Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.603787
Title: Studies of structure-function relationships in the 5-HT₃ and GABAc receptors
Author: Harrison, Neil John
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2005
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Abstract:
5-HT3 and GABAc receptors are members of the Cys-loop family of ligand-gated ion channels, which also includes nACh, GABAA and glycine receptors. The ligand binding site of this family of receptors is located in the extracellular domain, which is believed to be structurally conserved. The similarity was tested in this dissertation by exchanging the critical binding residues of the 5-HT3A receptor for those of the ionotropic GABA receptors. Residues were introduced according to alignment with the AChBP (homologous to the extracellular domain of the nACh α7 receptor), yet GABA binding was not achieved. Analyses of the mutant 5-HT3A receptors by radioligand binding and electrophysiology, however, revealed the changed residues were of importance in binding and/or gating. Elements of the 5-HT3A and GABAC p1 receptors were also combined in chimaeric studies, with the fusion of these two subunits at the extracellular/transmembrane domain junction, and subsequently at the M3/M3-M4 loop junction. These chimaeric receptors assembled at the cell surface, but did not function, likely resulting from structural perturbations caused from merging the two subunits. A study of the GABAC p1 receptor ensued, encompassing both the examination of the use of the FlexStation, which proved to be a suitable method of characterising this receptor, and a study of its extracellular domain. To guide this later study, a model of the extracellular domain of the GABAC p1 receptor was created, using AChBP as a template. GABA was computationally docked into this model, revealing three potential orientations. The most accurate model was chosen based on the experimental data available and probed using mutagenesis studies. Mutation at each residue proposed to be 5Ǻ GABA caused significant changes in GABA efficacy, indicating all of these residues are important for correct binding and/or gating.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.603787  DOI: Not available
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