Agonist Gating and Isoflurane Potentiation in the Human γ-Aminobutyric Acid Type A Receptor Determined by the Volume of a Second Transmembrane Domain Residue

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Abstract
γ-Aminobutyric acid type A (GABAA )receptors are targets for allosteric modulation by general anesthetics. Mutation of Ser270 within the second transmembrane domain of the GABAAreceptor α subunit can ablate the modulation of the receptor by the anesthetic ether isoflurane. To investigate further the function of this critical amino acid residue, we made multiple amino acid substitutions at Ser270 and analyzed the concentration-dependent gating by GABA and regulation by isoflurane in each mutant receptor. There is a strong negative correlation between the EC50 for GABA and the molecular volume of the amino acid residue at position 270. Replacement of Ser by large residues such as His and Trp produced a shift of the GABA concentration-response curve to the left, whereas replacement of Ser with Gly had the opposite effect. There also was a strong negative association between the molecular volume of the amino acid residue at 270 and the degree of enhancement of submaximal GABA responses by isoflurane. These results indicate the significance of the amino acid at position α270 in gating of the GABAAreceptor. In addition, the data on isoflurane are consistent with the existence of a cavity of finite size in the region of α270 that may be filled by the anesthetic molecule or by the side chain of a larger residue at α270. The introduction of isoflurane, or of a large residue, into this cavity may stabilize the open state of the GABAA receptor relative to the closed state.