Thesis (Ph.D)


Engineering and functional characterisation of pentameric concatenated (alpha 4)2 (beta 2)3 and (alpha 4)3 (beta 2)2 nicotinic acetylcholine receptors

Abstract

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 stoichiometricspecific 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.

Attached files

Authors

Carbone, A L

Oxford Brookes departments

Faculty of Health and Life Sciences
Department of Biological and Medical Sciences

Dates

Year: 2009


© Carbone, A L
Published by Oxford Brookes University
All rights reserved. Copyright © and Moral Rights for this thesis are retained by the author and/or other copyright owners. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the copyright holder(s). The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the copyright holders.

Details

  • Owner: Unknown user
  • Collection: eTheses
  • Version: 1 (show all)
  • Status: Live
  • Views (since Sept 2022): 24