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Ethanol\u2010insensitive mutant NMDA receptors would be valuable molecular tools for evaluating the roles of NMDA receptors in ethanol actions. However, mutations that decrease ethanol sensitivity also usually alter ion channel gating, which would likely affect glutamatergic synaptic transmission and central nervous system (CNS) function. We selected candidate ethanol\u2010insensitive mutations in the GluN2A and GluN2B subunits with relatively low disruption of ion channel gating: isoleucine substitution at two positions (F636\/F637) in M3 of GluN2A and tryptophan substitution at G826 in M4 of GluN2B. Our strategy was to introduce additional mutations in the ligand\u2010binding domain (LBD), if needed, attempting to restore normal receptor kinetics while preserving low alcohol sensitivity.<\/jats:p><\/jats:sec><jats:sec><jats:title>Experimental Approach<\/jats:title><jats:p>We determined the alcohol sensitivity and gating parameters of the mutant subunits using whole\u2010cell patch\u2010clamp and outside\u2010out patch concentration\u2010jump recording in a cultured cell line.<\/jats:p><\/jats:sec><jats:sec><jats:title>Key Results<\/jats:title><jats:p>While the majority of GluN2A LBD mutations disrupted receptor function, the GluN2A(E413D\/F636I\/F637I) mutant subunit had approximately normal glutamate potency while retaining low alcohol sensitivity. LBD mutations were unnecessary in the GluN2B(G826W) subunit because glutamate potency and desensitization were unchanged compared to the wild\u2010type subunit. In concentration\u2010jump experiments, both mutant subunits yielded simulated synaptic currents similar to wild\u2010type currents.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion and Implications<\/jats:title><jats:p>The GluN2A(E413D\/F636I\/F637I) and GluN2B(G826W) mutant subunits exhibit nearly normal physiology and low alcohol sensitivity. These studies also provide evidence that changes in NMDA receptor ethanol sensitivity are not dependent upon changes in ion channel gating.<\/jats:p><\/jats:sec>","DOI":"10.1111\/bph.70107","type":"journal-article","created":{"date-parts":[[2025,6,24]],"date-time":"2025-06-24T20:08:30Z","timestamp":1750795710000},"page":"5214-5225","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Identification of ethanol\u2010insensitive <i>N<\/i>\u2010methyl\u2010<scp>d<\/scp>\u2010aspartate receptor GluN2A and GluN2B subunit mutants with minimal alterations in ion channel gating"],"prefix":"10.1111","volume":"182","author":[{"given":"Claire M.","family":"Fellbaum","sequence":"first","affiliation":[{"name":"Department of Biomedical Sciences Marquette University  Milwaukee Wisconsin USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"John 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