Allosteric signal initiation and communication in neuromuscular acetylcholine receptors

Acetylcholine receptors (AChRs) expressed at the nerve-muscle synapses are prototypic allosteric receptors that shuttle between a resting C losed (C) and active O pen (O) states. ACh binding at the neurotransmitter binding sites (TBS) leads to opening of the ‘gate’ in the channel pore that regulates ion flow. How agonist binding to the TBS communicates the allosteric signal to the channel gate located ∼50 Å away is not understood. In the absence of agonists, the wild-type and mutant AChRs, including those causing congenital myasthenia syndrome, show constitutive gating. However, whether allosteric activation pathways in the presence vs absence of agonists are identical, is debatable. Here, by using a combination of kinetic modelling, phi (ϕ)- and activation energy (ΔΔG ^ǂ ) estimation for >60 residues from single channel current recordings and molecular dynamics simulations, we show the existence of parallel gating pathways (‘major’ and ‘minor’) in the unliganded AChRs and activation pathways are non-identical for the liganded vs unliganded AChRs. Kinetic analysis of the minor gating and correlation between the state residence probabilities vs C-loop conformations suggest, 1. the C-loop capping triggers allosteric communication independent of the presence of an agonist and, 2. the minor gating is a pre-existing allosteric pathway which is preferentially chosen in the presence of agonists. Further, we show the presence of a continuous ‘live-wire’ like allosteric network in the liganded receptor between the TBS and the gate constituted by residues which lower the activation energy barrier by >-4 kcal/mol. In contrast, in the unliganded AChRs, the major gating seems to initiate from ‘hub’ residues in the allosteric network. The results presented here provide novel insight into the mechanism of allosteric signal initiation and communication in AChRs.