Identification of Acetylcholine Receptor Channel-Lining Residues in the M1 Segment of the β-Subunit

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Abstract
The substituted cysteine accessibility method (SCAM) was applied to the first membrane-spanning segment (M1) of the mouse-muscle acetylcholine (ACh) receptor β subunit. One at a time, each residue from βR219 to βP247, except βC233, was mutated to Cys, and the mutant β subunits were expressed together with wild-type α, γ, and δ 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 M1, βF224C, βY225C, and βL226C, reacted both in the absence and presence of ACh, and one deeper residue, βV229C reacted only in the presence of ACh. βV229C also reacted with 2-aminoethyl-2-aminoethanethiosulfonate (AEAETS) and with 2-hydroxyethyl MTS (MTSEH). The rate constants for the reactions of βV229C 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 βV229C in the open channel. The N-terminal third of βM1, like that of αM1, contributes to the lining of the channel and undergoes structural changes during gating.