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Title: Comparative molecular physiology of novel P2X receptors : identification, cloning and functional characterisation
Author: Hanmer, Stuart B.
ISNI:       0000 0004 5352 6049
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2014
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P2X receptors are ATP-gated ion channels with myriad roles in humans and other higher vertebrates. Research over the past decade has described the cloning, pharmacology, and physiological role of this receptor family in a number of invertebrate organisms, as well as in unicellular amoeba and algae. However, questions remain regarding the extant of P2X receptor phylogeny and function in many invertebrates. A greater understanding of invertebrate P2X receptor pharmacology and function may provide insights into structure-function relationships in “higher” homologues, as well as novel roles for this ligand-gated ion channel family. This thesis investigated P2X receptor expression and function within eukaryotic phylogeny, with particular emphasis on invertebrate animals. Homology searching of transcriptomic and genomic datasets identified a number of candidate P2X receptor sequences across a range of phyla. Notably, homologues were bioinformatically identified within orders of the class Insecta, where they had been previously thought to be absent. Homologous sequences were also identified in a multicellular alga of the Charophycea, a class of the Viridiplantae division Charophyta, considered to represent the closest extant taxon to terrestrial plants. Following the identification of a P2X receptor homologue in the cnidarian Hydra vulgaris (AEP) (‘aepP2X’) by bioinformatics approaches, total RNA preparations were used for cDNA synthesis to generate templates for PCR to yield aepP2X clones. These clones encoded proteins that exhibited concentration-dependent ATP-evoked inward currents when expressed heterologously in HEK293 cells (EC50 ca. 120 μM; holding potential of -60 mV). Co-application of the classic P2 receptor antagonists pyridoxalphosphate-6-azophenyl-2', 5'-disulphonic acid (PPADS) and suramin was ineffective, as were phenol red and brilliant blue G (BBG). The synthetic ATP analogues α,β-methylene ATP and β,γ-methylene ATP (1 mM) did not evoke currents at aepP2X. Consistent with most mammalian homologues, aepP2X was found to be a non-selective cation channel with negligible chloride ion permeability. Immunohistochemistry using a custom polyclonal antiserum raised against a C- terminal epitope of aepP2X suggested the expression of this receptor occurs in developing and late-stage nematocysts in whole Hydra polyps. A P2X receptor from the microcrustacean Daphnia pulex (DpuP2XB) was also expressed heterologously in HEK293 cells, and was found to be largely insensitive to extracellular ATP (EC50 > 1 mM), although reduction of total divalent cation concentration of perfusing extracellular solution partially restored ATP sensitivity in DpuP2XB. Whilst a P2X receptor homologue from the placazoan, Trichoplax adhaerens (TadP2XA) did not respond to extracellular ATP in HEK293 cells, a P2X receptor from the lancelet Branchiostoma floridae (BfloP2X) does respond in a concentration-dependent manner to A TP . Currents evoked through BfloP2X displayed rundown with repeated 1 mM ATP applications.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QL Zoology ; QP Physiology