Allosteric contributions of muscle nicotinic acetylcholine receptor residues in small-molecule interactions, disease and subunit assembly

The nicotinic acetylcholine receptor (nAChR) is a member of the Cys-loop receptor superfamily of heteropentameric ligand-gated ion channels. The nAChR subfamily has 17 different possible subunits in vertebrates that are found in multiple stoichiometric combinations. Compared to other nAChRs, muscle type nAChRs possess a large subunit repertoire with respect to other nAChRs being comprised of four different subunits- namely alpha, beta, delta and either epsilon or gamma in adult or foetal varieties respectively. The endogenous neurotransmitter of nAChRs, acetylcholine (ACh), binds at the interfaces of alpha(+)delta(-) and alpha(+)epsilon(-) or alpha(+) gamma(-) subunits. By controlling the safety margin of neuromuscular transmission, nAChRs maintain high-fidelity muscle contraction under a range of physiological conditions. The interference of this process as a result of organophosphorus nerve agent (OPNA) exposure or from genetic disorders such as congenital myasthenic syndrome (CMS) can therefore have deleterious consequences. OPNAs work by covalent modification of acetylcholinesterase, preventing the breakdown of acetylcholine leading to desensitising block of nAChRs. No antidotes that interact with nAChRs currently exist, however a class of non-oxime bispyridinium compounds (BPDs) have been shown to be efficacious in this regard. In the first part of this project, we sought to rationalise the structure activity relationship data of a series of congeneric BPDs using molecular dynamics (MD) simulations, followed by mutagenesis and electrophysiology to elucidate molecular determinants for their interactions. In part II of this thesis we identify a novel CMS mutation located at the muscle nAChR alpha subunit transmembrane domain and use enhanced sampling MD simulations and patch-clamp electrophysiology to reveal a new CMS pathomechanism caused by a swap in charge selectivity from cationic to anionic. In the final part of this thesis, we sought to further explore observations from part I regarding functional differences between orthosteric interfaces as well as discern the structural correlates responsible for the fixed stoichiometric assembly of muscle nAChRs. In the first instance, by integrating MD simulation, evolutionary data and electrophysiology we determine the structural correlates for muscle nAChR subunit assembly and show functional differences between WT and ’double delta’ human muscle nAChR subtypes . Further to this, we explore the relative importance of individual domains of the nAChR delta subunit in contributing to this fixed stoichiometric assembly by generating a range of chimeric epsilon and delta subunits and assessing their cell-surface expression with I125-alpha-BuTx binding assays.