Identification of Ethanol-Insensitive N-Methyl-D-Aspartate Receptor Glun2A and Glun2B Subunit Mutants with Minimal Alterations in Ion Channel Gating
Document Type
Article
Publication Date
11-2025
Publisher
Wiley
Source Publication
British Journal of Pharmacology
Source ISSN
0007-1188
Original Item ID
DOI: 10.1111/bph.70107
Abstract
Background and Purpose
Ethanol inhibits N-methyl-d-aspartate (NMDA) receptors through actions on positions in the membrane-associated (M) domains. Ethanol-insensitive 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-insensitive 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-binding domain (LBD), if needed, attempting to restore normal receptor kinetics while preserving low alcohol sensitivity.
Experimental Approach
We determined the alcohol sensitivity and gating parameters of the mutant subunits using whole-cell patch-clamp and outside-out patch concentration-jump recording in a cultured cell line.
Key Results
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-type subunit. In concentration-jump experiments, both mutant subunits yielded simulated synaptic currents similar to wild-type currents.
Conclusion and Implications
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.
Recommended Citation
Fellbaum, Claire M.; McKee, John L.; Zhao, Yulin; Harris, Josephy M.; Padua, Danicamae; Katti, Rohan; Mitra, Tarini; Levahn, John L.; Katti, Priya; Ren, Hong; and Peoples, Robert W., "Identification of Ethanol-Insensitive N-Methyl-D-Aspartate Receptor Glun2A and Glun2B Subunit Mutants with Minimal Alterations in Ion Channel Gating" (2025). Biomedical Sciences Faculty Research and Publications. 267.
https://epublications.marquette.edu/biomedsci_fac/267
Comments
British Journal of Pharmacology, Vol. 182, No. 21 (November 2025): 5214-5225.DOI.