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Title: Engineering and functional characterisation of pentameric concatenated (α4)₂(β2)₃ and (α4)₃(β2)₂ nicotinic acetylcholine receptors
Author: Carbone, Anna Lisa
ISNI:       0000 0004 2668 5335
Awarding Body: Oxford Brookes University
Current Institution: Oxford Brookes University
Date of Award: 2009
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Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that influence neurotransmitter release, hence constituting a key component of the physiological mechanisms of neuronal signalling. This thesis is concerned with the properties of the a4P2 nAChR, the most abundant nAChR in the brain, and the major contributor to the central effects of nicotine. The a4P2 nAChR is made up of five subunits, which in heterologous systems can assemble into at least two different stoichiometries: the high sensitivity (HS) (a4h(P2)3 stoichiometry and the low sensitivity (LS) (a4)3(p2)2 stoichiometry, which might both exist in native tissues. Despite the attractiveness of the a.4P2 nAChR as a target for therapeutic intervention, progress in the development of a4P2 nAChR-selective drugs has been slowed, partly because of the lack of stoichiometric-specific receptor models. This study presents a strategy to express homogenous populations of a4P2 nAChRs with fixed stoichiometry. By using standard molecular biological techniques, pentameric concatenated (a4)2(P2)3 and (a4)3(P2)2 nAChRs were engineered. These receptors were expressed in Xenopus laevis oocytes and functional studies showed that their functional properties resembled those of their non-linked counterparts. Subsequent site-directed mutagenesis in combination with functional analysis allowed the identification of the agonist-binding subunits in both concatamers. Concatenated receptors proved to be suitable for comparative studies of the effects of receptor mutation linked to autosomal dominant nocturnal frontal lobe epilepsy. Studies carried out on non-linked receptors, showed that the properties of the (a4)3(p2)2 stoichiometry were affected more markedly than those of the (a4)2(p2)3 stoichiometry. Insertion of the mutation in concatenated receptors revealed that the mutation not only affected the functional properties of a.4P2 nAChRs but also altered the subunit composition of the receptor. These studies show that pentameric concatenated constructs are a powerful tool to study the function and structure of receptors that assemble in multimeric types in expression systems.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available