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

摘要

Charged residues in the β10-M1 linker region ("pre-Ml") are important in the expression and function of neuro-muscular acetylcholine receptors (AChRs). The perturbation of a salt bridge between pre-Ml 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-Ml (positions M207-P211) plus E45 in loop 2 in the mouse α_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). O values for R209 (on the E45A background), L210, and E45 were 0.74, 0.35, and 0.80, respectively. Φ 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 a subunits, combined). Pre-Ml 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.