The muscle-type acetylcholine (ACh) receptor has the composition (α2βγδ. The subunits are arranged quasisymmetrically around a central, ion-conducting, water-filled channel. Each subunit has four membrane-spanning segments, M1-M4, and the channel through the membrane is formed among these segments. Substituting cysteine for each of the residues in and flanking the αM2 segment, we previously found that, at 10 of the 21 mutated positions, the cysteine was accessible to a small, positively charged, sulfhydryl-specific reagent, methanethiosulfonate ethylammonium (MTSEA), and inferred that the residues at these positions are exposed in the channel lumen. We have now applied the substitutedcysteine- accessibility method to αM1. We analyzed 15 consecutive residues, starting at αPro211 at the extracellular end of M1. Wild-type a contains Cys222, which is inaccessible to MTSEA. We mutated each of the other 14 residues to cysteine and expressed the mutant a subunits, together with wild-type β, γ, and δ subunits, in Xenopus oocytes. Thirteen of the fourteen mutants gave robust ACh-induced currents. MTSEA irreversibly altered the ACh-induced response of seven cysteine-substitution mutants: αY213C was susceptible to MSTEA added in the presence or the absence of ACh, αP211C, αI215C, αV216C, αN217C, and αI220C were susceptible in the absence of ACh, and αV218C was susceptible in the presence of ACh. These results imply that Ml is exposed in the channel, and its exposure changes during gating or desensitization. From the pattern of exposure of structure of the extracellular half of Ml is irregular.
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