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New chemical tools for studying Endolysosomal Two-pore Channels

Yuan, Yu; (2021) New chemical tools for studying Endolysosomal Two-pore Channels. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Two-pore channels (TPCs) are endolysosomal ion channels of physiological and pathophysiological significance. However, fundamental properties concerning ion permeability and activation mechanisms are ambiguous. Also, as the likely targets for Ca2+-mobilizing messenger NAADP, the role of TPCs in cell-wide Ca2+ signalling is ill-defined. Importantly, their pharmacology is limited to cell-impermeable activators and a few non-selective inhibitors, which brings challenges for characterizing TPCs. In this thesis, I address the above issues. I began by examining the mechanism of action of the lysosomotropic agent, glycyl-L- phenylalanine 2-naphthylamide (GPN), which has long been appreciated for mediating Ca2+ signals from lysosomes and for probing TPC function. Its action on lysosomes has recently been questioned. However, using fibroblasts, here I show that GPN mobilises Ca2+ from acidic organelles. I move on to characterise two cell-permeable and selective TPC2 activators (A1 and H07). Additionally, I confirm that approved drugs targeting estrogen and dopamine receptors are selective TPC2 inhibitors. I go on to show that A1 and H07 activate TPC2 differentially. A1 induced larger and quicker Ca2+ signals than H07 but similar Na+ signals. A1 and H07 targeted distinct sites on TPC2. Besides, H07 but not A1-induced Ca2+ signals were regulated by external (luminal) pH. The implication is that TPC2 may be regulated in an agonist- specific manner. Finally, by using GPN and inhibiting TPC activity with novel inhibitors or siRNA knockdown, I show that TPCs are required for histamine- but not bradykinin-induced Ca2+ signals. More specifically, histamine-mediated Ca2+ signals were reduced upon TPC2 but not TPC1 knockdown. Thus, TPCs are implicated in global Ca2+ signalling evoked by physiological stimuli likely in an isoform-dependent manner. Collectively, this research has provided novel TPC modulators with which to further characterize fundamental properties and physiological roles of TPCs.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: New chemical tools for studying Endolysosomal Two-pore Channels
Event: UCL (University College London)
Language: English
Additional information: Copyright © The Author 2021. Original content in this thesis is licensed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request.
UCL classification: UCL
UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Cell and Developmental Biology
URI: https://discovery.ucl.ac.uk/id/eprint/10121424
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