University of Leicester
Browse

The Molecular Basis of Antagonism at Cardiovascular P2X1 and P2X4 Receptors

Download (11.95 MB)
thesis
posted on 2017-09-05, 12:12 authored by Louise Katie Farmer
Structural information for the zebrafish P2X4 receptor in both an agonist bound and unbound resting state provided a major advance in understanding agonist action and has given insight into movement that occurs in the receptor upon ATP binding. Despite agonist action now being well characterised, the molecular basis of antagonism is poorly understood. In this thesis the mechanism of antagonist action at the hP2X1 receptor has been investigated through determining properties of chimeras and mutant receptors based on differences between antagonist sensitive and insensitive P2X receptors. The antagonists suramin, NF449 and PPADS potently inhibit the human P2X1 receptor but have little or no action at the rat P2X4 receptor. The extracellular loop of the hP2X1 receptor was shown to determine antagonist sensitivity and was therefore split into four sections, residues of which were swapped with corresponding residues of the antagonist insensitive rP2X4 receptor and vice versa. Sub-chimeras and point mutations were then made to identify particular residues and regions which contribute to antagonist action. These experiments identified two regions important for NF449 binding at the receptor. These are a cluster of four positively charged residues at the base of the cysteine rich head region (136-140) and three residues located just below them (T216, H224 and Q231). An NF449 bound model of the hP2X1 receptor has been generated. The introduction of the four positively charged residues at the base of the cysteine rich head region to the rP2X4 receptor introduced suramin and PPADS sensitivity to this previously insensitive receptor. This mutation is thought to cause a conformational change which allows the antagonist to bind at residues which are already present in the wildtype receptor. In summary this thesis has advanced the understanding of antagonist action at the hP2X1 receptor and the antagonist insensitivity of the rP2X4 receptor.

History

Supervisor(s)

Evans, Richard; Mahaut-Smith, Martyn

Date of award

2014-08-01

Author affiliation

Department of Cell Physiology and Pharmacology

Awarding institution

University of Leicester

Qualification level

  • Doctoral

Qualification name

  • PhD

Language

en

Usage metrics

    University of Leicester Theses

    Categories

    No categories selected

    Keywords

    Exports

    RefWorks
    BibTeX
    Ref. manager
    Endnote
    DataCite
    NLM
    DC