A Novel Alcohol-Sensitive Site in the M3 Domain of the NMDA Receptor GluN2A Subunit

Accumulating studies have demonstrated that the N-methyl-D-aspartate receptor is one of the most important targets of ethanol in the central nervous system. Previous studies from this laboratory have found that one position in the third (F637) and two positions in the fourth (M823 and A825) membrane-associated (M) domains of the N-methyl-D-aspartate receptor GluN2A subunit modulate alcohol action and ion channel gating. Using site-directed mutagenesis and whole-cell patch-clamp recording, we have found an additional position in M3 of the GluN2A subunit, F636, which significantly influences ethanol sensitivity and functionally interacts with F637. Tryptophan substitution at F636 significantly decreased the ethanol IC50, decreased both peak and steady-state glutamate EC50, and altered agonist deactivation and apparent desensitization. There was a significant correlation between steadystate: peak current ratio, a measure of desensitization, and ethanol IC50 values for a series of mutants at this site, raising the possibility that changes in ethanol sensitivity may be secondary to changes in desensitization. Mutant cycle analysis revealed a significant interaction between F636 and F637 in regulating ethanol sensitivity. Our results suggest that F636 in the M3 domain of the GluN2A subunit not only influences channel gating and agonist potency, but also plays an important role in mediating the action of ethanol. These studies were supported by grants R01 AA015203-01A1 and AA015203-06A1 from the NIAAA to R.W.P