The Unliganded Gating Mechanism Of Nicotinic Acetylcholine Receptors

Abstract

The nicotinic acetylcholine receptor (AChR) switches between C (low agonist affinity and low conductance) and O (high agonist affinity and high conductance) conformations (‘gating’). The probability of channel opening is very low in the absence of agonist, but when agonists are present at the two transmitter-binding-sites opening increases rapidly (∼20μs), transiently to a high probability (∼0.95). We observe that ‘gain-of-function’ mutants that increase the diliganded gating equilibrium constant (without affecting agonist binding to C) also increase the frequency of spontaneous openings. Unliganded openings occur in clusters in AChRs having several of such mutations. We analyzed the intra-cluster interval durations to estimate that the unliganded gating equilibrium constant is ∼1.15 × 10-7 (mouse, α2βδε, -100 mV). The agonist affinity ratios (C vs. O) for acetylcholine, carbamylcholine, tetramethylammonium and choline are ∼15,600, ∼6700, ∼6700 and ∼600. The monoliganded (with ACh) gating equilibrium constant is ∼1.7 × 10-3. Acetylcholine provides only ∼0.9 kBT more binding energy per site than tetramethylammonium, but ∼3.1 kBT more than choline. Mutations of binding site residue αW149 increase unliganded gating, and 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. The mechanisms of the gating conformational change and of desensitization are similar with and without ligands at the transmitter binding sites.