Identification of acetylcholine receptor channel-lining residues in the M1 segment of the β-subunit

The substituted cysteine accessibility method (SCAM) was applied to the first membrane-spanning segment (M1) of the mouse-muscle acetylcholine (ACh) receptor beta subunit. One at a time, each residue from beta R219 to beta P247, except beta C233, was mutated to Cys, and the mutant beta subunits were expressed together with wild-type alpha, gamma, and delta in Xenopus oocytes. All 28 mutants yielded functional receptors. The accessibility of the substituted Cys to the methanethiosulfonate (MTS) derivatives, MTS ethylammonium (MTSEA), MTS ethyltrimethylammonium (MTSET), and MTS ethylsulfonate (MTSES), added extracellularly in the absence or the presence of ACh, was inferred from their irreversible effects on ACh-induced current, Three consecutive residues close to the extracellular end of Mi, beta F224C, beta Y225C, and beta L226C, reacted both in the absence and presence of ACh, and one deeper residue, beta V229C reacted only in the presence of ACh. beta V229C also reacted with 2-aminoethyl-2-aminoethanethiosulfonate (AEAETS) and with 2-hydroxyethyl MTS (MTSEH). The rate constants for the reactions of beta V229C with MTSEA, which permeates the open channel, and with MTSEH, which is uncharged, were independent of membrane potential. The rate constant for the reaction of the doubly positively charged AEAETS, however, was dependent on membrane potential, consistent with the exposure of beta V229C in the open channel. The N-terminal third of beta M1, like that of alpha M1, contributes to the lining of the channel and undergoes structural changes during gating.