Positions in the N‐methyl‐D‐aspartate Receptor GluN2C Subunit M3 and M4 Domains Regulate Alcohol Sensitivity and Receptor Kinetics

Background Alcohol alters synaptic transmission in the brain. The N‐methyl‐D‐aspartate (NMDA) receptor (NMDAR), a subtype of glutamate‐gated ion channel, is an important synaptic target of alcohol in the brain. We and others have previously identified 4 alcohol‐sensitive positions in the third and fourth membrane‐associated (M) domains, designated M31‐2and M41‐2, of the GluN1, GluN2A, and GluN2B NMDAR subunits. In the present study, we tested whether the corresponding positions in the GluN2C subunit also regulate alcohol sensitivity and ion channel gating. Methods We performed alanine‐ and tryptophan‐scanning mutagenesis in the GluN2C subunit followed by expression in HEK 293 cells and electrophysiological patch‐clamp recording. Results Alanine substitution at the M31 (F634) and M41‐2 (M821 and M823) positions did not alter ethanol (EtOH) sensitivity, whereas substitution of alanine at the M32 position (F635) yielded nonfunctional receptors. Tryptophan substitution at the M31‐2 positions did not change EtOH sensitivity, whereas tryptophan substitution at the M41 position increased, and at the M42 position decreased, EtOH sensitivity. The increased EtOH sensitivity of the tryptophan mutant at M41 is in marked contrast to previous results observed in the GluN2A and GluN2B subunits. In addition, this mutant exhibited increased desensitization, but to a much lesser extent compared to the corresponding mutations in GluN2A and GluN2B. A series of mutations at M41 altered EtOH sensitivity, glutamate potency, and desensitization. Seven amino acid substitutions (of 15 tested) at this position yielded nonfunctional receptors. Among the remaining mutants at M41, EtOH sensitivity was not significantly correlated with hydrophobicity, molecular volume, or polarity of the substituent, or with glutamate EC50 values, but was correlated with maximal steady‐state‐to‐peak current ratio, a measure of desensitization. Conclusions The identity and characteristics of alcohol‐sensitive positions in the GluN2C subunit differ from those previously reported for GluN2A and GluN2B subunits, despite the high homology among these subunits