Background: The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor is involved in a variety of processes that regulate neuronal plasticity and is an important target for the acute and chronic effects of ethanol. However, the specific sites where ethanol interacts with the receptor protein have yet to be fully elucidated. We previously demonstrated that a phenylalanine to alanine mutation in the third transmembrane domain (TM3) of the NR1 subunit decreased the ethanol inhibition of NMDA receptors expressed in HEK293 cells. In this study, we characterized the ethanol inhibition of NMDA receptors containing additional mutations within the TM3 and TM4 domains of the NR1 subunit.
Methods: Site-directed mutagenesis was used to alter specific amino acid residues in the TM3 and TM4 domains of the NR1 subunit. Mutant NR1 subunits were coexpressed with the NR2A subunit in HEK293 cells and examined for alterations in ethanol sensitivity using whole-cell voltage-clamp electrophysiology.
Results: Replacing phenylalanine at TM3 position 639 in the NR1 subunit (F639) with 9 different amino acids produced functional receptors when coexpressed with the NR2A subunit. All mutants showed a concentration-dependent inhibition by ethanol (10-100 mM), with the alanine and serine mutants being significantly less sensitive to ethanol. Amino acid substitutions at the F639 site also produced variable changes in the concentration-response relationship to glycine. However, no significant correlation between glycine EC(50) values and the magnitude of ethanol inhibition was observed. Alanine mutations at TM4 positions 813 (M813A) and 819 (L819A), but not at 817 (F817A), of the NR1 subunit enhanced ethanol inhibition. Substitution of tryptophan for TM4 residues in the NR1 subunit (positions 820-822) that are homologous to a site in the NR2A subunit shown to reduce ethanol inhibition (A825W) had no effect on ethanol sensitivity. However, these NR1 TM4 tryptophan mutants restored the ethanol inhibition of the NR1 TM3 F639A mutant to wild-type levels in a stepwise fashion.
Conclusions: These results indicate that the ethanol sensitivity of NMDA receptors may be regulated by discrete sites within the TM3 and TM4 domains of the NR1 subunit.