Ethanol inhibition of N-methyl-D-aspartate receptors is reduced by site-directed mutagenesis of a transmembrane domain phenylalanine residue

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 Phe(639) 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.