AMINO-ACIDS LINING THE CHANNEL OF THE GAMMA-AMINOBUTYRIC-ACID TYPE-A RECEPTOR IDENTIFIED BY CYSTEINE SUBSTITUTION

The binding of gamma-aminobutyric acid (GABA) to gamma-aminobutyric ac id type A (GABA(A)) receptors triggers the opening of an anion-selecti ve channel. To identify amino acid residues that line the channel, we combined cysteine mutagenesis and covalent chemical modification. We m utated, one at a time, four consecutive residues (268-271) in the M2 m embrane-spanning segment of the rat GABA(A) receptor alpha1 subunit to cysteine and expressed the mutant alpha1 subunits, together with eith er the beta1 subunit or the beta1 and gamma2 subunits, in Xenopus oocy tes. We probed the susceptibility of the cysteine substitution mutants to covalent modification by charged, sulfhydryl reagents added extrac ellularly. We assumed that among the residues in membrane-spanning seg ments, only those lining the channel would be susceptible to modificat ion by polar reagents and that such modification would irreversibly al ter conduction. We infer that the residues Thr-268 and Ile-271 are exp osed in the channel in both the open and closed states but that Leu-26 9 and Ser-270 are not exposed. The susceptibility of Thr-268 and Ile-2 71 in the closed state implies that the gate must be closer to the cyt oplasmic end of the channel than Thr-268.