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 (E-0) 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) E-0 for mouse, wild-type alpha(2)beta delta epsilon AChRs is approximate to 1.15 x 10(-7); (ii) unliganded AChRs open for approximate to 80 mu s, once every approximate to 15 min; (iii) the affinity for ACh of the O(pen) conformation is approximate to 10 nM, or approximate to 15,600 times greater than for the C(losed) conformation; (iv) the ACh-monoliganded gating equilibrium constant is approximate to 1.7 x 10(-3); (v) the C -> O isomerization reduces substantially ACh dissociation, but only slightly increases association; and ( vi) ACh provides only approximate to 0.9 k(B) T more binding energy per site than carbamylcholine but approximate to 3.1 k(B)T more than choline, mainly because of a low O conformation affinity. Most mutations of binding site residue alpha W149 increase E-0. We estimate that the mutation alpha W149F 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 (Phi 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.