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Previous studies have shown that amino acid residues in trans-membrane (TM) segments 1, 2 and 3 of the alpha subunit are critical for the enhancement of GABA(A) receptor function by inhaled anesthetics. In this study we used tryptophan (Trp) scanning mutagenesis between Ile 406 and Asn 417 in the alpha1 subunit to determine the effects of Trp substitution in the fourth transmembrane segment (TM4) on receptor gating and anesthetic modulation. Wild-type and mutant alpha1 subunits were transiently expressed in HEK 293 cells with wild-type beta2 and gamma2s subunits and GABA-activated currents were recorded using whole-cell voltage clamp. The potentiation by three inhaled anesthetics (isoflurane, halothane and chloroform) of responses elicited by a submaximal concentration of GABA were also examined.EC(50) values for GABA at the mutant receptors were in the range 4-60 microM (wild-type=20 microM), indicating that Trp substitution can alter the apparent affinity of the receptor for GABA positively or negatively, dependent on position. The variation of the calculated EC(50) value for GABA exhibited an interesting periodicity, with the cycle length for each repeat corresponding to approximately 3.6 amino acids. These data are consistent with an alpha-helical structure for the TM4 segment of the alpha subunit. Several of these Trp point mutations altered the ability of one or more of the three inhaled anesthetics to modulate receptor function; four of the 12 mutations abolished receptor modulation by one or more of the anesthetics tested. These data are consistent with a role for these residues at the extracellular end of TM4 in anesthetic modulation of GABA(A) receptors.

The GABA(A) receptor is an important target for a variety of general anesthetics (Franks and Lieb, 1994) and for benzodiazepines such as diazepam. Specific point mutations in the GABA(A) receptor selectively abolish regulation by benzodiazepines (Rudolph et al., 1999; McKernan et al., 2000) and by anesthetic ethers (Mihic et al., 1997; Krasowski et al., 1998; Koltchine et al., 1999), suggesting the existence of discrete binding sites on the GABA(A) receptor for these drugs. Using anesthetics of different molecular size (isoflurane > halothane > chloroform) together with complementary mutagenesis of specific amino acid side chains, we estimate the volume of a proposed anesthetic binding site as between 250 and 370 A(3). The results of the "cutoff" analysis suggest a common site of action for the anesthetics isoflurane, halothane, and chloroform on the GABA(A) receptor. Moreover, the data support a crucial role for Leu232, Ser270, and Ala291 in the alpha subunit in defining the boundaries of an amphipathic cavity, which can accommodate a variety of small general anesthetic molecules.