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Title: Characterisation of the human two-pore channels
Author: Funnell, Timothy
ISNI:       0000 0004 2717 3374
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2011
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The Ca²⁺-mobilising messenger NAADP has been shown to play a key role in the regulation of mammalian physiology. Recently, the two-pore channels (TPCs) have been proposed as an NAADP-gated Ca²⁺ channel. Chapter 1 introduces the TPCs as the major candidates in governing NAADP-mediated Ca²⁺-release from acidic stores. Chapter 2 explains the methodologies developed and used. Chapter 3 demonstrates the successful immunopurification of HsTPC2 and its incorporation into an artificial lipid bilayer. K⁺ and Ca²⁺ currents were seen in reponse to nM - μM concentrations of NAADP; with the open probability (P₀) fitting a bell-shaped concentration-response curve. Ligand sensitivity was shown to be regulated by luminal [Ca²⁺], whereby a 20-fold increase in [Ca²⁺] lumen (10 μM to 200 μM) caused a 100-fold reduction in the EC50 from ≈ 500 nM to 5 nM. Furthermore, a reduction in luminal pH from 7.2 to 4.8 reduced the P₀ but 1 μM Ned-19 inhibiting all channel activity. Chapter 4 investigates the in situ properties of HsTPC2 by the purification and patch clamp of intact lysosomes from cells overexpressing HsTPC2. Three methods of purification were compared: differential centrifugation, whole lysosome immunoprecipitation and magnetic chromatography. Techniques involving lysosomal swelling and whole cell homogenisation were also optimised to ensure minimal contamination by non-lysosomal proteins. Whole lysosome patch clamping revealed NAADP-induced, Ca²⁺-specific currents in response to NAADP, but not cADPR, IP₃ or Ned-19. High concentrations of NAADP (mM) and Ned-19 (μM) showed prolonged ≈ 5 minutes) inhibition of channel activity. Chapter 5 explores the protein-protein interactions of the purified HsTPC2 and identifies a heterodimeric interaction between HsTPC1 and HsTPC2 was further dissected by coimmunoprecipitation, colocalisation and FRET studies. Despite clear evidence that both isoforms independently form homodimers, it is likely that heterodimerisation is a dynamic interaction only seen in a subset of the channel population. Chapter 6 discusses the results obtained in the wider context of cell physiology.
Supervisor: Galione, Antony Sponsor: British Pharmacological Society
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Pharmacology ; two-pore channels ; calcium