Mapping the Allosteric Pathway Leading from a Mutation in the Nicotinic Acetylcholine Receptor to a Congenital Myasthenic Syndrome

Abstract

The peripheral and highly lipid-exposed M4 α-helix, although distant from the agonist binding site, channel gate, and other important gating structures, is involved in modulating function of the nicotinic acetylcholine receptor. M4 "senses" changes in the surrounding lipid environment and may consequently affect receptor function by altering specific interactions between the M4 C-terminus and the Cys-loop. An example of this lipid sensing ability is demonstrated by a lipid-facing Cys418 to Trp substitution on αM4 (αM4 C418W) of the muscle-type receptor, which subtly alters protein-lipid interactions and potentiates channel function 16-fold, leading to a slow-channel congenital myasthenic syndrome. Through the use of mutational studies and mutant cycle analysis, I determine that, contrary to previous studies, M4–Cys-loop interactions are not critical to wild-type muscle-type receptor function, nor are they involved in C418W-induced potentiation. Instead, C418W potentiates channel activity by enhancing local M4-M1 interactions mediated by three polar side-chains, which are absolutely critical to potentiation. I show that altered M4-M1 interactions are ultimately translated to two important gating structures, which work in tandem to stabilize the open conformation of the receptor. These studies highlight how altered protein-lipid interactions can affect channel function and contribute to our understanding of the underlying gating mechanism of the muscle-type receptor.