAanalysis of variancePLSDprotected least significant difference. receptor, a subtype of receptor for the major excitatory neurotransmitter glutamate, is thought to be of particular importance in mediating the effects of alcohols in the mammalian brain. Ethanol inhibits NMDA-activated current in central neurons (1.Lovinger D.M. White G. Weight F.F. Science. 1989; 243: 1721-1724Crossref PubMed Scopus (1249) Google Scholar, 2.Lovinger D.M. White G. Weight F.F. J. Neurosci. 1990; 10: 1372-1379Crossref PubMed Google Scholar) as well as NMDA-evoked Ca2+ flux, cyclic GMP production, and neurotransmitter release (3.Dildy J.E. Leslie S.W. Brain Res. 1989; 499: 383-387Crossref PubMed Scopus (248) Google Scholar, 4.Hoffman P.L. Moses F. Tabakoff B. Neuropharmacology. 1989; 28: 1239-1243Crossref PubMed Scopus (40) Google Scholar, 5.Hoffman P.L. Rabe C.S. Moses F. Tabakoff B. J. Neurochem. 1989; 52: 1937-1940Crossref PubMed Scopus (492) Google Scholar, 6.Göthert M. Fink K. Naunyn-Schmiedeberg's Arch. Pharmacol. 1989; 340: 516-521Crossref PubMed Scopus (115) Google Scholar). Studies using in vivo electrophysiological techniques have also reported ethanol inhibition of NMDA receptors at relevant concentrations (7.Simson P.E. Criswell H.E. Johnson K.B. Hicks R.E. Breese G.R. J. Pharmacol. Exp. Ther. 1991; 257: 225-231PubMed Google Scholar). Ethanol appears to interact with a novel allosteric site, which is independent of the recognition site for the agonist glutamate or coagonist glycine (6.Göthert M. Fink K. Naunyn-Schmiedeberg's Arch. Pharmacol. 1989; 340: 516-521Crossref PubMed Scopus (115) Google Scholar, 8.Peoples R.W. White G. Lovinger D.M. Weight F.F. Br. J. Pharmacol. 1997; 122: 1035-1042Crossref PubMed Scopus (84) Google Scholar, 9.Mirshahi T. Woodward J.J. Neuropharmacology. 1995; 34: 347-355Crossref PubMed Scopus (139) Google Scholar, 10.Peoples R.W. Weight F.F. Brain Res. 1992; 571: 342-344Crossref PubMed Scopus (82) Google Scholar, 11.Gonzales R.A. Woodward J.J. J. Pharmacol. Exp. Ther. 1990; 253: 1138-1144PubMed Google Scholar, 12.Chu B. Anantharam V. Treistman S.N. J. Neurochem. 1995; 65: 140-148Crossref PubMed Scopus (135) Google Scholar), and reduces the mean open time and opening frequency of the channel (13.Lima-Landman M.T.R. Albuquerque E.X. FEBS Lett. 1989; 247: 61-67Crossref PubMed Scopus (183) Google Scholar, 14.Wright J.M. Peoples R.W. Weight F.F. Brain Res. 1996; 738: 249-256Crossref PubMed Scopus (95) Google Scholar). N-methyl-d-aspartate 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid human embryonic kidney γ-aminobutyric acid, type A analysis of variance protected least significant difference. Presumed alcohol binding sites have been identified on a small number of receptors and ion channels, but the location and nature of these sites vary considerably. Sites of alcohol interaction have been reported to be located in the ion-channel lumen of nicotinic acetylcholine receptors from muscle (15.Forman S.A. Miller K.W. Yellen G. Mol. Pharmacol. 1995; 48: 574-581PubMed Google Scholar) in a cytoplasmic loop of a Drosophila potassium channel (16.Covarrubias M. Vyas T.B. Escobar L. Wei A. J. Biol. Chem. 1995; 270: 19408-19416Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), in the cytoplasmic C terminus of G-protein-coupled inwardly rectifying potassium channels (17.Lewohl J.M. Wilson W.R. Mayfield R.D. Brozowski S.J. Morrisett R.A. Harris R.A. Nat. Neurosci. 1999; 2: 1084-1090Crossref PubMed Scopus (209) Google Scholar), and in transmembrane domains of the GABAA and glycine receptors (18.Mihic S.J. Ye Q. Wick M.J. Koltchine V.V. Krasowski M.A. Finn S.E. Mascia M.P. Valenzuela C.F. Hanson K.K. Greenblatt E.P. Harris R.A. Harrison N.L. Nature. 1997; 389: 385-389Crossref PubMed Scopus (1102) Google Scholar, 19.Ueno S. Wick M.J. Ye Q. Harrison N.L. Harris R.A. Br. J. Pharmacol. 1999; 127: 377-382Crossref PubMed Scopus (59) Google Scholar, 20.Wick M.J. Mihic S.J. Ueno S. Mascia M.P. Trudell J.R. Brozowski S.J. Ye Q. Harrison N.L. Harris R.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6504-6509Crossref PubMed Scopus (192) Google Scholar). The identity of alcohol binding sites is also variable among alcohol-sensitive proteins. No consensus sequence or structure has yet been defined for an alcohol binding site. A recent study has demonstrated that a phenylalanine residue in the third membrane-associated domain (M3) of the NR1 subunit influences ethanol sensitivity (21.Ronald K.M. Mirshahi T. Woodward J.J. J. Biol. Chem. 2001; 276: 44729-44735Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), but the magnitude of the variation in ethanol sensitivity among mutants at this site suggests that it is not the sole site of alcohol action. Previous work in our laboratory has shown that the site of alcohol action on the NMDA receptor is located on a region of the protein that is accessible only from the extracellular environment (22.Peoples R.W. Stewart R.R. Neuropharmacology. 2000; 39: 1681-1691Crossref PubMed Scopus (49) Google Scholar). We hypothesized that there may be an additional site or sites of alcohol action in one of the membrane-associated domains that participates in the regulation of ion-channel gating and that is accessible from the extracellular environment. We have previously identified a methionine residue in M4 of the NR2A subunit (Met823), which has a predicted location close to the extracellular face of the membrane and which exerts a powerful influence on NMDA receptor ion-channel gating (23.Ren H. Honse Y. Karp B.J. Lipsky R.H. Peoples R.W. J. Biol. Chem. 2003; 278: 276-283Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). We report here that this residue influences NMDA receptor alcohol sensitivity in a manner that is related both to desensitization of the ion channel and to the molecular volume and hydrophobicity of the substituent amino acid. Mutagenesis—Plasmids containing NR1-1a and NR2A subunit cDNA were kindly provided by Drs. D. R. Lynch (University of Pennsylvania) and D. M. Lovinger (National Institute on Alcohol Abuse and Alcoholism). Site-directed mutagenesis was performed using the QuikChange kit (Stratagene, La Jolla, CA), and all of the mutations were verified by DNA sequencing. Cell Culture and Transfection—Human embryonic kidney (HEK 293) cells obtained from the American Type Culture Collection (Manassas, VA) were cultured in minimum essential medium containing 2 mm l-glutamine, Earle's balanced salt solution, 0.1 mm non-essential amino acids, 1.0 mm sodium pyruvate, and 10% heat-inactivated horse serum at 37 °C in 5% CO2 and 95% air. Cells were plated at a density of 106 cells/ml in 35-mm dishes and allowed to grow to 70–95% confluence prior to transient transfection using LipofectAMINE 2000 or calcium phosphate (both from Invitrogen). The cDNA plasmid ratio for NR1 and NR2A subunits and green fluorescent protein (pGreen Lantern, Invitrogen) was 2:2:1 (total amount of DNA, 2.5–6 μg). During and after the transfection, 100 μm ketamine and 200 μm dl-2-amino-5-phosphonovaleric acid were added to the culture medium to enhance survival of transfected cells. Within 18–72 h following transfection, cells were mechanically dissociated and plated at a low density in 35-mm dishes for use in experiments. Electrophysiological Recording—Whole-cell patch clamp recording was performed using an Axopatch 1D or 200B (Axon Instruments Inc., Union City, CA) amplifier at room temperature. Electrodes with open tip resistances of 1–5 megohms were used. After establishing whole-cell mode, series resistances of 2–10 megohms were compensated by 80%. Cells were voltage-clamped at –50 mV and superfused in an extracellular containing mm mm mm mm and mm was to with and was to with Ca2+ was used to NMDA receptor Y. V. S. B. Mol. Pharmacol. 1991; Google Scholar). The mm 2 mm mm and mm was to with and was to with were at 2 and at on a using a and (Axon of and ethanol were to cells by using a used the by the and were in extracellular Ethanol from was obtained from and all of the were obtained from of of of amino were using Inc., CA) following using the used for amino acid and were reported previously Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, J. J. Mol. Biol. PubMed Scopus Google Scholar, J.M. R. J. Biol. PubMed Scopus Google Scholar). reported for IC50 and were obtained from of to is the inhibition and is the IC50 values for inhibition of current were used because these not significantly from for peak current in mutants IC50 values obtained from from cells were using analysis of variance by the protected least significant In analysis the were at and or in were at were and were with a of the as shown in is the density at frequency is the and is the were obtained from the correlation and analysis were performed using the Inc., of the values as the mean S.E. at NR2A(Met823) Ethanol have previously identified a site in the M4 region of the NR2A that NMDA receptor ion-channel gating (23.Ren H. Honse Y. Karp B.J. Lipsky R.H. Peoples R.W. J. Biol. Chem. 2003; 278: 276-283Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). this site in ethanol inhibition of NMDA we ethanol sensitivity of substitution mutants at this site. we have reported a number of amino acid at this site receptors (23.Ren H. Honse Y. Karp B.J. Lipsky R.H. Peoples R.W. J. Biol. Chem. 2003; 278: 276-283Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). of the functional NR2A(Met823) substitution receptors were by ethanol in a manner The of the ethanol not significantly among the mutants. the values for ethanol IC50 varied significantly among the from to mm of the subunits or with NR1 subunits receptors with the sensitivity to ethanol, and of NR1 subunits with or subunits in receptors with the sensitivity to of ethanol on NMDA-activated current in cells NR2A(Met823) by μm NMDA and μm glycine in the absence and presence of 100 mm ethanol in cells NR1 and type NR2A or NR2A(Met823) substitution for ethanol inhibition of current by μm NMDA and μm glycine in cells type or substitution S.E. of and shown to IC50 values for ethanol in cells the NR2A(Met823) IC50 values that significantly and from the IC50 for type Ethanol in NR2A(Met823) that the mutations not a in the mechanism of action of ethanol on the receptor, we used analysis to mean open in NMDA receptors containing subunits in the absence and the presence of 100 mm ethanol. of density for NMDA-activated current in cells and we have previously (23.Ren H. Honse Y. Karp B.J. Lipsky R.H. Peoples R.W. J. Biol. Chem. 2003; 278: 276-283Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), time from of to density that the mean open time of NMDA receptors was with that in type ethanol a in the mean open time in these receptors to that in type 100 mm ethanol mean open time by in receptors containing subunits in the absence and presence of ethanol, were obtained in cells subunits inhibition of mean open in the absence and presence of ethanol, to obtained for type receptors in neurons in a study J.M. Peoples R.W. Weight F.F. Brain Res. 1996; 738: 249-256Crossref PubMed Scopus (95) Google Scholar). In a variance analysis of NMDA-activated current that the of the ion channel was not in the presence of ethanol in receptors containing or subunits not of Ethanol to the of ethanol on the NMDA receptor may be to sensitivity to the coagonist glycine (21.Ronald K.M. Mirshahi T. Woodward J.J. J. Biol. Chem. 2001; 276: 44729-44735Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, Morrisett R.A. Mol. Pharmacol. 1995; 48: Google Scholar), we mutations that ethanol sensitivity the glycine We no significant in values of glycine for of peak and current in cells and subunits in in a of Ethanol to Desensitization and subunits no were for ethanol of peak current not the mean open time and to peak current ratio varied widely among mutants we of these be related to the in ethanol Ethanol IC50 was significantly correlated with to peak current ratio There was also a significant correlation between ethanol sensitivity and mean open time for the series of NR2A(Met823) mutants. this correlation was dependent on the mean open time in the tryptophan because the correlation this was from the analysis the that both mean open time and ethanol IC50 varied significantly among the remaining mutants. Ethanol and of the at the in ethanol sensitivity among the NR2A(Met823) subunits were to a interaction of ethanol with this site, one that this be in a significant relation of ethanol sensitivity with one or of the amino acid substituent at the site. the of the of the amino acid residue at this site to alcohol we performed analysis of alcohol sensitivity molecular and of the substituent all of the substitution mutants were in the there was no significant relation between ethanol IC50 and molecular volume or of the of ethanol and molecular volume a trend among the mutants with the exception of tryptophan was from the significant were between ethanol IC50 and both and molecular volume of the substituent. The of the study that mutations at the conserved methionine (Met823) in the fourth membrane-associated domain of the NR2A subunit influence sensitivity of the NMDA channel to ethanol. The range over which ethanol IC50 values varied among the mutants is the range we have previously among type subunits not Although it is not at to that the mechanism of action of ethanol was not among the the that at this position not the of the ethanol or the of inhibition by ethanol of mean open time is with this We previously reported that both the and of desensitization as well as mean open time significantly among the mutants at an important role for this residue in the regulation of ion channel gating (23.Ren H. Honse Y. Karp B.J. Lipsky R.H. Peoples R.W. J. Biol. Chem. 2003; 278: 276-283Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). A recent report has also shown a role for the M4 domain in desensitization S. D. J. Neurosci. 2003; PubMed Google Scholar). Although ethanol inhibits the NMDA receptor by mean open time of the ion there was only a weak correlation in the study between ethanol sensitivity and mean open time of NR2A(Met823) mutants that was dependent on the inclusion of the value for the tryptophan mutant. this value was from the there was no trend toward a correlation of ethanol sensitivity with mean open time among the remaining the that there were significant among these mutants in both ethanol IC50 and mean open In ethanol sensitivity among the NR2A(Met823) mutants in the study was correlated with the to peak current a of the of that the the desensitization in a the its ethanol A for this be that NR2A(Met823) influences ethanol in this is not by of in type NMDA receptors in which ethanol is not at agonist concentrations (6.Göthert M. Fink K. Naunyn-Schmiedeberg's Arch. Pharmacol. 1989; 340: 516-521Crossref PubMed Scopus (115) Google Scholar, 8.Peoples R.W. White G. Lovinger D.M. Weight F.F. Br. J. Pharmacol. 1997; 122: 1035-1042Crossref PubMed Scopus (84) Google Scholar, 11.Gonzales R.A. Woodward J.J. J. Pharmacol. Exp. Ther. 1990; 253: 1138-1144PubMed Google Scholar). that ethanol in part by NMDA receptor desensitization J.M. Peoples R.W. Weight F.F. Brain Res. 1996; 738: 249-256Crossref PubMed Scopus (95) Google Scholar), but no in desensitization in the presence of ethanol was in of the receptors in the from of the of to peak ratio ethanol it is that there is not an of ethanol sensitivity on the to peak of the and substitution mutants were but the ethanol IC50 values of these subunits considerably. Although the of the study a role for desensitization in the action of ethanol, a for these may be that molecular at NR2A(Met823) influence both in desensitization and in alcohol have that alcohols and to to a site by in the and domains of GABAA and glycine receptors and channel in a manner that is dependent volume of the site (18.Mihic S.J. Ye Q. Wick M.J. Koltchine V.V. Krasowski M.A. Finn S.E. Mascia M.P. Valenzuela C.F. Hanson K.K. Greenblatt E.P. Harris R.A. Harrison N.L. Nature. 1997; 389: 385-389Crossref PubMed Scopus (1102) Google Scholar, 20.Wick M.J. Mihic S.J. Ueno S. Mascia M.P. Trudell J.R. Brozowski S.J. Ye Q. Harrison N.L. Harris R.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6504-6509Crossref PubMed Scopus (192) Google Scholar, M.P. Trudell J.R. Harris R.A. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar, A. Greenblatt E.P. A. A. A. A. Trudell J.R. Harrison N.L. J. Neurosci. 2001; Google Scholar). In this we not a relation between ethanol sensitivity and molecular volume of the substitution at the value for the tryptophan was from the there was a significant relation of ethanol sensitivity with molecular tryptophan is the amino acid, this may that the action of ethanol a volume in a in part by this site in the receptor and that the presence of a tryptophan residue in the disrupts its ability to interact with ethanol. In in this study we that ethanol sensitivity of the with the exception of tryptophan, was related to the at this A number of have demonstrated that the action of alcohols on NMDA receptors (1.Lovinger D.M. White G. Weight F.F. Science. 1989; 243: 1721-1724Crossref PubMed Scopus (1249) Google Scholar, K. M. J. Pharmacol. 1990; PubMed Scopus Google Scholar, R.A. Neuropharmacology. 1991; PubMed Scopus Google Scholar, R.W. Weight F.F. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar, R.W. H. Mol. Pharmacol. PubMed Scopus Google Scholar). The of a relation between ethanol sensitivity and of the substituent at NR2A(Met823) in this study suggests that ethanol interacts with this site in a manner that The of this study with a role of NR2A(Met823) in mediating part of the effect of ethanol on the NMDA receptor. In the that ethanol sensitivity was significantly related to the molecular volume and hydrophobicity of the substituent part of the for a site of alcohol action. Previous on alcohol-sensitive ion channels have reported between the molecular volume of alcohols and and for ion-channel A. Greenblatt E.P. A. A. A. A. Trudell J.R. Harrison N.L. J. Neurosci. 2001; Google Scholar, Harris R.A. J. Pharmacol. Exp. Ther. 2001; Google Scholar, K. A. Harrison N.L. Neuropharmacology. 2001; PubMed Scopus Google Scholar). In in one of these the molecular volume which ion-channel is was dependent the molecular volume of the substituent at a site of alcohol action A. Greenblatt E.P. A. A. A. A. Trudell J.R. Harrison N.L. J. Neurosci. 2001; Google Scholar, Harris R.A. J. Pharmacol. Exp. Ther. 2001; Google Scholar, K. A. Harrison N.L. Neuropharmacology. 2001; PubMed Scopus Google Scholar). Although be for this site on the NMDA receptor on the of a R.W. Weight F.F. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar), we have shown that the for alcohol inhibition of the NMDA receptor is not dependent the molecular volume of the alcohol R.W. H. Mol. Pharmacol. PubMed Scopus Google Scholar). in of our we that the of the most with a in which NR2A(Met823) forms part of a site of alcohol action that is as a a In this alcohols in the of the and NR2A(Met823) is in a region that is to the of the The correlation with molecular volume of the amino acid at this site in the suggests that part of the alcohol interacts with NR2A(Met823) to influence channel to this in the of the amino acid to the of the in alcohol to it that in the molecular volume of the amino acid or the alcohol this is it that in the molecular volume of amino at to NR2A(Met823) the binding the of alcohols with this The importance of NR2A(Met823) in regulation of ion-channel (23.Ren H. Honse Y. Karp B.J. Lipsky R.H. Peoples R.W. J. Biol. Chem. 2003; 278: 276-283Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) is in to the that mutations at a site of ethanol action on a protein not its In of the that ethanol produces its effects at concentrations in the for ethanol interacts with low with sites and one or of these sites that important in regulation of the sites of ethanol action. appears to be of alcohol and binding sites on glycine and GABAA receptors (18.Mihic S.J. Ye Q. Wick M.J. Koltchine V.V. Krasowski M.A. Finn S.E. Mascia M.P. Valenzuela C.F. Hanson K.K. Greenblatt E.P. Harris R.A. Harrison N.L. Nature. 1997; 389: 385-389Crossref PubMed Scopus (1102) Google Scholar, 19.Ueno S. Wick M.J. Ye Q. Harrison N.L. Harris R.A. Br. J. Pharmacol. 1999; 127: 377-382Crossref PubMed Scopus (59) Google Scholar, 20.Wick M.J. Mihic S.J. Ueno S. Mascia M.P. Trudell J.R. Brozowski S.J. Ye Q. Harrison N.L. Harris R.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6504-6509Crossref PubMed Scopus (192) Google Scholar), because a recent study has reported that a in at least one of these sites ion-channel gating M. S.A. J. Neurosci. PubMed Google Scholar). be that NR2A(Met823) not to be to for ethanol because ethanol sensitivity was not in of the mutants both NR1 and subunit is for NMDA it is that sites of ethanol action located on both subunit In this an site in the third membrane-associated domain (M3) of the NR1 subunit has been reported (21.Ronald K.M. Mirshahi T. Woodward J.J. J. Biol. Chem. 2001; 276: 44729-44735Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). mutations at this site in influence glycine (21.Ronald K.M. Mirshahi T. Woodward J.J. J. Biol. Chem. 2001; 276: 44729-44735Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) and to ion-channel gating as Honse and R. Ethanol may inhibit the NMDA receptor by in at that important in the regulation of NMDA receptor of the magnitude of the of of these sites in the NR1 and subunits to the alcohol sensitivity of the NMDA receptor. We and Lipsky for and