Acetylcholine Receptor Gating at Extracellular Transmembrane Domain Interface: the “Pre-M1” Linker

Charged residues in the beta10-M1 linker region ("pre-M1") are important in the expression and function of neuromuscular acetylcholine receptors (AChRs). The perturbation of a salt bridge between pre-M1 residue R209 and loop 2 residue E45 has been proposed as being a principle event in the AChR gating conformational "wave." We examined the effects of mutations to all five residues in pre-M1 (positions M207-P211) plus E45 in loop 2 in the mouse alpha(1)-subunit. M207, Q208, and P211 mutants caused small (approximately threefold) changes in the gating equilibrium constant (K(eq)), but the changes for R209, L210, and E45 were larger. Of 19 different side chain substitutions at R209 on the wild-type background, only Q, K, and H generated functional channels, with the largest change in K(eq) (67-fold) from R209Q. Various R209 mutants were functional on different E45 backgrounds: H, Q, and K (E45A), H, A, N, and Q (E45R), and K, A, and N (E45L). Phi values for R209 (on the E45A background), L210, and E45 were 0.74, 0.35, and 0.80, respectively. Phi values for R209 on the wt and three other backgrounds could not be estimated because of scatter. The average coupling energy between 209/45 side chains (six different pairs) was only -0.33 kcal/mol (for both alpha subunits, combined). Pre-M1 residues are important for expression of functional channels and participate in gating, but the relatively modest changes in closed- vs. open-state energy caused mutations, the weak coupling energy between these residues and the functional activity of several unmatched-charge pairs are not consistent with the perturbation of a salt bridge between R209 and E45 playing the principle role in gating.