Biophys J, November 1999, p. 2563-2574, Vol. 77, No. 5

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

 ^*Center for Molecular Recognition,  ^#Integrated Program in Cellular, Molecular, and Biophysical Studies, and  ^§Department of Physiology and Cellular Biophysics and Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032 USA

-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 ₁M3 membrane-spanning segment are water-accessible. Cysteine was substituted, one at a time, for each M3 residue from ₁Ala²⁹¹ to ₁Val³⁰⁷. The ability of these mutants to react with the water-soluble, sulfhydryl-specific reagent pCMBS was assayed electrophysiologically. Cysteines substituted for ₁Ala²⁹¹ and ₁Tyr²⁹⁴ reacted with pCMBS applied both in the presence and in the absence of GABA. Cysteines substituted for ₁Phe²⁹⁸, ₁Ala³⁰⁰, ₁Leu³⁰¹, and ₁Glu³⁰³ 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 ₁Ala²⁹¹, 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.

Biophys J, November 1999, p. 2563-2574, Vol. 77, No. 5
© 1999 by the Biophysical Society   0006-3495/99/11/2563/12  $2.00

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