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.
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