Penny, CJ; (2016) The Molecular Physiology of Two-pore Channels. Doctoral thesis , UCL (University College London).

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Abstract

Ca2+ ions play a vital signalling role in a vast range of physiological and pathological processes. NAADP is a major component of the intracellular Ca2+ signalling milieu, releasing Ca2+ from acidic organelles to generate both local and global responses. Over the past 7 years, Two-pore Channels (TPCs) have emerged as the likely target for NAADP. However, unlike many of the other channels within their voltage-gated ion channel superfamily, TPCs remain poorly characterised at a molecular level. Their pharmacology is also limited to a handful of non-selective inhibitors of other channels. Furthermore, the functional interplay between TPC-mediated local and global Ca2+ signalling remains ill-defined. This thesis takes a highly interdisciplinary approach to address these shortcomings- to investigate the molecular physiology of the TPCs. First, I dissected both of the divergent pore regions from human TPC2 and expressed them individually in HeLa cells, where they depleted the ER of Ca2+. After heterologous expression in E. coli cells, I purified a stable, folded and functional pore- only tetramer. My results suggest that both pore regions from TPC2 assemble and function even in the absence of their partner, retaining several key properties of the wildtype channel. Second, to diversify the TPC pharmacology away from non- selective channel inhibitors, I virtually screened a library of FDA-approved drugs against a structural model of TPC2. By cross-referencing these data with in vitro drug screens against a TPC-mediated disease, I identified eight novel, putative TPC inhibitors with reasonable potency and selectivity. Finally, I generated a computational model of local lysosome-ER Ca2+ signalling, guided by experimental observations. The model suggests that Ca2+ microdomains can both drive and modulate global signalling responses, depending on the ‘expression’ pattern of TPCs. Together this research provides a set of tools and an experimental framework with which to further characterise the molecular physiology of TPCs.

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