| Čoahkkáigeassu: | <p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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 I<sup>125</sup>-alpha-BuTx binding assays.</p>
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