Ca2+ -activated Cl- channels (CaCCs) are activated by an increase in intracellular [Ca2+] and as vascular smooth muscle cells (VSMCs) activity accumulate Cl- ions, opening of these channels leads to Cl- ion efflux and membrane depolarisation. There is very little known on how these channels are regulated, the aim of this project was to investigate the regulation of CaCCs in VSMCs. Initial experiments were carried out to characterise CaCC currents (lClca) in isolated rat pulmonary artery smooth muscle cells (PASMCs). Native CaCCs are proposed to be encoded by TMEM16A. The presence of this gene in PASMCs shown using quantitative PCR and protein expression with Western Blot. Furthermore novel modulators of currents produced by overexpression of TMEM16A were shown to modulate native lClca. The majority of the work, investigated the novel finding that CaCCs are regulated by the membrane phospholipid phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2). Using whole cell electrophysiology, a reduction in PI(4,5)P2 levels via either phospholipase C activation, PI4K inhibition, PIP2 scavenging or absorption, increased lC/ca. Conversely intracellular enrichment of PI(4,5)P2 inhibited lClca. Furthermore, PI(4,5)P2and other phospholipids were shown to directly bind to TMEM16A isolated from rat pulmonary artery and TMEM16A-eGFP expressed in HEK293 cells. This data suggest that PI(4,5)P2 plays a negative role on CaCC activation. Finally the interaction of CaCCs and the cystic fibrosis transmembrane conductance regulator (CFTR) channel in mesenteric artery smooth muscle (MASMCs) was studied. TMEM16A and CFTR proteins were shown to locate <40 nm of each other with proximity ligation assay. In isometric tension recordings TMEM16A (T16inh- AOl) and CFTR (oxo-CFTR-172) inhibitors caused potent relaxation on preconstricted vessels, with oxo-CFTR-172 showing paradoxical effect. Increasing CFTR activity either by directly or indirectly decreased the potency of T16Ainh-AOl, whereas decreasing CFTR activity increased the potency of T16Ainh-AOl. This suggests that CFTR negatively regulates TMEM16A.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:677182 |
| Date | January 2015 |
| Creators | Pritchard, Harry Anthony Toomey |
| Publisher | St George's, University of London |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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