Energy and structure of the M2 helix in acetylcholine receptor-channel gating.

We studied single-channel currents from neuromuscular acetylcholine receptor-channels with mutations in the pore-lining, M2 helix of the epsilon-subunit. Three parameters were quantified: 1), the diliganded gating equilibrium constant (E(2)), which reflects the energy difference between C(losed) and O(pen) conformations; 2), the correlation between the opening rate constant and E(2) on a log-log scale (Phi), which illuminates the energy character of the residue (C- versus O-like) within the C<-->O isomerization process; and 3), the open-channel current amplitude (i(0)), which reports whether a mutation alters the energetics of ion permeation. The largest E(2) changes were observed in the cytoplasmic half of epsilonM2 (5', 9', 12', 13', and 16'), with smaller changes apparent for residues > or =17'. Phi was approximately 0.54 for most epsilonM2 residues, but was approximately 0.32 at the positions that had largest E(2) changes. An arginine substitution reduced i(0) significantly at six positions, with the magnitude of the reduction increasing, 16'-->2'. The measurements suggest that the 9', 12', and 13' residues experience large and late free-energy changes in the channel-opening process. We speculate that in the gating isomerization the pore-facing residues >6' and <16' experience multiple energy perturbations associated with changes in protein structure and, perhaps, hydration.