Interaction of picrotoxin with GABA(A) receptor channel-lining residues probed in cysteine mutants

We used the substituted-cysteine-accessibility method to identify the channel-lining residues in a region (257-261) near the putative cytoplasmic end of the M2 membrane-spanning segment of the rat gamma-aminobutyric acid type A (GABA(A)) receptor alpha(1) subunit. The residues alpha(1)Val257 and alpha(1)Thr261 were accessible to charged, sulfhydryl-specific reagents applied extracellularly in both the open and closed states. The accessibility of alpha(1)V257C and alpha(1)T261C in the closed state implies that the gate must be at least as close to the cytoplasmic end of the channel as alpha(1)Val257. Also, the positively charged reagent methanethiosulfonate ethylammonium penetrated from the extracellular end of the channel to alpha(1)T261C, with which it reacted, indicating that the anion-selectivity filter is closer to the cytoplasmic end of the channel than this residue is. Go-application of picrotoxin prevented the sulfhydryl reagents from reacting with alpha(1)V257C but did not prevent reaction with the more extracellular residue alpha(1)T261C. Picrotoxin protection of alpha(1)V257C may be due to steric block by picrotoxin bound in the channel at the level of alpha(1)Val257; however, if this protection is allosteric, it is not due to the induction of the resting closed state in which alpha(1)V257C was accessible to sulfhydryl reagent.