Profiling the Effects of L9' Mutations on the Function of the Human Adult Muscle Nicotinic Acetylcholine Receptor

Abstract

The nicotinic acetylcholine receptor (nAChR) is a pentameric ligand-gated ion channel (pLGIC) and is a core component of the neuromuscular junction, facilitating fast synaptic transmission leading to muscle contraction. Mutations to the human adult muscle nAChR lead to various forms of congenital myasthenic syndrome (CMS), a disease characterized by progressive fatigable muscle weakness. A central channel pore constriction formed by a ring of five leucine residues (L9’) forms part of the nAChR channel gate. CMS-causing mutations in the L9’ residues lead to a form of CMS that results in longer channel opening times and a delayed signal decay. To understand better how L9’ mutations in the human adult muscle nAChR influence channel function, I used two-electrode voltage clamp electrophysiology to perform a comprehensive mutant screen of all L9’ residues in each subunit of the human adult muscle nAChR. This resulted in a total of 76 unique mutations: 19 L9’ mutations consisting of every possible natural amino acid substitution in each subunit (α, β, ε, δ). The results of this screen show that while the polarity and size of a substituted residue contribute to its effect on channel function, increasing the polarity of the side chain typically has a more potentiating effect on channel function than does a change in size. The subunit in which the mutation is expressed also tailors the effect of a given mutation on channel function, with several δL9’ mutations producing qualitatively different effects than equivalent mutations in other subunits. Because the majority of L9’ mutations resulted in a gain-of-function, I originally postulated that interactions between L9’ and surrounding residues stabilize the resting state with the elimination of such interaction through mutations destabilizing the resting state to promote channel gating. Using a double mutant cycle, I explored interactions between the L9’ and adjacent non-L9’ residues but found that there are only weak or no interactions that contribute to channel function. Instead, my data support the hypothesis that the nAChR operates via a hydrophobic gating mechanism, and that adjacent L9’ residues are driven together by the hydrophobic effect to form a closed pore. L9’ mutations that either increase the polarity or decrease residue size likely reduce the hydrophobic driving forces that stabilizes the resting state, thus leading to an enhancement in channel function.