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.
|Original language||English (US)|
|Number of pages||4|
|Journal||Journal of Biological Chemistry|
|State||Published - 1993|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology