Two-pore channels and disease Patel, Sandip; Kilpatrick, Bethan S.
Biochimica et biophysica acta. Molecular cell research,
11/2018, Volume:
1865, Issue:
11
Journal Article
Peer reviewed
Open access
Two-pore channels (TPCs) are Ca2+-permeable endo-lysosomal ion channels subject to multi-modal regulation. They mediate their physiological effects through releasing Ca2+ from acidic organelles in ...response to cues such as the second messenger, NAADP. Here, we review emerging evidence linking TPCs to disease. We discuss how perturbing both local and global Ca2+ changes mediated by TPCs through chemical and/or molecular manipulations can induce or reverse disease phenotypes. We cover evidence from models of Parkinson's disease, non-alcoholic fatty liver disease, Ebola infection, cancer, cardiac dysfunction and diabetes. A need for more drugs targeting TPCs is identified.
Two-pore channels (TPCs) are Ca2+-permeable ion channels localised to the endo-lysosomal system where they regulate trafficking of various cargoes including viruses. As a result, TPCs are emerging as ...important drug targets. However, their pharmacology is ill-defined. There are no approved drugs to target them. And their mechanism of ligand activation is largely unknown. Here, we identify a number of FDA-approved drugs as TPC pore blockers. Using a model of the pore of human TPC2 based on recent structures of mammalian TPCs, we virtually screened a database of ~1500 approved drugs. Because TPCs have recently emerged as novel host factors for Ebola virus entry, we reasoned that Ebola virus entry inhibitors may exert their effects through inhibition of TPCs. Cross-referencing hits from the TPC virtual screen with two recent high throughput anti-Ebola screens yielded approved drugs targeting dopamine and estrogen receptors as common hits. These compounds inhibited endogenous NAADP-evoked Ca2+ release from sea urchin egg homogenates, NAADP-mediated channel activity of TPC2 re-routed to the plasma membrane, and PI(3,5)P2-mediated channel activity of TPC2 expressed in enlarged lysosomes. Mechanistically, single channel analyses showed that the drugs reduced mean open time consistent with a direct action on the pore. Functionally, drug potency in blocking TPC2 activity correlated with inhibition of Ebola virus-like particle entry. Our results expand TPC pharmacology through the identification of approved drugs as novel blockers, support a role for TPCs in Ebola virus entry, and provide insight into the mechanisms underlying channel regulation. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
•Identification of approved drugs as TPC2 pore blockers through virtual screening•Prioritisation of drugs that inhibit Ebola virus entry•Blockade of endogenous and recombinant TPC2 channel activity by select drugs•Similar potency for inhibition of channel activity and Ebola virus entry
Membrane contact sites are regions of close apposition between organelles that facilitate information transfer. Here, we reveal an essential role for Ca2+ derived from the endo-lysosomal system in ...maintaining contact between endosomes and the endoplasmic reticulum (ER). Antagonizing action of the Ca2+-mobilizing messenger NAADP, inhibiting its target endo-lysosomal ion channel, TPC1, and buffering local Ca2+ fluxes all clustered and enlarged late endosomes/lysosomes. We show that TPC1 localizes to ER-endosome contact sites and is required for their formation. Reducing NAADP-dependent contacts delayed EGF receptor de-phosphorylation consistent with close apposition of endocytosed receptors with the ER-localized phosphatase PTP1B. In accord, downstream MAP kinase activation and mobilization of ER Ca2+ stores by EGF were exaggerated upon NAADP blockade. Membrane contact sites between endosomes and the ER thus emerge as Ca2+-dependent hubs for signaling.
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•NAADP/TPC1 signaling maintains endo-lysosomal morphology•TPC1 localizes to contacts between late endosomes and the endoplasmic reticulum•NAADP/TPC1 signaling regulates contact site formation•NAADP tempers EGF receptor-mediated signaling
Endosomes form junctions with the ER, but how this contact is regulated remains unclear. Kilpatrick et al. find that Ca2+ release by an endosomal ion channel facilitates inter-organellar coupling to temper signals mediated by an internalized growth factor receptor. Endosome-ER contact sites thus emerge as Ca2+-dependent signaling hubs.
Transient receptor potential (TRP) mucolipins (TRPMLs), encoded by the MCOLN genes, are patho-physiologically relevant endo-lysosomal ion channels crucial for membrane trafficking. Several lines of ...evidence suggest that TRPMLs mediate localised Ca
release but their role in Ca
signalling is not clear. Here, we show that activation of endogenous and recombinant TRPMLs with synthetic agonists evoked global Ca
signals in human cells. These signals were blocked by a dominant-negative TRPML1 construct and a TRPML antagonist. We further show that, despite a predominant lysosomal localisation, TRPML1 supports both Ca
release and Ca
entry. Ca
release required lysosomal and ER Ca
stores suggesting that TRPMLs, like other endo-lysosomal Ca
channels, are capable of 'chatter' with ER Ca
channels. Our data identify new modalities for TRPML1 action.
Accumulating evidence implicates acidic organelles of the endolysosomal system as mobilisable stores of Ca(2+) but their relationship to the better-characterised endoplasmic reticulum (ER) Ca(2+) ...store remains unclear. Here we show that rapid osmotic permeabilisation of lysosomes evokes prolonged, spatiotemporally complex Ca(2+) signals in primary cultured human fibroblasts. These Ca(2+) signals comprised an initial response that correlated with lysosomal disruption and secondary long-lasting spatially heterogeneous Ca(2+) oscillations that required ER-localised inositol trisphosphate receptors. Electron microscopy identified extensive membrane contact sites between lysosomes and the ER. Mobilisation of lysosomal Ca(2+) stores is thus sufficient to evoke ER-dependent Ca(2+) release probably through lysosome-ER membrane contact sites, and akin to the proposed mechanism of action of the Ca(2+) mobilising messenger nicotinic acid adenine dinucleotide phosphate (NAADP). Our data identify functional and physical association of discrete Ca(2+) stores important for the genesis of Ca(2+) signal complexity.
Acidic organelles form an important intracellular Ca(2+) pool that can drive global Ca(2+) signals through coupling with endoplasmic reticulum (ER) Ca(2+) stores. Recently identified lysosome-ER ...membrane contact sites might allow formation of Ca(2+) microdomains, although their size renders observation of Ca(2+) dynamics impractical. Here, we generated a computational model of lysosome-ER coupling that incorporated a previous model of the inositol trisphosphate (IP3) receptor as the ER Ca(2+) 'amplifier' and lysosomal leaks as the Ca(2+) 'trigger'. The model qualitatively described global Ca(2+) responses to the lysosomotropic agent GPN, which caused a controlled but substantial depletion of small solutes from the lysosome. Adapting this model to physiological lysosomal leaks induced by the Ca(2+) mobilising messenger NAADP demonstrated that lysosome-ER microdomains are capable of driving global Ca(2+) oscillations. Interestingly, our simulations suggest that the microdomain Ca(2+) need not be higher than that in the cytosol for responses to occur, thus matching the relatively high affinity of IP3 receptors for Ca(2+). The relative distribution and overall density of the lysosomal leaks dictated whether microdomains triggered or modulated global signals. Our data provide a computational framework for probing lysosome-ER Ca(2+) dynamics.
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