Molecular characterisation of nicotinic acetylcholine receptor subtypes: interactions with agonists and allosteric modulators.
Doctoral thesis, UCL (University College London).
Nicotinic acetylcholine receptors (nAChRs) are members of the ligand-gated ion channel family. They are also members of the sub-family of Cys-loop ligandgated ion channels, which includes closely related receptors for the neurotransmitters γ-aminobutyric acid (GABA), glycine, and 5- hydroxytryptamine (serotonin). There is a wealth of evidence demonstrating that nAChRs are involved in mediating fast excitatory transmission, and also that they modulate the release of other neurotransmitters. They have been implicated in several neurological disorders such as Alzheimer’s disease, Parkinson’s disease, and schizophrenia, and also mediate the effects of nicotine associated with tobacco smoking. They are therefore viewed as potentially useful targets for the development of therapeutic drugs. In addition to the endogenous neurotransmitter acetylcholine, nAChRs are targets for a diverse collection of naturally occurring ligands. These include agonists and antagonists isolated from plants, freshwater algae, marine worms, frogs, the venoms of snakes and predatory marine snails. An increasing collection of synthetic ligands have also been produced which act upon the nAChR. The aim of work presented in this thesis is to investigate recently developed ligands of the nAChR and to characterise their action. Work is presented aimed at characterising TMAQ, a β4-selective nAChR agonist. Strikingly, this agonist exhibits activity only upon nAChRs containing the human β4 subunit whilst showing no activity on receptors containing the rat β4 subunit. Evidence is presented which, using hybrid receptors and chimeric subunits, identifies the extracellular portion of the human β4 subunit as being critical to this species-selectivity. More specifically, the work demonstrates that a region of the extracellular domain (loop D) of the β4 subunit is responsible for this species selectivity. Additionally, it is shown that, although acting as an agonist of human β4-containing nAChRs, TMAQ acts as an antagonist of rat β4-containing nAChRs. A homology model of the β4 subunit is also presented which identifies the location of the important residues; rationale is provided for their effects. Work is also reported which characterises positive allosteric modulators of α7 nAChRs, another class of nAChR ligand. Two classes of positive allosteric modulators are examined utilising prototypes from each class. It was found that type I positive allosteric modulators, such as LY-2087101, increase the magnitude of peak response to agonists. Type II positive allosteric modulators, such as PNU- 120596, not only increase the magnitude of peak response to agonist but also slow the receptor’s rate of desensitisation. Chimeric α7/5HT3A subunits were used to identify domains crucial to the site of action for PNU-120596; the first three transmembrane domains were found to be essential. Mutation of individual residues within the α7 transmembrane domains into the equivalent amino acids in the 5HT3A subunit identified several key residues. Homology modelling and computational docking of allosteric modulators helped to support the theory that these residues line the binding site for PNU-120596 and LY-2087101. Due to this site’s similarity to modulatory sites found in other closely related receptors it is argued that it represents a conserved modulatory site across the Cys-loop family of receptors.
|Title:||Molecular characterisation of nicotinic acetylcholine receptor subtypes: interactions with agonists and allosteric modulators|
|Additional information:||Authorisation for digitisation not received|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Life Sciences > Biosciences (Division of) > Neuroscience, Physiology and Pharmacology|
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