SummaryAccumulating 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.

Two-pore channels (TPCs) are endolysosomal ion channels implicated in Ca2+ signalling from acidic organelles. The relevance of these ubiquitous proteins for human disease, however, is unclear. Here, we report that lysosomes are enlarged and aggregated in fibroblasts from Parkinson disease patients with the common G2019S mutation in LRRK2. Defects were corrected by molecular silencing of TPC2, pharmacological inhibition of TPC regulators [Rab7, NAADP and PtdIns(3,5)P2] and buffering local Ca2+ increases. NAADP-evoked Ca2+ signals were exaggerated in diseased cells. TPC2 is thus a potential drug target within a pathogenic LRRK2 cascade that disrupts Ca2+-dependent trafficking in Parkinson disease.

SummaryMembrane 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.

A B S T R A C TTwo-pore channels (TPCs) are Ca 2+ -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 Ca 2+ release from sea urchin egg homogenates, NAADP-mediated channel activity of TPC2 re-routed to the plasma membrane, and PI(3,5)P 2 -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.

Graphical abstract