| Summary: | <p>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.</p><p>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.</p><p>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⁺] <sub>lumen</sub> (10 μM to 200 μM) caused a 100-fold reduction in the EC<sub>50</sub> 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.</p><p>Chapter 4 investigates the <em>in situ</em> 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.</p><p>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.</p><p>Chapter 6 discusses the results obtained in the wider context of cell physiology.</p>
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