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Ethanol Inhibition ofN-Methyl-d-aspartate Receptors Is Reduced by Site-directed Mutagenesis of a Transmembrane Domain Phenylalanine Residue

2001; Elsevier BV; Volume: 276; Issue: 48 Linguagem: Inglês

10.1074/jbc.m102800200

1083-351X

Kimberly M. Ronald, Tooraj Mirshahi, John J. Woodward,

Ion channel regulation and function

N-Methyl-d-aspartate (NMDA) receptors (NRs) are ionotropic receptors activated by glutamate and the co-agonist glycine. Ethanol inhibits NMDA receptor function, although its site of action is undefined. We hypothesized that ethanol acts at specific amino acids contained within the transmembrane (TM) domains of the receptor. In this study, NR1 and NR2A subunits were altered by mutagenesis and tested for sensitivity to ethanol. Three NR1 mutants (W636A, F817A, and L819A) and one NR2A mutant (F637A) failed to generate functional receptors. Pre-TM1 (I546A, L551A, F554A, and F558A), TM1 (W563A), and TM2 (W611A) NR1 mutations did not affect ethanol sensitivity of heteromeric receptors. In contrast, altering a TM3 phenylalanine to alanine (F639A) reduced the ethanol inhibition of NMDA receptors expressed in oocytes and human embryonic kidney 293 cells. Mutation of the nearby methionine (M641) to alanine did not affect ethanol sensitivity, whereas changing Phe639 to tryptophan slightly enhanced ethanol inhibition. NR1(F639A) did not alter the agonist potency of glutamate but did produce a leftward shift in the glycine concentration response for receptors containing NR2A and NR2B subunits. NR1(F639A) also reduced the potency of the competitive glycine antagonist 5,7-dichlorokynurenic acid and increased the efficacy of the glycine partial agonist 3-amino-1-hydroxy-2-pyrrolidinone ((+)-HA-966). These results suggest that ethanol may interact with amino acids contained in the TM3 domain of NMDA subunits that are involved in transducing agonist binding to channel opening. N-Methyl-d-aspartate (NMDA) receptors (NRs) are ionotropic receptors activated by glutamate and the co-agonist glycine. Ethanol inhibits NMDA receptor function, although its site of action is undefined. We hypothesized that ethanol acts at specific amino acids contained within the transmembrane (TM) domains of the receptor. In this study, NR1 and NR2A subunits were altered by mutagenesis and tested for sensitivity to ethanol. Three NR1 mutants (W636A, F817A, and L819A) and one NR2A mutant (F637A) failed to generate functional receptors. Pre-TM1 (I546A, L551A, F554A, and F558A), TM1 (W563A), and TM2 (W611A) NR1 mutations did not affect ethanol sensitivity of heteromeric receptors. In contrast, altering a TM3 phenylalanine to alanine (F639A) reduced the ethanol inhibition of NMDA receptors expressed in oocytes and human embryonic kidney 293 cells. Mutation of the nearby methionine (M641) to alanine did not affect ethanol sensitivity, whereas changing Phe639 to tryptophan slightly enhanced ethanol inhibition. NR1(F639A) did not alter the agonist potency of glutamate but did produce a leftward shift in the glycine concentration response for receptors containing NR2A and NR2B subunits. NR1(F639A) also reduced the potency of the competitive glycine antagonist 5,7-dichlorokynurenic acid and increased the efficacy of the glycine partial agonist 3-amino-1-hydroxy-2-pyrrolidinone ((+)-HA-966). These results suggest that ethanol may interact with amino acids contained in the TM3 domain of NMDA subunits that are involved in transducing agonist binding to channel opening. N-methyl-d-aspartate γ-aminobutyric acid human embryonic kidney transmembrane 7-DCK, 5,7-dichlorokynurenic acid NMDA receptor 3-amino-1-hydroxy-2-pyrrolidinone N-Methyl-d-aspartate (NMDA)1 receptors are calcium-permeable ion channels expressed by neurons and require both glutamate and glycine for activation. Combinations of NMDA receptor 1 (NR1) and NR2 subunits yield receptors with different biophysical and pharmacological properties such as differences in desensitization and sensitivity to agonists and antagonists. NMDA receptors play an important role in neuronal development and are required for some forms of synaptic plasticity such as associative long-term potentiation that may underlie some forms of learning and memory (1Feldmeyer D. Cull-Candy S. J. Neurocytol. 1996; 25: 857-867Crossref PubMed Google Scholar). NMDA receptors are also involved in the excitotoxic effects of glutamate that accompany traumatic brain injury and stroke-induced ischemia.Ethanol inhibits native NMDA receptor function in vitro andin vivo (2Lovinger D.M. White G. Weight F.F. Science. 1989; 243: 1721-1724Crossref PubMed Scopus (1237) Google Scholar, 3Dildy J.E. Leslie S.W. Brain Res. 1989; 499: 383-387Crossref PubMed Scopus (246) Google Scholar, 4Hoffman P.L. Rabe C.S. Moses F. Tabakoff B. J. Neurochem. 1989; 52: 1937-1940Crossref PubMed Scopus (484) Google Scholar, 5Gothert M. Fink K. Naunyn Schmiedebergs Arch. Pharmacol. 1989; 340: 516-521Crossref PubMed Scopus (115) Google Scholar, 6Gonzales R.A. Woodward J.J. J. Pharmacol. Exp. Ther. 1990; 252: 1138-1144Google Scholar, 7Simson P.E. Criswell H.E. Johnson K.B. Hicks R.E. Breese G.R. J. Pharmacol. Exp. Ther. 1991; 257: 225-231PubMed Google Scholar). Chronic exposure of neurons to ethanol results in up-regulation of NMDA receptor function and enhanced glutamate-mediated excitotoxicity (8Iorio K.R. Tabakoff B. Hoffman P.L. Eur. J. Pharmacol. 1993; 248: 209-212Crossref PubMed Scopus (81) Google Scholar, 9Chandler L.J. Newsom H. Sumners C. Crews F. J. Neurochem. 1993; 60: 1578-1581Crossref PubMed Scopus (187) Google Scholar, 10Blevins T. Mirshahi T. Woodward J.J. Neurosci. Lett. 1995; 200: 214-218Crossref PubMed Scopus (52) Google Scholar). NMDA antagonists block the seizures associated with ethanol withdrawal (11Morrisett R.A. Rezvani A.H. Overstreet D. Janowsky D.S. Wilson W.A. Swartzwelder H.S. Eur. J. Pharmacol. 1990; 176: 103-105Crossref PubMed Scopus (150) Google Scholar, 12Grant K.A. Valverius P. Hudspith M. Tabakoff B. Eur. J. Pharmacol. 1990; 176: 289-296Crossref PubMed Scopus (388) Google Scholar), and human alcoholics report ethanol-like subjective effects after administration of ketamine, a nondissociative anesthetic that inhibits NMDA channel function (13Krystal J.H. Petrakis I.L. Webb E. Cooney N.L. Karper L.P. Namanworth S. Stetson P. Trevisan L.A. Charney D.S. Arch. Gen. Psychiatry. 1998; 55: 354-360Crossref PubMed Scopus (142) Google Scholar). Despite the wealth of knowledge indicating that the NMDA receptor is an important target for ethanol in the brain, there is no consensus as to how ethanol inhibits receptor function. Ethanol behaves as a noncompetitive and voltage-independent antagonist of the receptor, and attempts to correlate its inhibitory actions with any of the known modulatory sites on the receptor have been largely negative (14Woodward J.J. J. Neurochem. 1994; 62: 987-991Crossref PubMed Scopus (43) Google Scholar, 15Peoples R.W. White G. Lovinger D.M. Weight F.F. Br. J. Pharmacol. 1997; 122: 1035-1042Crossref PubMed Scopus (82) Google Scholar, 16Chu B. Anantharam V. Treistman S.N. J. Neurochem. 1995; 65: 140-148Crossref PubMed Scopus (132) Google Scholar). In single-channel studies, the inhibitory effects of ethanol were best accounted for by decreases in the mean open time and frequency of channel opening, effects consistent with an allosteric reduction in agonist-induced channel gating (17Wright J.M. Peoples R.W. Weight F.F. Brain Res. 1996; 738: 249-256Crossref PubMed Scopus (91) Google Scholar).Studies with recombinant NMDA receptors have shown that receptors containing NR1/2A or NR1/2B subunits are generally more sensitive to ethanol inhibition than NR1/2C or NR1/2D receptors (16Chu B. Anantharam V. Treistman S.N. J. Neurochem. 1995; 65: 140-148Crossref PubMed Scopus (132) Google Scholar, 18Masood K. Wu C. Brauneis U. Weight F.F. Mol. Pharmacol. 1994; 45: 324-329PubMed Google Scholar, 19Mirshahi T. Woodward J.J. Neuropharmacology. 1995; 34: 347-355Crossref PubMed Scopus (137) Google Scholar). In addition, ethanol inhibition of NMDA-induced currents in oocytes expressing NR1, NR2A, and NR2C subunits was less than that observed with NR1 and NR2A receptors, suggesting that subunit composition significantly influences overall ethanol sensitivity (19Mirshahi T. Woodward J.J. Neuropharmacology. 1995; 34: 347-355Crossref PubMed Scopus (137) Google Scholar). Recent studies from this laboratory have also shown that ethanol inhibition of NR1/2A receptors expressed in human embryonic kidney 293 (HEK293) cells is reduced by Fyn tyrosine kinase-mediated phosphorylation of the NR2A subunit as well as by conditions that block calcium-dependent inactivation of NR1/2A receptors (20Anders D.L. Blevins T. Sutton G. Swope S. Chandler L.J. Woodward J.J. J. Neurochem. 1999; 72: 1389-1393Crossref PubMed Scopus (50) Google Scholar,21Anders D.L. Blevins T.L. Smothers C.T. Woodward J.J. J. Biol. Chem. 2000; 275: 15019-15024Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). However, these manipulations only partially reduce ethanol inhibition of receptor function and C-terminal truncated NMDA subunits retain substantial sensitivity to inhibition by ethanol (21Anders D.L. Blevins T.L. Smothers C.T. Woodward J.J. J. Biol. Chem. 2000; 275: 15019-15024Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 22Mirshahi T. Anders D.L. Ronald K.M. Woodward J.J. J. Neurochem. 1998; 71: 1095-1107Crossref PubMed Scopus (44) Google Scholar, 23Peoples R.W. Stewart R.R. Neuropharmacology. 2000; 39: 1681-1691Crossref PubMed Scopus (49) Google Scholar). Overall, these data suggest that although C-terminal modifications may influence the ethanol sensitivity of the NMDA receptor, it is unlikely that these intracellular domains represent the major site of action for ethanol.Results from recent mutagenesis studies with alcohol- and anesthetic-sensitive γ-aminobutyric acid A (GABAA) and glycine receptors have shown that mutation of a serine residue in the second transmembrane (TM) domain or an alanine residue in the TM3 greatly affected the potentiation of GABAA and glycine channel function by ethanol and volatile anesthetics (24Mihic 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 (1096) Google Scholar). The magnitude of this effect was correlated with the molecular volume of the substituted amino acid, with larger amino acids producing inhibition by ethanol and volatile anesthetics and smaller amino acids producing enhanced potentiation. These results suggested that specific amino acids in these subunits may define an alcohol- and anesthetic-sensitive site (25Koltchine V.V. Finn S.E. Jenkins A. Nikolaeva N. Lin A. Harrison N.L. Mol. Pharmacol. 1999; 56: 1087-1093Crossref PubMed Scopus (81) Google Scholar). Although the sequence identity and structural homology between GABAA and glycine receptors and glutamate receptors is extremely low, we hypothesized that NMDA receptors also possess an ethanol-sensitive site that is defined by specific amino acids contained in one or more of the TM domains of the receptor. We reasoned that these amino acids would be relatively large and conserved between various NMDA subunits and not face the pore of the ion channel.In this study, a series of amino acids fitting these criteria were altered by site-directed mutagenesis, and the resulting mutant receptors were tested for their ethanol sensitivity. The results demonstrate that substitution of a single phenylalanine residue in the TM3 domain markedly reduces ethanol inhibition of NMDA receptor currents and that this effect is influenced by amino acid volume. A preliminary report of these findings has been presented in abstract form (26Ronald K.M. Anders D.L. Blevins T. Woodward J.J. Alcohol. Clin. Exp. Res. 2000; 24 (abstr.).: 9Google Scholar).RESULTSFig. 1 shows the sequences of the TM domains of the NR1 and NR2 NMDA receptor subunits. Asterisksindicate residues shown in the NR1 subunit that have been previously assigned as pore-facing by cysteine scanning mutagenesis (29Kuner T. Wollmuth L.P. Karlin A. Seeburg P.H. Sakmann B. Neuron. 1996; 17: 343-352Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 30Beck C. Wollmuth L.P. Seeburg P.H. Sakmann B. Kuner T. Neuron. 1999; 22: 559-570Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Residues in the NR1 subunit shown in boldface were mutated to alanine in the present study and tested for ethanol sensitivity. Most of these mutant receptor subtypes gave rise to large NMDA-mediated currents when expressed in Xenopus oocytes (TableI). However, some of the NR1 mutants (W636A, F817A, and L819A) and NR2A mutants (F637A) tested did not yield reproducible currents and were not further investigated. None of the mutants tested appeared to be tonically active in the absence of agonist based on analysis of holding currents at −80 mV. Ethanol inhibition of wild-type NMDA receptors was determined the same day as that of mutant receptors to control for any seasonal variability. These control data were pooled where appropriate to give an average value of ethanol inhibition and are consistent with previously published results using oocytes (19Mirshahi T. Woodward J.J. Neuropharmacology. 1995; 34: 347-355Crossref PubMed Scopus (137) Google Scholar, 22Mirshahi T. Anders D.L. Ronald K.M. Woodward J.J. J. Neurochem. 1998; 71: 1095-1107Crossref PubMed Scopus (44) Google Scholar).Table IMean current amplitudes of wild-type and mutant NMDA receptors expressed in Xenopus oocytesReceptor subtypeMean current amplitudeNo. testednARI/2A1258 ± 15875RI(I546A)/2A598 ± 12012RI(L551A)/2A688 ± 19214RI(F554A)/2A1012 ± 244 8RI(F558A)/2A159 ± 3411RI(W563A)/2A181 ± 30 9RI(W611A)/2A552 ± 143 6RI(W636A)/2A1-aReceptor did reliably produce currents >50 nA. —RI(F639A)/2A1778 ± 28023RI(F639W)/2A279 ± 54 9RI(M641A)/2A211 ± 3620RI(F817A)/2A1-aReceptor did reliably produce currents >50 nA. —RI(L819A)/2A1-aReceptor did reliably produce currents >50 nA. —RI/2A(F637A)1-aReceptor did reliably produce currents >50 nA. —RI(F639A)/2A(F637A)1-aReceptor did reliably produce currents >50 nA. —Data are shown as mean ± S.E.M. Oocytes injected with the indicated receptor subunits were stimulated with 100 μm glycine in barium-containing recording solution.1-a Receptor did reliably produce currents >50 nA. Open table in a new tab Mutations made at four different sites within the pre-TM1 domain of the NR1 subunit resulted in functional and ethanol-sensitive NMDA receptors when co-expressed with the wild-type NR2A subunit. Receptors composed of NR1 I546A, L551A, F554A, or F558A plus the NR2A subunit were all inhibited to the same extent by 25–100 mm ethanol (Fig.2 A). The range of ethanol sensitivity among these mutants was not different from that determined for wild-type NR1/2A receptors. Similarly, selected mutations made in either the TM1 (W563A) or TM2 (W611A) domain of the NR1 receptor resulted in functional NMDA receptors that showed normal sensitivity to ethanol (Fig. 3, A andB).Figure 2Ethanol sensitivity of NMDA receptors carrying mutations in the pre-TM1 domain of the NR1 subunit. Oocytes expressing the NR2A subunit and either wild-type or mutant NR1 subunits were stimulated with 100 μm NMDA and 10 μm glycine in the absence or presence of ethanol. Data represent the mean ± S. E. percent inhibition by ethanol and are from 4–14 (mutants) or 28–30 (wild-type) oocytes for each ethanol concentration.View Large Image Figure ViewerDownload (PPT)Figure 3Ethanol sensitivity of wild-type NR1/2A, TM1 mutant NR1(W563A)/2A (A), and TM2 mutant NR1(W611A)/2A (B) expressed in oocytes. Oocytes were stimulated with 100 μm NMDA and 10 μmglycine in the absence or presence of ethanol. Data represent the mean ± S. E. percent ethanol inhibition and are from 5–16 oocytes for each ethanol concentration.View Large Image Figure ViewerDownload (PPT)In contrast, expression of the TM3 mutant NR1(F639A) with the NR2A subunit yielded receptors that were significantly less sensitive to ethanol than wild type (Fig.4 A). This effect was manifested as a rightward and downward shift in the ethanol dose-response curve and persisted over ethanol concentrations from 10 to 200 mm (Fig. 4 B). Concentrations of ethanol >200 mm produced unstable responses in voltage-clamped oocytes and were not tested. Mutation of the nearby methionine (Met641) to alanine did not alter the inhibitory effect of ethanol compared with wild-type receptors (Fig. 4 C). In addition, mutation of NR1(F639A) to the larger tryptophan residue (F639W) produced receptors that were slightly more sensitive to ethanol than wild-type receptors (Fig. 4 D).Figure 4Ethanol inhibition of NMDA receptors carrying mutations in the TM3 domain of the NR1 subunit. A, representative currents from wild-type (NR1/2A) and TM3 mutant NR1(F639A)/2A receptors expressed in oocytes. Currents were stimulated with 100 μm NMDA and 10 μm glycine (horizontal bar) in the absence and presence of 100 mm ethanol. B, dose-response relationship for ethanol inhibition of NR1/2A and NR1(F639A)/2A receptors expressed in oocytes. Data are mean ± S. E. percent ethanol inhibition from 4–12 oocytes at each ethanol concentration. Asteriskindicates value significantly different from corresponding control value, p < 0.05, analysis of variance with Tukey's posthoc test. C and D, dose-response relationship for ethanol inhibition of NR1/2A and NR1(M641A)/2A or NR1(F639W)/2A receptors expressed in oocytes. Data are mean ± S. E. percent ethanol inhibition from 8–16 oocytes at each ethanol concentration.View Large Image Figure ViewerDownload (PPT)To determine whether the effects of the F639A mutation on ethanol sensitivity were NR2 subunit-dependent, NR1(F639A) was co-expressed with either NR2B or NR2C subunits. The ethanol sensitivity of both NR1(F639A)/2B and NR1 (F639A)/2C receptors was also significantly less than that determined for their respective wild-type counterparts (Fig. 5, A andB). Expression of NR1(F639A) with NR2A, NR2B, or NR2C subunits in HEK293 cells also significantly reduced the inhibitory effects of 100 mm ethanol compared with wild-type receptors (Fig. 5 C).Figure 5Ethanol inhibition in oocytes or HEK293 cells by various NR2 subunits and the NR1 TM3 mutant NR1(F639A). Shown are dose-response relationships for ethanol inhibition of NR1/2B and NR1(F639A)/2B (A) or NR1/2C and NR1(F639A)/2C (B) receptors expressed in oocytes. Data are mean ± S. E. percent ethanol inhibition from five to nine oocytes at each ethanol concentration. Asterisk indicates value significantly different from corresponding control value, p < 0.05, analysis of variance with Tukey's posthoc test. C, Inhibition of wild-type and NR1(F639A) NMDA receptors expressed in HEK293 cells. Data shown represent mean ± S. E. percent ethanol inhibition from five to eight cells for each receptor combination tested. Representative traces show currents activated by a 2-s pulse with 200 μm glutamate and 50 μm glycine in the absence (control and washout) and presence of 100 mm ethanol.View Large Image Figure ViewerDownload (PPT)Current-voltage experiments revealed no differences in the reversal potential or slope conductance between NR1(F639A)/2A receptors and wild-type receptors (data not shown). In addition, expression of NR1(F639A) with the NR2A subunit did not significantly alter the ability of the physiological agonist glutamate to activate the receptor (Fig. 6 A). However, the F639A substitution in the NR1 subunit shifted the concentration response for glycine to the left of that of the wild-type receptor (Fig. 6 B). Calculated EC50 values for the wild-type (NR1/2A) and mutant (NR1(F639A)/2A) receptors were 0.94 μm (Hill slope, 1.58) and 0.38 μm (Hill slope, 1.40), respectively. This effect of the F639A mutation on glycine potency was even more pronounced in receptors co-expressing NR2B subunits (Fig. 6 C). The glycine EC50 value for wild-type NR1/2B receptors was 0.18 μm (Hill slope, 2.00). When NR1(F639A) was co-expressed with the NR2B subunit, significant receptor activation was observed even in the absence of added glycine. This effect prevented an accurate calculation of the EC50 value for this subunit combination. The activation of NR1(F639A)/2B receptors in solutions lacking added glycine was blocked by the glycine site antagonist 5,7-dichlorokynurenic acid (5,7-DCK; data not shown). The glycine sensitivity of receptors expressing NR1(F639A) and NR2C subunits (EC50, 0.18 μm; Hill slope, 1.44) was not significantly different from wild-type NR1/2C receptors (EC50, 0.31 μm; Hill slope, 1.96; Fig. 6 D).Figure 6Agonist sensitivity of NR1 TM3 mutant NR1(F639A)/2A receptors expressed in oocytes. Shown are dose-response curves for glutamate (A) and glycine (B–D) in oocytes expressing either wild-type NR1/2A or NR1(F639A) plus an NR2 subunit. For the glutamate dose response, the glycine concentration was held at 10 μm, whereas glycine dose responses were obtained using 100 μm NMDA. Data represent the mean ± S. E. from 5–10 oocytes for each condition and are expressed as percent maximal current obtained in each oocyte at a saturating concentration of agonist.View Large Image Figure ViewerDownload (PPT)To investigate possible mechanisms underlying the shift in apparent glycine sensitivity with the mutant receptor, the sensitivity of wild-type and mutant receptors to a competitive glycine antagonist and a glycine partial agonist were determined. At a fixed glycine concentration of 10 μm, the competitive antagonist, 5,7-DCK dose-dependently inhibited NMDA-stimulated currents from oocytes expressing wild-type NR1/2A receptors with an IC50 value of 0.68 μm (Fig.7 A). Expression of NR1(F639A)/2A receptors shifted the concentration-response curve for 5,7-DCK to the right and increased the IC50 value to 2.37 μm.Figure 7Modulation of wild-type NR1/2A and NR1 TM3 mutant NR1(F639A)/2A receptors by glycine site compounds. A, dose-response relationship for inhibition of wild-type and NR1(F639A)/2A receptors by the competitive glycine site antagonist 5,7-DCK. Currents were activated by 100 μm NMDA and 10 μm glycine in the presence of increasing concentrations of 5,7-DCK. Data represent the mean ± S. E. obtained from 6–11 oocytes at each concentration and are expressed as percent control current obtained in the absence of antagonist for each oocyte.B, dose-response relationship for the glycine site partial agonist (+)-HA-966 in oocytes expressing wild-type NR1/2A or NR1(F639A)/2A receptors. Currents were activated by 100 μm NMDA and increasing concentrations of (+)-HA-966 in the absence of any added glycine. Data represent the mean ± S. E. obtained from 8–12 oocytes at each concentration and are expressed as percent maximum current obtained in each oocyte with 100 μm NMDA and 10 μm glycine.View Large Image Figure ViewerDownload (PPT)The effect of the F639A mutant on glycine efficacy was examined by using (+)-HA-966, a high-affinity, low-efficacy agonist at the glycine site. Oocytes expressing either wild-type NR1/2A or NR1(F639A)/2A receptors were stimulated with NMDA (100 μm) and increasing concentrations of (+)-HA-966 in the absence of any added glycine. The currents obtained in the presence of each concentration of (+)-HA-966 were normalized to the current produced in each oocyte by a maximum concentration of NMDA and glycine. In the absence of any added glycine, NMDA application resulted in currents from both wild-type and NR1(F639A)/2A receptors that were ∼5–10% of the response obtained in the presence of saturating concentrations of NMDA and glycine (Fig.7 B). Addition of (+)-HA-966 up to 300 μm to NMDA-containing solutions lacking added glycine did not significantly increase the amplitude of currents from wild-type receptors. In contrast, (+)-HA-966 dose-dependently increased the amplitude of NMDA-stimulated currents in oocytes expressing NR1(F639A)/2A subunits, reaching a maximum of ∼30% at 300 μm.DISCUSSIONThe major goal of this study was to test the hypothesis that the inhibition of NMDA receptor currents by ethanol is mediated via an interaction with one or more amino acids contained within transmembrane domains of the receptor. Because ethanol inhibition of NMDA receptor currents does not resemble that of channel-blocking drugs such as MK801 or ketamine, we initially selected amino acids that were not thought to be pore-facing (29Kuner T. Wollmuth L.P. Karlin A. Seeburg P.H. Sakmann B. Neuron. 1996; 17: 343-352Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Subsequent cysteine scanning studies revealed that some of the pre-TM1 residues tested (Phe554 and Phe558) were accessible to sulfhydryl-modifying agents, suggesting that the pre-TMI domain contains amino acids that contribute to the outer vestibule of the channel (30Beck C. Wollmuth L.P. Seeburg P.H. Sakmann B. Kuner T. Neuron. 1999; 22: 559-570Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). The pre-TM1 domain of NR2 subunits also contains amino acids involved in regulating part of the glycine-independent desensitization of the NR2A- and NR2B-containing receptors (31Krupp J.J. Vissel B. Heinemann S.F. Westbrook G.L. Neuron. 1998; 20: 317-327Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). In the present study, none of the NR1 subunit pre-TMI mutants tested modified the NMDA receptor sensitivity to ethanol inhibition. Although it is possible that changes in ethanol sensitivity may have been seen by modifying NR2 residues specifically involved in regulating glycine-independent desensitization, nondesensitizing receptor subtypes (e.g. NR1/2C) are still significantly inhibited by ethanol, suggesting that these amino acids are not likely sites of ethanol interaction.Mutation of Phe639 to alanine in the TM3 domain of the NR1 subunit significantly decreased the ethanol inhibition of NMDA receptors expressed in either oocytes or HEK293 cells. Substitution of the slightly larger tryptophan residue at Phe639 resulted in receptors that were slightly more sensitive to ethanol inhibition than wild-type receptors, suggesting that some physical or chemical property of the amino acid substitution at this position may be an important determinant of ethanol sensitivity.This has been more carefully studied in mutagenesis experiments on GABAA and glycine receptors. In those receptors, residues in TM2 and TM3 have been shown to influence the degree of potentiation of receptor function by ethanol and volatile anesthetics such as isoflurane (24Mihic 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 (1096) Google Scholar, 25Koltchine V.V. Finn S.E. Jenkins A. Nikolaeva N. Lin A. Harrison N.L. Mol. Pharmacol. 1999; 56: 1087-1093Crossref PubMed Scopus (81) Google Scholar). Replacement of the conserved TM2 serine with amino acids of different volumes resulted in receptors that were either potentiated (small amino acids), inhibited (large amino acids), or insensitive to these compounds (25Koltchine V.V. Finn S.E. Jenkins A. Nikolaeva N. Lin A. Harrison N.L. Mol. Pharmacol. 1999; 56: 1087-1093Crossref PubMed Scopus (81) Google Scholar, 32Ye Q. Koltchine V.V. Mihic S.J. Mascia M.P. Wick M.J. Finn S.E. Harrison N.L. Harris R.A. J. Biol. Chem. 1998; 273: 3314-3319Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Whether these amino acids are involved in defining a binding site or pocket for ethanol or whether they indirectly affect ethanol sensitivity by altering channel gating is unknown. However, replacement of the TM2 serine in GABAAand glycine receptors with amino acids that reduce ethanol potentiation are often associated with a significant leftward shift in the agonist dose-response curves for these receptors. This mutation-induced leftward shift in agonist potency often reduces further potentiation by alcohols and inhaled volatile anesthetics.In the present study, the F639A mutation caused a significant leftward shift in the potency of the receptor for glycine, with less effect on that for glutamate. This was NR2 subunit-dependent and was especially marked for receptors containing the NR2B subunit. The reduction in glycine but not glutamate potency is consistent with previous studies suggesting that NR1 subunits contribute the glycine binding site of the receptor, whereas NR2 subunits provide the glutamate binding site. The leftward shift in the concentration-response curve for glycine may have resulted from a change in the affinity of the NR1 subunit for glycine or in an increase in the efficacy of glycine as a receptor agonist. Results obtained with the glycine site competitive antagonist 5,7-DCK were not conclusive, because the reduced sensitivity of F639A mutant receptors to this antagonist may have resulted from an increase in the binding affinity for glycine. However, for several reasons, it seems unlikely that the effect of the F639A mutation on glycine potency is attributable to a change in glycine affinity. First, amino acids that regulate glycine binding to the NMDA receptor are located in the extracellular S1 and S2 lobes of the NR1 subunit. Most of these map to homologous positions shown by x-ray crystallography to define the glutamate binding site of the GluR2 subunit (33Armstrong N. Sun Y. Chen G.-Q. Gouaux E. Nature. 1998; 395: 913-917Crossref PubMed Scopus (603) Google Scholar). Second, in the present study, oocytes expressing NR1(F639A)/2A recept