IDENTIFICATION OF ACETYLCHOLINE-RECEPTOR CHANNEL-LINING RESIDUES IN THE ENTIRE M2 SEGMENT OF THE ALPHA-SUBUNIT

Each residue in and flanking the M2 membrane-spanning segment of the a subunit, from Glu-241 to Glu-262, was mutated to cysteine, and the mu tant subunits were expressed together with wild-type beta, gamma, and delta subunits in Xenopus oocytes. Cysteines substituted for Glu-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, sulfhy dryl-specific reagent methanethiosulfonate ethylammonium (MTSEA), adde d extracellularly. These 10 residues, therefore, are exposed in the ch annel lumen. The pattern of exposure is compatible with an a helix, in terrupted by an extended structure from Leu-250 to Ser-252. Acetylchol ine caused subtle changes in the accessibilities of some of the engine ered cysteines. Since all 10 residues are accessible to MTSEA in the c losed state of the channel, the channel gate is at least as cytoplasmi c as Glu-241, the most cytoplasmic of the residues tested.