Considerable controversy surrounds the location of the closed channel gate in members of the Cys-loop receptor family of neurotransmitter-gated ion channels that includes the GABAA, glycine, acetylcholine, and 5-HT3 receptors. Cysteine-accessibility studies concluded that the gate is near the cytoplasmic end of the channel in acetylcholine and GABA A receptors but in the middle of the 5-HT3A receptor channel. Zn2+ accessibility studies in a chimeric 5-HT 3-ACh receptor suggested the gate is near the channel's cytoplasmic end. In the 4-Å resolution structure of the acetylcholine receptor closed state determined by cryoelectron microscopy, the narrowest region, inferred to be the gate, is in the channel's midsection from 9′ to 14′ but the M1-M2 loop residues at the channel's cytoplasmic end were not resolved in that structure. We used blocker trapping experiments with picrotoxin, a GABA A receptor open channel blocker, to determine whether a gate exists at a position more extracellular than the picrotoxin binding site, which is in the vicinity of α1 Val257 (2′) near the channel's cytoplasmic end. We show that picrotoxin can be trapped in the channel after removal of GABA. By using the state-dependent accessibility of engineered cysteines as reporters for the channel's structural state we infer that after GABA washout, with picrotoxin trapped in the channel, the channel appears to be in the closed state. We infer that a gate exists between the picrotoxin binding site and the channel's extracellular end, consistent with a closed channel gate in the middle of the channel. Given the homology with acetylcholine and 5-HT3 receptors there is probably a similar gate in those channels as well. This does not preclude the existence of an additional gate at a more cytoplasmic location.
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