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 M3 1-2 and M4 1-2 , 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 M3 1 (F634) and M4 1-2 (M821 and M823) positions did not alter ethanol (EtOH) sensitivity, whereas substitution of alanine at the M3 2 position (F635) yielded nonfunctional receptors. Tryptophan substitution at the M3 1-2 positions did not change EtOH sensitivity, whereas tryptophan substitution at the M4 1 position increased, and at the M4 2 position decreased, EtOH sensitivity. The increased EtOH sensitivity of the tryptophan mutant at M4 1 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 M4 1 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 M4 1 , EtOH sensitivity was not significantly correlated with hydrophobicity, molecular volume, or polarity of the substituent, or with glutamate EC 50 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.