Mutations at F637 in the NMDA receptor NR2A subunit M3 domain influence agonist potency, ion channel gating and alcohol action

Background and purpose: NMDA receptors are important molecular targets of ethanol action in the CNS. Previous studies have identified a site in membrane-associated domain 3 ( M3) of the NR1 subunit and two sites in M4 of the NR2A subunit that influence alcohol action; the sites in NR2A M4 also regulate ion channel gating. The purpose of this study was to determine whether mutations at the site in the NR2A subunit corresponding to the NR1 M3 site influence alcohol action and ion channel gating. Experimental approach: We investigated the effects of mutations at phenylalanine ( F) 637 of the NR2A subunit using whole-cell and single-channel patch-clamp electrophysiological recording in transiently-transfected HEK 293 cells. Key results: Mutations at F637 in the NR2A subunit altered peak and steady-state glutamate EC50 values, maximal steady- state to peak current ratios ( I-ss: I-p), mean open time, and ethanol IC50 values. Differences in glutamate potency among the mutants were not due to changes in desensitization. Ethanol IC50 values were significantly correlated with glutamate EC50 values, but not with maximal I-ss: I-p or mean open time. Ethanol IC50 values were linearly and inversely related to molecular volume of the substituent. Conclusions and implications: These results demonstrate that NR2A(F637) influences NMDA receptor affinity, ion channel gating, and ethanol sensitivity. The changes in NMDA receptor affinity are likely to be the result of altered ion channel gating. In contrast to the cognate site in the NR1 subunit, the action of ethanol does not appear to involve occupation of a critical volume at NR2A(F637).