γ-Aminobutyric Acid Increases the Water Accessibility of M3 Membrane-Spanning Segment Residues in γ-Aminobutyric Acid Type A Receptors

Abstract

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 α 1M3 membrane-spanning segment are water-accessible. Cysteine was substituted, one at a time, for each M3 residue from α 1Ala 291 to α 1Val 307. The ability of these mutants to react with the water-soluble, sulfhydryl-specific reagent pCMBS − was assayed electrophysiologically. Cysteines substituted for α 1Ala 291 and α 1Tyr 294 reacted with pCMBS − applied both in the presence and in the absence of GABA. Cysteines substituted for α 1Phe 298, α 1Ala 300, α 1Leu 301, and α 1Glu 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 α 1Ala 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.