Function of the M1 π‐helix in endplate receptor activation and desensitization

Key points A conserved proline in M1 causes a kink between α and π helical segments. The kink is under greater tension in the resting versus active conformation. The kink and the agonist do not interact directly. The π‐helix separates the gating functions of the extracellular and transmembrane domains. Mutations of the conserved proline and propofol increase desensitization.Abstract Nicotinic acetylcholine receptors (AChRs) switch on/off to generate transient membrane currents (C↔O; closed‐open ‘gating’) and enter/recover from long‐lived, refractory states (O↔D; ‘desensitization’). The M1 transmembrane helix of the muscle endplate AChR is linked to a β‐strand of the extracellular domain that extends to a neurotransmitter binding site. We used electrophysiology to measure the effects of mutations of amino acids that are located at a proline kink in M1 that separates π and α helices, in both α (N217, V218 and P221) and non‐α subunits. In related receptors, the kink is straighter and more stable in O vs . C structures (gating is ‘spring‐loaded’). None of the AChR kink mutations had a measureable effect on agonist affinity but many influenced the allosteric gating constant substantially. Side chains in the M1 α‐helix experience extraordinarily large energy differences between C and O structures, probably because of a ∼2 Å displacement and tilt of M2 relative to M1. There is a discrete break in the character of the gating transition state between αN217 and αV218, indicating that the π‐helix is a border between extracellular‐ and transmembrane‐domain function. Mutations of the conserved M1 proline, and the anaesthetic propofol, increase a rate constant for desensitization. The results suggest that straightening of the M1 proline kink triggers AChR desensitization.