The identification of five predicted proteins (dP2XA-E) with homology to vertebrate P2X receptors in Dictyostelium discoideum proved a unique opportunity to explore purinergic receptor function in a simple unicellular eukaryote from outside the animal kingdom. dP2XE was shown to be expressed as a trimer and trafficked to the cell surface in Xenopus oocytes. However, no currents were detected to extracellularly applied ligands. This lack of function was potentially due to inaccurate post-translational modifications since dP2XE expressed in oocytes and D. discoideum displayed different molecular weights. Unexpectedly for a potential ligand-gated ion channel, dP2XE-eGFP displayed a solely intracellular distribution in D. discoideum, localising to the endolysosomal and contractile vacuole systems. Ablation of the p2xE gene by homologous recombination revealed a role for dP2XE in axenic growth in suspension, associated with a modest defect in cytokinesis, and a potential involvement in the calcium signalling and homeostasis functions of the contractile vacuole. Both this distribution and disruption phenotype were mirrored for p2xA. The existence of purinergic signalling in D. discoideum was demonstrated by utilising an apoaequorin expressing strain to show that extracellular ATP and ADP evoked increases in intracellular Caˆ{2,+}. Indicative of P2X receptor activation, responses were rapid and transient, required extracellular Caˆ{2,+}, inhibited by Gdˆ{3,+}, modified by extracellular pH and remained unaffected by deletion of either the single heterotrimeric G\beta or iplA genes. ATP/ADP responses were unaffected by ablation of either the p2xA or p2xE genes leaving dP2XB-D as potential candidates. Inhibition of the large P2X-like response with Znˆ{2,+} revealed the presence of a much smaller response with a slower time course indicating that P2Y-like receptors may also be present. The work presented in this thesis demonstrates that D. discoideum possesses cell surface purinergic receptors for extracellular ATP/ADP and extends our knowledge of the intracellular role of purinergic signalling in this organism.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:509266 |
| Date | January 2009 |
| Creators | Ludlow, Melanie Joanna |
| Contributors | Ennion, Steve |
| Publisher | University of Leicester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/2381/7450 |
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