Two adjacent phenylalanines in the NMDA receptor GluN2A subunit M3 domain interactively regulate alcohol sensitivity and ion channel gating

The N-methyl-d-aspartate (NMDA) receptor is a key target of ethanol action in the central nervous system. Alcohol inhibition of NMDA receptor function involves small clusters of residues in the third and fourth membrane-associated (M) domains. Previous results from this laboratory have shown that two adjacent positions in the M3 domain, F636 and F637, can powerfully regulate alcohol sensitivity and ion channel gating. In this study, we report that these positions interact with one another in the regulation of both NMDA receptor gating and alcohol action. Using dual mutant cycle analysis, we detected interactions among various substitution mutants at these positions with respect to regulation of glutamate EC50, steady-state to peak current ratios (Iss:Ip), mean open time, and ethanol IC50. This interaction apparently involves a balancing of forces on the M3 helix, such that the disruption of function due to a substitution at one position can be reversed by a similar substitution at the other position. For example, tryptophan substitution at F636 or F637 increased or decreased channel mean open time, respectively, but tryptophan substitution at both positions did not alter open time. Interestingly, the effects of a number of mutations on receptor kinetics and ethanol sensitivity appeared to depend upon subtle structural differences, such as those between the isomeric amino acids leucine and isoleucine, as they could not be explained on the basis of sidechain molecular volume or hydrophilicity. •Two phenylalanine residues in the NMDA receptor GluN2A subunit M3 domain influence alcohol action and ion channel gating.•Mutations at these positions interactively affect alcohol sensitivity and receptor function.•A mutation at one position can reverse the effect of a similar mutation at the other position.•These effects may arise from hydrophobic and aromatic interactions with surrounding side chains.