Intersubunit Interactions at Putative Sites of Ethanol Action in the M3 and M4 Domains of the NMDA Receptor GluN1 and GluN2B Subunits

Background and Purpose: The N-methyl-D-aspartate (NMDA) receptor is an important target of alcohol action in the brain. Recent studies in this laboratory have demonstrated that alcohol-sensitive positions in the intersubunit interfaces of the M3 and M4 domains of GluN1 and GluN2A subunits interact with respect to ethanol sensitivity and receptor kinetics, and that alcohol-sensitive positions in the M domains of GluN2A and GluN2B subunits differ. In this study we tested for interactions among alcohol-sensitive positions at the M domain intersubunit interfaces in GluN1/GluN2B NMDA receptors. Experimental Approach: We used whole-cell patch-clamp recording in tsA201 cells expressing tryptophan substitution mutants at ethanol-sensitive positions in the GluN1 and GluN2B NMDA receptor subunits to test for interactions among positions. Key Results: Six pairs of positions in GluN1/GluN2B significantly interacted to regulate ethanol inhibition: Gly638/Met824, Gly638/Leu825, Phe639/Leu825, Phe639/Gly826, Met818/Phe637 and Val820/Phe637. Tryptophan substitution at Met824 or Leu825 in GluN2B did not alter ethanol sensitivity but interacted with positions in the GluN1 M3 domain to regulate ethanol action, whereas tryptophan substitution at Gly638, which is the cognate of an ethanol-sensitive position in GluN2A, did not alter ethanol sensitivity or interact with positions in GluN1. Two and three pairs of positions interacted to regulate glutamate steady-state and peak current EC50, respectively, and one pair interacted with respect to macroscopic desensitization. Conclusions: Despite highly-conserved M domain sequences and similar ethanol sensitivity in the GluN2A and GluN2B subunits, the manner in which these subunits interact with the GluN1 subunit to regulate ethanol sensitivity and receptor kinetics differs

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Different Sites of Alcohol Action in the NMDA Receptor GluN2A and GluN2B Subunits

The NMDA receptor is a major target of alcohol action in the CNS, and recent behavioral and cellular studies have pointed to the importance of the GluN2B subunit in alcohol action. We and others have previously characterized four amino acid positions in the third and fourth membrane-associated (M) domains of the NMDA receptor GluN2A subunit that influence both ion channel gating and alcohol sensitivity. In this study, we found that substitution mutations at two of the four corresponding positions in the GluN2B subunit, F637 and G826, influence ethanol sensitivity and ion channel gating. Because position 826 contains a glycine residue in the native protein, we focused our attention on GluN2B(F637). Substitution mutations at GluN2B(F637) significantly altered ethanol IC50 values, glutamate EC50 values for peak (Ip) and steady-state (Iss) current, and steady-state to peak current ratios (Iss:Ip). Changes in apparent glutamate affinity were not due to agonist trapping in desensitized states, as glutamate Iss EC50 values were not correlated with Iss:Ip values. Ethanol sensitivity was correlated with values of both Ip and Iss glutamate EC50, but not with Iss:Ip. Values of ethanol IC50, glutamate EC50, and Iss:Ip for mutants at GluN2B(F637) were highly correlated with the corresponding values for mutants at GluN2A(F636), consistent with similar functional roles of this position in both subunits. These results demonstrate that GluN2B(Phe637) regulates ethanol action and ion channel function of NMDA receptors. However, despite highly conserved M domain sequences, ethanol\u27s actions on GluN2A and GluN2B subunits differ