P2X4 receptors (P2X4R) are ligand-gated ion channels activated by ATP and with a high permeability to Ca2+. They are predominantly localised to lysosomes and from there can traffic to the cell surface. ATP levels within the lysosome are high but P2X4Rs are inhibited by the acidic pH. Previously, it was shown that the alkalinisation of lysosomes using pharmacological reagents was sufficient to activate P2X4Rs, which promoted homotypic lysosome fusion. The main aim of this study was to identify physiological regulators of lysosomal P2X4Rs and to examine their role in lysosome Ca2+ signalling and fusion. The first candidate I investigated was P2X7R, which is typically co-expressed with P2X4R in immune and epithelial cells, and which has already been shown to induce changes in lysosome properties upon activation. I co-expressed these two receptors in normal rat kidney (NRK) cells and in HeLa cells and looked for a synergistic interaction between them in promoting lysosome fusion, as assessed by measuring the size of lysosomes. My results showed a significant increase in lysosome size following activation of P2X7R but only in the presence of P2X4R. Neither receptor alone was sufficient to promote lysosome fusion in response to the agonist BzATP. LAMP-GECO was used to measure changes in cytosolic [Ca2+] within the vicinity of the lysosome. Fusion of the Ca2+ reporter (GECO) to the C-terminus of LAMP-1 targets GECO to the cytosolic surface of the lysosome. Co-expression of P2X4R with P2X7R augmented the P2X7R-induced Ca2+ signal suggesting that P2X4Rs mediate lysosomal Ca2+ efflux downstream of P2X7R stimulation. Next, I showed that the expression of P2X4R was sufficient to enhance the cytosolic Ca2+ response to the activation of endogenous histamine H1 receptors and to promote lysosome fusion. Similar results were obtained with P2Y2R stimulation, which also couples to the phospholipase C pathway. Further experiments were conducted to look at differences in the trafficking behaviour of human and rat P2X4Rs and to examine a role for P2X4Rs in autophagic flux. My results suggest a synergistic interaction between P2X4R and P2X7R which inhibits autophagic flux, similar to the effect of bafilomycin treatment. Therefore, the effect of P2X4/7R in autophagy may be mediated by the alkalinisation of lysosomes. Altogether the results of my project improve our understanding of how the P2X4R Ca2+ channel regulates lysosome function.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:725602 |
| Date | January 2017 |
| Creators | Tan, Sin Lih |
| Contributors | Smith, Ewan ; Murrell-Lagnado, Ruth |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.repository.cam.ac.uk/handle/1810/267925 |
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