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Each residue in and flanking the M2 membrane-spanning segment of the a subunit, from Clu-241 to Clu-262, was mutated to cysteine, and the mutant subunits were expressed together with wild-type g, y, and 8 subunits in Xenopus oocytes. Cysteines substituted for Clu-262, Leu-258, Val-255, Ser-252, Leu-251, Leu-250, Ser-248, Leu-245, Thr-244, and Glu-241 reacted with the positively charged, hydrophilic, sulfhydryl-specific reagent methanethiosulfonate ethylammonium (MTSEA), added extracellularly. These 10 residues, therefore, are exposed in the channel lumen. The pattern of exposure is compatible with an a helix, interrupted by an extended structure from Leu-250 to Ser-252. Acetylcholine caused subtle changes in the accessibilities of some of the engineered cysteines. Since all 10 residues are accessible to MTSEA in the closed state of the channel, the channel gate is at least as cytoplasmic as Clu-241 …