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NAADP calcium mobilization Three signalling molecules cause increases in intracellular Ca 2+ levels by triggering release of Ca 2+ from intracellular stores due to their action on specific Ca 2+ -permeable receptors: inositol-1,4,5-trisphosphate binds to and opens the sarcoplasmic reticulum InsP 3 receptor; cyclic ADP ribose activates the endoplasmic reticulum ryanodine receptor; but the molecular identity and location of the nicotinic acid adenine dinucleotide phosphate (NAADP) receptor is unknown. Here Calcraft et al . show that the lysosomal two-pore channel, TPC2, is the molecular target of NAADP. Ca 2+ mobilization from intracellular stores represents an important cell signalling process that is regulated, in mammalian cells, by inositol-1,4,5-trisphosphate (InsP 3 ), cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate (NAADP). While the nature of the receptors for InsP 3 and cyclic ADP ribose are known, here the lysosomal two-pore channel, TPC2, is shown to be the molecular target of NAADP. Ca 2+ mobilization from intracellular stores represents an important cell signalling process 1 that is regulated, in mammalian cells, by inositol-1,4,5-trisphosphate (InsP 3 ), cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate (NAADP). InsP 3 and cyclic ADP ribose cause the release of Ca 2+ from sarcoplasmic/endoplasmic reticulum stores by the activation of InsP 3 and ryanodine receptors (InsP 3 Rs and RyRs). In contrast, the nature of the intracellular stores targeted by NAADP and the molecular identity of the NAADP receptors remain controversial 1 , 2 , although evidence indicates that NAADP mobilizes Ca 2+ from lysosome-related acidic compartments 3 , 4 . Here we show that two-pore channels (TPCs) comprise a family of NAADP receptors, with human TPC1 (also known as TPCN1) and chicken TPC3 (TPCN3) being expressed on endosomal membranes, and human TPC2 (TPCN2) on lysosomal membranes when expressed in HEK293 cells. Membranes enriched with TPC2 show high affinity NAADP binding, and TPC2 underpins NAADP-induced Ca 2+ release from lysosome-related stores that is subsequently amplified by Ca 2+ -induced Ca 2+ release by InsP 3 Rs. Responses to NAADP were abolished by disrupting the lysosomal proton gradient and by ablating TPC2 expression, but were only attenuated by depleting endoplasmic reticulum Ca 2+ stores or by blocking InsP 3 Rs. Thus, TPCs form NAADP receptors that release Ca 2+ from acidic organelles, which can trigger further Ca 2+ signals via sarcoplasmic/endoplasmic reticulum. TPCs therefore provide new insights into the regulation and organization of Ca 2+ signals in animal cells, and will advance our understanding of the physiological role of NAADP.

Significance The formation of new blood vessels (neoangiogenesis) accompanies tissue regeneration and healing, but is also crucial for tumor growth, hence understanding how capillaries are stimulated to grow in response to local cues is essential for the much sought-after aim of controlling this process. We have elucidated a Ca ²⁺ signaling pathway involving NAADP, TPCs, and lysosomal Ca ²⁺ release activated in vascular endothelial cells by VEGF, the main angiogenic growth factor, and we show that the angiogenic response can be abolished, in cultured cells and in vivo, by inhibiting components of this signaling cascade. The specificity of this pathway in terms of VEGF receptor subtype, intracellular messengers, target channels and Ca ²⁺ storage organelles, offers new targets for novel antiangiogenic therapeutic strategies. Vascular endothelial growth factor (VEGF) and its receptors VEGFR1/VEGFR2 play major roles in controlling angiogenesis, including vascularization of solid tumors. Here we describe a specific Ca ²⁺ signaling pathway linked to the VEGFR2 receptor subtype, controlling the critical angiogenic responses of endothelial cells (ECs) to VEGF. Key steps of this pathway are the involvement of the potent Ca ²⁺ mobilizing messenger, nicotinic acid adenine-dinucleotide phosphate (NAADP), and the specific engagement of the two-pore channel TPC2 subtype on acidic intracellular Ca ²⁺ stores, resulting in Ca ²⁺ release and angiogenic responses. Targeting this intracellular pathway pharmacologically using the NAADP antagonist Ned-19 or genetically using Tpcn2 ⁻/⁻ mice was found to inhibit angiogenic responses to VEGF in vitro and in vivo. In human umbilical vein endothelial cells (HUVECs) Ned-19 abolished VEGF-induced Ca ²⁺ release, impairing phosphorylation of ERK1/2, Akt, eNOS, JNK, cell proliferation, cell migration, and capillary-like tube formation. Interestingly, Tpcn2 shRNA treatment abolished VEGF-induced Ca ²⁺ release and capillary-like tube formation. Importantly, in vivo VEGF-induced vessel formation in matrigel plugs in mice was abolished by Ned-19 and, most notably, failed to occur in Tpcn2 ⁻/⁻ mice, but was unaffected in Tpcn1 ⁻/⁻ animals. These results demonstrate that a VEGFR2/NAADP/TPC2/Ca ²⁺ signaling pathway is critical for VEGF-induced angiogenesis in vitro and in vivo. Given that VEGF can elicit both pro- and antiangiogenic responses depending upon the balance of signal transduction pathways activated, targeting specific VEGFR2 downstream signaling pathways could modify this balance, potentially leading to more finely tailored therapeutic strategies.

Ion selectivity is a defining feature of a given ion channel and is considered immutable. Here we show that ion selectivity of the lysosomal ion channel TPC2, which is hotly debated (Calcraft et al., 2009; Guo et al., 2017; Jha et al., 2014; Ruas et al., 2015; Wang et al., 2012), depends on the activating ligand. A high-throughput screen identified two structurally distinct TPC2 agonists. One of these evoked robust Ca2+-signals and non-selective cation currents, the other weaker Ca2+-signals and Na+-selective currents. These properties were mirrored by the Ca2+-mobilizing messenger, NAADP and the phosphoinositide, PI(3,5)P2, respectively. Agonist action was differentially inhibited by mutation of a single TPC2 residue and coupled to opposing changes in lysosomal pH and exocytosis. Our findings resolve conflicting reports on the permeability and gating properties of TPC2 and they establish a new paradigm whereby a single ion channel mediates distinct, functionally-relevant ionic signatures on demand.

Research into the biological role of the Ca 2+ -releasing second messenger NAADP (nicotinic acid adenine dinucleotide phosphate) has been hampered by a lack of chemical probes. To find new chemical probes for exploring NAADP signaling, we turned to virtual screening, which can evaluate millions of molecules rapidly and inexpensively. We used NAADP as the query ligand to screen the chemical library ZINC for compounds with similar three-dimensional shape and electrostatic properties. We tested the top-ranking hits in a sea urchin egg bioassay and found that one hit, Ned-19, blocks NAADP signaling at nanomolar concentrations. In intact cells, Ned-19 blocked NAADP signaling and fluorescently labeled NAADP receptors. Moreover, we show the utility of Ned-19 as a chemical probe by using it to demonstrate that NAADP is a key causal link between glucose sensing and Ca 2+ increases in mouse pancreatic beta cells.

Our research introduces the natural flavonoid naringenin as a novel inhibitor of an emerging class of intracellular channels, Two-Pore Channel 2 (TPC2), as shown by electrophysiological evidence in a heterologous system, i.e. Arabidopsis vacuoles lacking endogenous TPCs. In view of the control exerted by TPC2 on intracellular calcium signaling, we demonstrated that naringenin dampens intracellular calcium responses of human endothelial cells stimulated with VEGF, histamine or NAADP-AM, but not with ATP or Angiopoietin-1 (negative controls). The ability of naringenin to impair TPC2-dependent biological activities was further explored in an established in vivo model, in which VEGF-containing matrigel plugs implanted in mice failed to be vascularized in the presence of naringenin. Overall, the present data suggest that naringenin inhibition of TPC2 activity and the observed inhibition of angiogenic response to VEGF are linked by impaired intracellular calcium signaling. TPC2 inhibition is emerging as a key therapeutic step in a range of important pathological conditions including the progression and metastatic potential of melanoma, Parkinson’s disease, and Ebola virus infection. The identification of naringenin as an inhibitor of TPC2-mediated signaling provides a novel and potentially relevant tool for the advancement of this field of research.

Significance Two-pore channels (TPCs) are a recently discovered family of endolysosomal ion channels, but their regulation is controversial. By defining the TPC interactome, we provide a community resource that illuminates TPC complex regulation and resolves associations with novel partners and processes. Physical interactions with endolysosomal trafficking regulators predominate, and Rab GTPases impart isoform-specific roles for TPCs in organelle proliferation and cellular pigmentation. These data imply a fundamental role for TPCs in trafficking that augurs significance for disease states exhibiting lysosomal proliferation where TPC dysregulation may drive pathogenesis.

Tetrandrine (Tet) is a potent inhibitor of Ebola virus replication by blocking NAADP-dependent calcium release through endolysosomal two-pore channels (TPCs) and a moderately potent anti-tumor agent. Using a clickable photoaffinity probe, we identify lysosomal integral membrane protein-2 (LIMP-2) as a direct target of Tet and a key regulator of this calcium signaling. Tet binds LIMP-2’s ectodomain, inhibiting lysosomal cholesterol and sphingosine transport, which alters lipid metabolism. Tet treatment and LIMP-2 depletion inhibit NAADP-dependent calcium release, reversible by removing lysosomal cholesterol and sphingosine. Sphingosine triggers lysosomal calcium release via TPCs and restores this signaling in Tet-treated or LIMP-2-deficient cells, revealing a LIMP-2-regulated, sphingosine-dependent lysosomal calcium pathway. At higher doses, Tet induces apoptosis through unfolded protein response activation independently of LIMP-2. These findings highlight Tet as a LIMP-2 inhibitor, elucidate its role in calcium signaling and cell death, and suggest therapeutic potential for Tet and LIMP-2 inhibitors in antiviral treatments. Tetrandrine is one of the most potent inhibitors of Ebola Virus infection. Here the authors identify LIMP-2 as a direct cellular target of Tetrandrine and establish a functional connection between lysosomal sphingosine homeostasis and calcium regulation.

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) stimulates calcium release from acidic stores such as lysosomes and is a highly potent calcium-mobilising second messenger. NAADP plays an important role in calcium signalling in the heart under basal conditions and following β-adrenergic stress. Nevertheless, the spatial interaction of acidic stores with other parts of the calcium signalling apparatus in cardiac myocytes is unknown. We present evidence that lysosomes are intimately associated with the sarcoplasmic reticulum (SR) in ventricular myocytes; a median separation of 20 nm in 2D electron microscopy and 3.3 nm in 3D electron tomography indicates a genuine signalling microdomain between these organelles. Fourier analysis of immunolabelled lysosomes suggests a sarcomeric pattern (dominant wavelength 1.80 μm). Furthermore, we show that lysosomes form close associations with mitochondria (median separation 6.2 nm in 3D studies) which may provide a basis for the recently-discovered role of NAADP in reperfusion-induced cell death. The trigger hypothesis for NAADP action proposes that calcium release from acidic stores subsequently acts to enhance calcium release from the SR. This work provides structural evidence in cardiac myocytes to indicate the formation of microdomains between acidic and SR calcium stores, supporting emerging interpretations of NAADP physiology and pharmacology in heart.

Nicotinic acid adenine dinucleotide 2’-phosphate (NAADP) is a naturally occurring nucleotide that has been shown to be involved in the release of Ca 2+ from intracellular stores in a wide variety of cell types, tissues and organisms. Current evidence suggests that NAADP may function as a trigger to initiate a Ca 2+ signal that is then amplified by other Ca 2+ release mechanisms. A fundamental question that remains unanswered is the identity of the NAADP receptor. Our recent studies have identified HN1L/JPT2 as a high affinity NAADP binding protein that is essential for the modulation of Ca 2+ channels.

We have developed a correlation between experimental and density functional theory-derived results of the hydride-donating power, or “hydricity”, of various ruthenium, rhenium, and organic hydride donors. This approach utilizes the correlation between experimental hydricity values and their corresponding calculated free-energy differences between the hydride donors and their conjugate acceptors in acetonitrile, and leads to an extrapolated value of the absolute free energy of the hydride ion without the necessity to calculate it directly. We then use this correlation to predict, from density functional theory-calculated data, hydricity values of ruthenium and rhenium complexes that incorporate the pbnHH ligand—pbnHH = 1,5-dihydro-2-(2-pyridyl)-benzo[ b ]-1,5-naphthyridine—to model the function of NADPH. These visible light-generated, photocatalytic complexes produced by disproportionation of a protonated-photoreduced dimer of a metal-pbn complex may be valuable for use in reducing CO ₂ to fuels such as methanol. The excited-state lifetime of photoexcited [Ru(bpy) ₂(pbnHH)] ²⁺ is found to be about 70 ns, and this excited state can be reductively quenched by triethylamine or 1,4-diazabicyclo[2.2.2]octane to produce the one-electron-reduced [Ru(bpy) ₂(pbnHH)] ⁺ species with half-life exceeding 50 μs, thus opening the door to new opportunities for hydride-transfer reactions leading to CO ₂ reduction by producing a species with much increased hydricity.