title: Gamma-aminobutyric acid increases the water accessibility of M3 membrane-spanning segment residues in gamma-aminobutyric acid type A receptors.
abstract: Gamma-aminobutyric acid type A (GABA(A)) receptors are members of the ligand-gated ion channel gene superfamily. Using the substituted cysteine accessibility method, we investigated whether residues in the alpha(1)M3 membrane-spanning segment are water-accessible. Cysteine was substituted, one at a time, for each M3 residue from alpha(1)Ala(291) to alpha(1)Val(307). The ability of these mutants to react with the water-soluble, sulfhydryl-specific reagent pCMBS(-) was assayed electrophysiologically. Cysteines substituted for alpha(1)Ala(291) and alpha(1)Tyr(294) reacted with pCMBS(-) applied both in the presence and in the absence of GABA. Cysteines substituted for alpha(1)Phe(298), alpha(1)Ala(300), alpha(1)Leu(301), and alpha(1)Glu(303) only reacted with pCMBS(-) applied in the presence of GABA. We infer that the pCMBS(-) reactive residues are on the water-accessible surface of the protein and that GABA induces a conformational change that increases the water accessibility of the four M3 residues, possibly by inducing the formation of water-filled crevices that extend into the interior of the protein. Others have shown that mutations of alpha(1)Ala(291), a water-accessible residue, alter volatile anesthetic and ethanol potentiation of GABA-induced currents. Water-filled crevices penetrating into the interior of the membrane-spanning domain may allow anesthetics and alcohol to reach their binding sites and thus may have implications for the mechanisms of action of these agents.
authors.affiliation: Center for Molecular Recognition, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.
authors.forename: D B
authors.initials: DB
authors.lastname: Williams
authors.forename: M H
authors.initials: MH
authors.lastname: Akabas
mesh_terms: D002731:4-Chloromercuribenzenesulfonate / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D003545:Cysteine; D057927:Hydrophobic and Hydrophilic Interactions; D015640:Ion Channel Gating / Q000187:drug effects; D008698:Mesylates / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D051381:Rats; D011963:Receptors, GABA-A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013499:Surface Properties; D014867:Water / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology*
chemical_list: D008698:Mesylates; D011963:Receptors, GABA-A; D013438:Sulfhydryl Compounds; D014867:Water; C074502:methanethiosulfonate; D005680:gamma-Aminobutyric Acid; D002731:4-Chloromercuribenzenesulfonate; D003545:Cysteine