Hooper, R. (2011) Molecular characterisation of NAADP-gated two-pore channels. Doctoral thesis, UCL (University College London).
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Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent intracellular calcium (Ca2+)-mobilising second messenger implicated in a variety of physiological processes. Unusually, NAADP mediates Ca2+ release from acidic organelles, such as the lysosome, and not the endoplasmic reticulum. Recently, members of the voltagegated ion channel super-family, the two-pore channels (TPCs), have been identified as molecular targets of NAADP. The aim of this thesis is to investigate the molecular properties of these poorly characterised ion channels. In this study, I present the cloning of a novel TPC isoform from the sea urchin, an extensively used model organism for NAADP signalling, and the subsequent characterisation of the complete ancestral sea urchin TPC family. Sea urchin TPCs appeared to be Nglycosylated in an isoform-specific manner, displayed anomalous migration upon fractionation, similar to endogenous NAADP-binding proteins, and localised to the endo-lysosomal system. To characterise the properties of human TPCs, antibodies suitable for Western blot and immunocytochemistry analyses were identified. The topology of human TPCs was examined using in silico prediction methods, combined with fluorescence protease protection assays and the mapping of TPC antibody epitopes and N-glycosylation sites. Human TPCs conformed to a twelve transmembrane region model with cytosolic termini. The quaternary structure of TPCs was investigated using FRET analysis, sucrose density gradients, gel filtration, co-immunoprecipitation, and chemical cross-linking of both full-length TPCs and individual hydrophobic domains. TPCs likely assemble as dimers possibly within a high molecular weight protein complex. Finally, I show that the N-terminus of TPC1 regulates NAADP-mediated Ca2+ release and identify a potential physiological role for TPC2 in pigmentation.
|Title:||Molecular characterisation of NAADP-gated two-pore channels|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Life Sciences > Biosciences (Division of) > Cell and Developmental Biology|
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