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Charged residues in the [beta]10-M1 linker region ('pre-M1') are important in the expression and traction 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].sub.1]-subunit. M207, Q208, and P211 mutants caused small (approximately threefold) changes in the gating equilibrium constant ([K.sub.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.sub.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.

Acetylcholine receptor channel gating is a propagated conformational cascade that links changes in structure and function at the transmitter binding sites in the extracellular domain (ECD) with those at a 'gate' in the transmembrahe domain (TMD). We used [PHI]-value analysis to probe the relative timing of the gating motions of [alpha]-subunit residues located near the ECD-TMD interface. Mutation of font of the seven amino acids in the M2-M3 linker (which connects the pore-lining M2 helix with the M3 helix), including three of the four residues in the core of the linker, changed the diliganded gating equilibrium constant ([K.sub.eq]) by up to 10,000-fold (P272 > I274 > A270 > G275). The average [PHI]-value for the whole linker was ~0.64. One interpretation of this result is that the gating motions of the M2-M3 linker are approximately synchronous with those of much of M2 (~0.64), but occur after those of the transmitter binding site region (~0.93) and loops 2 and 7 (~0.77). We also examined mutants of six cys-loop residues (V132, T133, H134, F135, P136, and F137). Mutation of V132, H134, and F135 changed [K.sub.eq] by 2800-, 10-, and 18-fold, respectively, and with an average [PHI]-value of 0.74, similar to those of other cys-loop residues. Even though V132 and 1274 are close, the energetic coupling between I and V mutants of these positions was small ([less than or equal to] 0.51 kcal [mol.sup.-1]). The M2-M3 linker appears to be the key moving part that couples gating motions at the base of the ECD with those in TMD. These interactions are distributed along an ~16-[Angstrom] border and involve about a dozen residues.