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Title: Molecular characterization of the channel properties of the 5-HT3 receptor
Author: Gunthorpe, M. J.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1998
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In this thesis I have used a combination of site-directed mutagenesis, radioligand binding and whole-cell patch clamp electrophysiology to study the 5-HT3 receptor expressed in HEK 293 cells. Alignment of the M2 domains of ligand-gated ion channel superfamily subunits indicates that a positively charged (lysine) residue within the M2 channel lining domain is a unique feature of the 5-HT3 receptor. To investigate the role of this residue in 5-HT3 receptor function I substituted it with alternative amino acids (arginine, glutamine, serine and glycine) which differ in charge and/or side chain length. Characterization of the resulting receptors indicated little or no effect upon agonist efficacy, rectification, or channel conductance (as determined by fluctuation noise analysis) suggesting that the structure of the mutant receptor proteins was probably not much altered. However, all of the substitutions reduced the rate of agonist-induced receptor desensitization compared to wild type. The results demonstrate that this residue is not the determinant of the low single-channel conductance of the recombinant receptor and establish a specific role for this lysine residue in the mechanism of 5-HT3 receptor desensitization. I have also investigated the molecular determinants of the ion selectivity of the 5-HT3 receptor. Construction of mutant receptors and their subsequent characterization in extracellular solutions of differing sodium and/or chloride concentrations established the determinants of cation versus anion selectivity for this receptor channel. The results show that three mutations, with M2 and the M1-M2 intracellular loop, result in a chloride permeable channel. These results support a role for the M1-M2 loop in forming part of the channel lining domain and highlight the importance of geometric constraints in determining the ion selectivity of the channel. The results of this study, in conjunction with similar results obtained for the α7 nicotinic acetylcholine receptor, suggest that the molecular mechanisms of channel function in these receptors, and by inference the other ligand-gated ion channel superfamily, are highly conserved.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available