Tryptophan and Cysteine Mutations in M1 Helices of &agr;1&bgr;3&ggr;2L &ggr;-Aminobutyric Acid Type A Receptors Indicate Distinct Intersubunit Sites for Four Intravenous Anesthetics and One Orphan Site

Background:&ggr;-Aminobutyric acid type A (GABAA) receptors mediate important effects of intravenous general anesthetics. Photolabel derivatives of etomidate, propofol, barbiturates, and a neurosteroid get incorporated in GABAA receptor transmembrane helices M1 and M3 adjacent to intersubunit pockets. However, photolabels have not been consistently targeted at heteromeric &agr;&bgr;&ggr; receptors and do not form adducts with all contact residues. Complementary approaches may further define anesthetic sites in typical GABAA receptors. Methods:Two mutation-based strategies, substituted tryptophan sensitivity and substituted cysteine modification–protection, combined with voltage-clamp electrophysiology in Xenopus oocytes, were used to evaluate interactions between four intravenous anesthetics and six amino acids in M1 helices of &agr;1, &bgr;3, and &ggr;2L GABAA receptor subunits: two photolabeled residues, &agr;1M236 and &bgr;3M227, and their homologs. Results:Tryptophan substitutions at &agr;1M236 and positional homologs &bgr;3L231 and &ggr;2L246 all caused spontaneous channel gating and reduced &ggr;-aminobutyric acid EC50. Substituted cysteine modification experiments indicated etomidate protection at &agr;1L232C and &agr;1M236C, R-5-allyl-1-methyl-5-(m-trifluoromethyl-diazirinylphenyl) barbituric acid protection at &bgr;3M227C and &bgr;3L231C, and propofol protection at &agr;1M236C and &bgr;3M227C. No alphaxalone protection was evident at the residues the authors explored, and none of the tested anesthetics protected &ggr;2I242C or &ggr;2L246C. Conclusions:All five intersubunit transmembrane pockets of GABAA receptors display similar allosteric linkage to ion channel gating. Substituted cysteine modification and protection results were fully concordant with anesthetic photolabeling at &agr;1M236 and &bgr;3M227 and revealed overlapping noncongruent sites for etomidate and propofol in &bgr;+–&agr;– interfaces and R-5-allyl-1-methyl-5-(m-trifluoromethyl-diazirinylphenyl) barbituric acid and propofol in &agr;+–&bgr;– and &ggr;+–&bgr;– interfaces. The authors’ results identify the &agr;+–&ggr;– transmembrane interface as a potentially unique orphan modulator site.

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