Unliganded gating of acetylcholine receptor channels

We estimated the unliganded opening and closing rate constants of neuromuscular acetylcholine receptor-channels (AChRs) having mutations that increased the gating equilibrium constant. For some mutant combinations, spontaneous openings occurred in clusters. For 25 different constructs, the unliganded gating equilibrium constant (E₀) was correlated with the product of the predicted fold-increase in the diliganded gating equilibrium constant caused by each mutation alone. We estimate that (i) E₀ for mouse, wild-type α₂βδε AChRs is ≈1.15 × 10^-7; (ii) unliganded AChRs open for ≈80 μs, once every ≈15 min; (iii) the affinity for ACh of the O(pen) conformation is ≈10 nM, or ≈15,600 times greater than for the C(losed) conformation; (iv) the ACh-monoliganded gating equilibrium constant is ≈1.7 × 10^-3; (v) the C→O isomerization reduces substantially ACh dissociation, but only slightly increases association; and (vi) ACh provides only ≈0.9 k _BT more binding energy per site than carbamylcholine but ≈3.1 k_BT more than choline, mainly because of a low O conformation affinity. Most mutations of binding site residue αW149 increase E ₀. We estimate that the mutation αW149F reduces the ACh affinity of C only by 13-fold, but of O by 190-fold. Rate-equilibrium free-energy relationships for different regions of the protein show similar slopes (Φ values) for un- vs. diliganded gating, which suggests that the conformational pathway of the gating structural change is fundamentally the same with and without agonists. Agonist binding is a perturbation that (like most mutations) changes the energy, but not the mechanism, of the gating conformational change.