Allosteric Activation of Unliganded AChRs: Multiple, Bumpy Pathways

Abstract

Acetylcholine receptors (AChRs) are allosteric proteins that isomerize (‘gates’) between resting and active conformations. We used single-channel electrophysiology to study gating of mouse adult-type endplate AChRs in the absence of agonists (free energy change, ΔG0). Many different point mutations in AChRs change ΔG0. For pairs of mutations, the net change in ΔG0 was about the same as the sum of the individual changes, indicating that mutations were independent and had mostly local effects. By correlating changes in ΔG0 with side chain partition coefficients, we could infer the hydrophobicity and volume of each residue's local environment. The kinetics of unliganded gating is complex, but was explained by schemes having 3-shut and 2-open states that all produced the same ΔG0. The longest-lived open component was eliminated by some mutations (aromatic residues at the agonist-binding site and in M1) and α-conotoxin (a specific blocker of the α-δ binding site), without affecting ΔG0. This suggests that long-lived unliganded openings are outside of the main activation sequence. In diliganded gating, in the α subunit there is a coarse-grained gradient in gating ϕ values (a conformational ‘wave’) that is less distinct in unliganded gating. Furthermore, without agonists the relative position of the gating transition state was more malleable compared to with agonists (larger Hammond shifts). The degree of shift (dϕ/dΔG0) was a function of the residue ϕ-value, and was largest at ϕ∼0.5. We hypothesize that without agonists, there are multiple activation pathways that traverse a rugged potential energy surface.