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 3-subunit. Three parameters were quantified: 1), the diliganded gating equilibrium constant (E₂), which reflects the energy difference between C(losed) and O(pen) conformations; 2), the correlation between the opening rate constant and E₂ on a log-log scale (Φ), 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 (i₀), which reports whether a mutation alters the energetics of ion permeation. The largest E ₂ changes were observed in the cytoplasmic half of εM2 (5′, 9′, 12′, 13′, and 16′), with smaller changes apparent for residues ≥17′. Φ was ∼0.54 for most εM2 residues, but was ∼0.32 at the positions that had largest E₂ changes. An arginine substitution reduced i₀ 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.