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 acetylcholin e receptors at the single-channel level. Hydrophobic mutations (Ile, Cys, a nd Phe) placed near the middle of the linker of the or subunit (alpha S269) prolong apparent openings elicited by low concentrations of acetylcholine (ACh), whereas hydrophilic mutations (Asp, Lys, and Gin) are without: effec t. Because the gating kinetics of the alpha S269I receptor (a congenital my asthenic syndrome mutant) in the presence of ACh are too fast, choline was used as the agonist. This revealed an similar to 92-fold increased gating e quilibrium constant, which is consistent with all similar to 10-fold decrea sed EC50 in the presence of ACh. With choline, this mutation accelerates ch annel opening similar to 28-fold, slows channel closing similar to 3-fold, but does not affect agonist binding to the closed state. These ratios sugge st that, with ACh, alpha S269I acetylcholine receptors open at a rate of si milar to 1.4 X 10(6) s(-1) and close at a rate of similar to 760 s(-1). The se gating rate constants, together with the measured duration of apparent o penings at low ACh concentrations, further suggest that ACh dissociates fro m the diliganded open receptor at a rate of similar to 140 s(-1). Ile mutat ions at positions flanking alpha S269 impair, rather than enhance, channel gating. Inserting or deleting one residue from this linker in the alpha sub unit increased and decreased, respectively, the apparent open time approxim ately twofold. Contrary to the alpha S269I mutation, Ile mutations at equiv alent positions of the beta, epsilon, and delta subunits do not affect appa rent open-channel lifetimes. However, in beta and epsilon, shifting the mut ation one residue to the NH2-terminal end enhances channel gating. The over all results indicate that this linker is a control element whose hydrophobi city determines channel gating in a position- and subunit-dependent manner. Characterization of the transition state of the gating reaction suggests t hat during channel opening the M2-M3L of the alpha subunit: moves before th e corresponding linkers of the beta and epsilon subunits.