title: Ethanol inhibition of constitutively open N-methyl-D-aspartate receptors.
abstract: N-Methyl-D-aspartate (NMDA) receptors gate a slow and calcium-rich component of the postsynaptic glutamate response. Like all ionotropic glutamate receptors, NMDA subunits contain a highly conserved motif (SYTANLAAF) in the transmembrane (TM) 3 domain that is critically involved in channel gating. Mutation of an alanine in this domain (A7; underlined above) results in constitutively open receptors that show reduced sensitivity to several allosteric modulators. In this study, we examined the effects of ethanol, a substance that inhibits NMDA currents via an unknown mechanism, on tonically active NMDA receptors expressed in human embryonic kidney 293 cells. Ethanol (100 mM) inhibited currents from GluN1(A7R)/GluN2A and GluN1(A7R)/GluN2B receptors by approximately 50%, whereas those from GluN1/GluN2B(A7R) receptors were reduced by less than 10%. In cysteine-substituted GluN1 and GluN2 A7 mutants, estimated ethanol IC₅₀ values for agonist-gated currents were 101, 117, 103, and 69 mM for GluN1(A7C)/GluN2A, GluN1(A7C)/GluN2B, GluN1/GluN2A(A7C), and GluN1/GluN2B(A7C) receptors, respectively. After exposure to the thiol-modifying reagent 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET), A7C mutants showed robust agonist-independent currents and reduced sensitivity to ethanol (IC₅₀ values of 371, 256, 715, and 958 mM, respectively, as above). In contrast, cysteine modification of the ligand-binding domain resulted in constitutively open receptors that showed robust ethanol inhibition. Ethanol inhibition of MTSET-treated GluN1(A7C) receptors was further reduced by TM3/TM4 mutations previously shown to reduce ethanol sensitivity of agonist-gated receptors. Overall, these results show that ethanol affects NMDA receptor function at a site distal from agonist binding and appears to exert greater effects via perturbation of GluN2 subunits.
authors: Department of Neurosciences, Medical University of South Carolina, Charleston, SC 29425, USA.
authors: Minfu
authors: M
authors: Xu
authors: C Thetford
authors: CT
authors: Smothers
authors: James
authors: J
authors: Trudell
authors: John J
authors: JJ
authors: Woodward
mesh_terms: D019943:Amino Acid Substitution; D001120:Arginine / Q000502:physiology; D002478:Cells, Cultured; D002492:Central Nervous System Depressants / Q000494:pharmacology*; D003545:Cysteine / Q000502:physiology; D018076:DNA, Complementary / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D055724:Electrophysiological Phenomena; D000431:Ethanol / Q000494:pharmacology*; D006801:Humans; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics / Q000502:physiology; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D014162:Transfection
chemical_list: D002492:Central Nervous System Depressants; D018076:DNA, Complementary; D004220:Disulfides; D016194:Receptors, N-Methyl-D-Aspartate; D000431:Ethanol; D001120:Arginine; D003545:Cysteine