Abstract

Abundant evidence supports a role for N-methyl-d-aspartate (NMDA) receptor inhibition in the behavioral actions of ethanol, but the underlying molecular mechanisms have not been fully elucidated. We recently found that clusters of five positions in the third and fourth membrane-associated domains (M3 and M4) at the intersubunit interfaces form putative sites of alcohol action. In the present study, we found that one of these positions, NMDA receptor subunit, GluN2A(F636), can strongly regulate ethanol sensitivity, glutamate potency, and apparent desensitization: ethanol IC₅₀ values, peak (I_p) and steady-state (I_ss) glutamate EC₅₀ values, and steady-state to peak current ratio (I_ss:I_p) values differed significantly among the mutants tested. Changes in glutamate affinity among the various mutants were not attributable to agonist trapping due to desensitization, as glutamate peak EC₅₀ values were correlated with values of both steady-state EC₅₀ and I_ss:I_p. The mean open times determined in selected mutants could be altered up to 4-fold but did not account for the changes in ethanol sensitivity. Ethanol sensitivity was significantly correlated with glutamate EC₅₀ and I_ss:I_p values, but the changes in ethanol IC₅₀ among mutants at this position do not appear to be secondary to changes in ion channel kinetics. Substitution of the isomeric amino acids leucine and isoleucine had markedly different effects on ethanol sensitivity, agonist potency, and desensitization, which is consistent with a stringent structural requirement for ion channel modulation by the side chain at this position. Our results indicate that GluN2A(F636) plays an important role in both channel function and ethanol inhibition in NMDA receptors.