The extracellular linker of muscle acetylcholine receptor channels is a gating control element

We describe the functional consequences of mutations in the linker between the second and third transmembrane segments (M2-M3L) of muscle acetylcholine receptors at the single-channel level. Hydrophobic mutations (Ile, Cys, and Phe) placed near the middle of the linker of the α subunit (αS269) prolong apparent openings elicited by low concentrations of acetylcholine (ACh), whereas hydrophilic mutations (Asp, Lys, and Gln) are without effect. Because the gating kinetics of the αS269I receptor (a congenital myasthenic syndrome mutant) in the presence of ACh are too fast, choline was used as the agonist. This revealed an ~92-fold increased gating equilibrium constant, which is consistent with an ~10-fold decreased EC₅₀ in the presence of ACh. With choline, this mutation accelerates channel opening ~28-fold, slows channel closing ~3-fold, but does not affect agonist binding to the closed state. These ratios suggest that, with ACh, αS269I acetylcholine receptors open at a rate of ~1.4 x 10⁶ s^-1 and close at a rate of ~760 s^-1. These gating rate constants, together with the measured duration of apparent openings at low ACh concentrations, further suggest that ACh dissociates from the diliganded open receptor at a rate of ~140 s^-1. Ile mutations at positions flanking αS269 impair, rather than enhance, channel gating. Inserting or deleting one residue from this linker in the α subunit increased and decreased, respectively, the apparent open time approximately twofold. Contrary to the αS269I mutation, Ile mutations at equivalent positions of the β, ε, and δ subunits do not affect apparent open-channel lifetimes. However, in β and ε, shifting the mutation one residue to the NH₂-terminal end enhances channel gating. The overall results indicate that this linker is a control element whose hydrophobicity determines channel gating in a position- and subunit-dependent manner. Characterization of the transition state of the gating reaction suggests that during channel opening the M2-M3L of the α subunit moves before the corresponding linkers of the β and ε subunits.