Asymmetric and independent contribution of the second transmembrane segment 12 ' residues to diliganded gating of acetylcholine receptor channels - Asingle-channel study with choline as the agonist

Mutagenesis studies have suggested that the second transmembrane segment (M 2) plays a critical role during acetylcholine receptor liganded gating. An adequate description of the relationship between gating and structure of th e M2 domain, however has been hampered by the fact that many M2 mutations i ncrease the opening rate constant to levels that, in the presence of acetyl choline, are unresolvably fast. Here, we show that the use of saturating co ncentrations of choline, a low-efficacy agonist, is a convenient tool to ci rcumvent this problem. In the presence of 20 mM choline: (a) single-channel currents occur in clusters; (b) fast blockade by choline itself reduces th e single-channel conductance by similar to 50%, yet the excess open-channel noise is only moderate; (c) the kinetics of gating are fitted best by a si ngle-step, C <-> O model; and (d) opening and closing rate constants are wi thin a well resolvable range. Application of this method to a series of rec ombinant adult mouse muscle M2 12' mutants revealed that: (a) the five homo logous M2 12' positions make independent. and asymmetric contributions to d iliganded gating, the delta subunit being the most sensitive to mutation; ( b) mutations at delta 12' increase the diliganded gating equilibrium consta nt in a manner that is consistent with the sensitivity of the transition st ate to mutation being similar to 30% like that of the open state and simila r to 70% like that of the closed state; (c) the relationship between delta 12' amino acid residue volume, hydrophobicity or alpha-helical tendency, an d the gating equilibrium constant of the corresponding mutants is not strai ghtforward; however (d) rate and equilibrium constants for the mutant serie s are linearly correlated (on log-log plots), which suggests that the confo rmational rearrangements upon mutation are mostly local and that the positi on of the transition state along the gating reaction coordinate is unaffect ed by these mutations.