Amino acids lining the channel of the γ-aminobutyric acid type A receptor identified by cysteine substitution

The binding of γ-aminobutyric acid (GABA) to γ-aminobutyric acid type A (GABA(A)) receptors triggers the opening of an anion-selective channel. To identify amino acid residues that line the channel, we combined cysteine mutagenesis and covalent chemical modification. We mutated, one at a time, four consecutive residues (268-271) in the M2 membrane-spanning segment of the rat GABA(A) receptor α₁ subunit to cysteine and expressed the mutant α₁ subunits, together with either the β₁ subunit or the β₁ and γ₂ subunits, in Xenopus oocytes. We probed the susceptibility of the cysteine substitution mutants to covalent modification by charged, sulfhydryl reagents added extracellularly. We assumed that among the residues in membrane- spanning segments, only those lining the channel would be susceptible to modification by polar reagents and that such modification would irreversibly alter conduction. We infer that the residues Thr-268 and Ile-271 are exposed in the channel in both the open and closed states but that Leu-269 and Ser- 270 are not exposed. The susceptibility of Thr-268 and Ile-271 in the closed state implies that the gate must be closer to the cytoplasmic end of the channel than Thr-268.