Abstract
Mutagenesis studies have suggested that the second transmembrane segment (M2) plays a critical role during acetylcholine receptor liganded gating. An adequate description of the relationship between gating and structure of the M2 domain, however, has been hampered by the fact that many M2 mutations increase the opening rate constant to levels that, in the presence of acetylcholine, are unresolvably fast. Here, we show that the use of saturating concentrations of choline, a low-efficacy agonist, is a convenient tool to circumvent this problem. In the presence of 20 mM choline: (a) single-channel currents occur in clusters; (b) fast blockade by choline itself reduces the single-channel conductance by approximately 50%, yet the excess open-channel noise is only moderate; (c) the kinetics of gating are fitted best by a single-step, C <--> O model; and (d) opening and closing rate constants are within a well resolvable range. Application of this method to a series of recombinant adult mouse muscle M2 12' mutants revealed that: (a) the five homologous M2 12' positions make independent and asymmetric contributions to diliganded gating, the delta subunit being the most sensitive to mutation; (b) mutations at delta12' increase the diliganded gating equilibrium constant in a manner that is consistent with the sensitivity of the transition state to mutation being approximately 30% like that of the open state and approximately 70% like that of the closed state; (c) the relationship between delta12' amino acid residue volume, hydrophobicity or alpha-helical tendency, and the gating equilibrium constant of the corresponding mutants is not straightforward; however, (d) rate and equilibrium constants for the mutant series are linearly correlated (on log-log plots), which suggests that the conformational rearrangements upon mutation are mostly local and that the position of the transition state along the gating reaction coordinate is unaffected by these mutations.