γ-Aminobutyric acid increases the water accessibility of M3 membrane- spanning segment residues in γ-aminobutyric acid type A receptors

γ-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 α₁M3 membrane-spanning segment are water-accessible. Cysteine was substituted, one at a time, for each M3 residue from α1Ala²⁹¹ to α₁Val³⁰⁷. The ability of these mutants to react with the water-soluble, sulfhydryl-specific reagent pCMBS^- was assayed electrophysiologically. Cysteines substituted for α₁Ala²⁹¹ and α₁Tyr²⁹⁴ reacted with pCMBS^- applied both in the presence and in the absence of GABA. Cysteines substituted for α₁Phe²⁹⁸, α₁Ala³⁰⁰, α₁Leu³⁰¹, and α₁Glu³⁰³ 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 α₁Ala²⁹¹, 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.