Agonist gating and isoflurane potentiation in the human gamma-aminobutyricacid type A receptor determined by the volume of a second transmembrane domain residue

gamma-Aminobutyric acid type A (GABA(A)) receptors are targets for alloster ic modulation by general anesthetics. Mutation of Ser270 within the second transmembrane domain of the GABA(A) receptor a subunit can ablate the modul ation of the receptor by the anesthetic ether isoflurane. To investigate fu rther the function of this critical amino acid residue, we made multiple am ino acid substitutions at Ser270 and analyzed the concentration-dependent g ating by GABA and regulation by isoflurane in each mutant receptor. There i s 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 la rge 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 op posite effect. There also was a strong negative association between the mol ecular volume of the amino acid residue at 270 and the degree of enhancemen t of submaximal GABA responses by isoflurane. These results indicate the si gnificance of the amino acid at position alpha 270 in gating of the GABA(A) receptor. In addition, the data on isoflurane are consistent with the exis tence of a cavity of finite size in the region of alpha 270 that may be fil led by the anesthetic molecule or by the side chain of a larger residue at alpha 270. The introduction of isoflurane, or of a large residue, into this cavity may stabilize the open state of the GABA(A) receptor relative to th e closed state.