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Two-pore channels (TPCs) are two-domain members of the voltage-gated ion channel superfamily that localize to acidic organelles. Their mechanism of activation (ligands such as NAADP/PI(3,5)P 2 versus voltage) and ion selectivity (Ca 2+ versus Na + ) is debated. Here we report that a cluster of arginine residues in the first domain required for selective voltage-gating of TPC1 map not to the voltage-sensing fourth transmembrane region (S4) but to a cytosolic downstream region (S4-S5 linker). These residues are conserved between TPC isoforms suggesting a generic role in TPC activation. Accordingly, mutation of residues in TPC1 but not the analogous region in the second domain prevents Ca 2+ release by NAADP in intact cells. Our data affirm the role of TPCs in NAADP-mediated Ca 2+ signalling and unite differing models of channel activation through identification of common domain-specific residues., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The two-pore channels (TPC1 and TPC2) belong to an ancient family of intracellular ion channels expressed in the endolysosomal system. Little is known about how regulatory inputs converge to modulate TPC activity, and proposed activation mechanisms are controversial. Here, we compiled a proteomic characterization of the human TPC interactome, which revealed that TPCs complex with many proteins involved in Ca(2+) homeostasis, trafficking, and membrane organization. Among these interactors, TPCs were resolved to scaffold Rab GTPases and regulate endomembrane dynamics in an isoform-specific manner. TPC2, but not TPC1, caused a proliferation of endolysosomal structures, dysregulating intracellular trafficking, and cellular pigmentation. These outcomes required both TPC2 and Rab activity, as well as their interactivity, because TPC2 mutants that were inactive, or rerouted away from their endogenous expression locale, or deficient in Rab binding, failed to replicate these outcomes. Nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca(2+) release was also impaired using either a Rab binding-defective TPC2 mutant or a Rab inhibitor. These data suggest a fundamental role for the ancient TPC complex in trafficking that holds relevance for lysosomal proliferative scenarios observed in disease.

TPCs (two-pore channels) are NAADP (nicotinic acid-adenine dinucleotide phosphate)-sensitive Ca2+-permeable ion channels expressed on acidic organelles. In the present study we show that deletion of the N-terminal region redirects TPC1 to the ER (endoplasmic reticulum). The introduction of fluorophores at the N-terminus of TPC1 does not affect its subcellular location, but does reversibly abolish NAADP sensitivity. Our results reveal a dual role for the N-terminus in localization and function of TPC1.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent second messenger that mobilizes Ca(2+) from the acidic endolysosomes by activation of the two-pore channels TPC1 and TPC2. The channel properties of human TPC1 have not been studied before, and its cellular function is not known. In the present study, we characterized TPC1 incorporated into lipid bilayers. The native and recombinant TPC1 channels are activated by NAADP. TPC1 activity requires acidic luminal pH and high luminal Ca(2+). With Ba(2+) as the permeable ion, luminal Ca(2+) activates TPC1 with an apparent K(m) of 180 μm. TPC1 operates in two tightly coupled conductance states of 47 ± 8 and 200 ± 9 picosiemens. Importantly, opening of the large conductance markedly increases the small conductance mean open time. Changes in membrane potential from 0 to -60 mV increased linearly both the small and the large conductances and NP(o), indicating that TPC1 is regulated by voltage. Intriguingly, the apparent affinity for activation of TPC1 by its ligand NAADP is not constant. Rather, hyperpolarization increases the apparent affinity of TPC1 for NAADP by 10 nm/mV. The concerted regulation of TPC1 activity by luminal Ca(2+) and by membrane potential thus provides a potential mechanism to explain NAADP-induced Ca(2+) oscillations. These findings reveal unique properties of TPC1 to explain its role in Ca(2+) oscillations and cell function.

CD38 is a multifunctional protein possessing ADP-ribosyl cyclase activity responsible for both the synthesis and the degradation of several Ca(2+)-mobilizing second messengers. Although a variety of functions have been ascribed to CD38, such as immune responses, insulin secretion, and social behavior in adults, nothing is known of its role during embryonic development when Ca(2+) signals feature prominently. Here, we report the identification and functional expression of CD38 from Xenopus laevis, a key model organism for the study of vertebrate development. We show that CD38 expression and endogenous ADP-ribosyl cyclase activity are developmentally regulated during cellular differentiation. Chemical or molecular inhibition of CD38 abolished ADP-ribosyl cyclase activity and disrupted elongation of the anterior-posterior axis and differentiation of skeletal muscle, culminating in embryonic death. Our data uncover a previously unknown role for CD38 as an essential regulator of embryonic development.

Mutations in the leucine-rich repeat kinase-2 (LRRK2) gene cause late-onset Parkinson's disease, but its physiological function has remained largely unknown. Here we report that LRRK2 activates a calcium-dependent protein kinase kinase-β (CaMKK-β)/adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathway which is followed by a persistent increase in autophagosome formation. Simultaneously, LRKR2 overexpression increases the levels of the autophagy receptor p62 in a protein synthesis-dependent manner, and decreases the number of acidic lysosomes. The LRRK2-mediated effects result in increased sensitivity of cells to stressors associated with abnormal protein degradation. These effects can be mimicked by the lysosomal Ca(2+)-mobilizing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) and can be reverted by an NAADP receptor antagonist or expression of dominant-negative receptor constructs. Collectively, our data indicate a molecular mechanism for LRRK2 deregulation of autophagy and reveal previously unidentified therapeutic targets.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is an agonist-generated second messenger that releases Ca(2+) from intracellular acidic Ca(2+) stores. Recent evidence has identified the two-pore channels (TPCs) within the endolysosomal system as NAADP-regulated Ca(2+) channels that release organellar Ca(2+) in response to NAADP. However, little is known about the mechanism coupling NAADP binding to calcium release. To identify the NAADP binding site, we employed a photoaffinity labeling method using a radioactive photoprobe based on 5-azido-NAADP ([(32)P-5N(3)]NAADP) that exhibits high affinity binding to NAADP receptors. In several systems that are widely used for studying NAADP-evoked Ca(2+) signaling, including sea urchin eggs, human cell lines (HEK293, SKBR3), and mouse pancreas, 5N(3)-NAADP selectively labeled low molecular weight sites that exhibited the diagnostic pharmacology of NAADP-sensitive Ca(2+) release. Surprisingly, we were unable to demonstrate labeling of endogenous, or overexpressed, TPCs. Furthermore, labeling of high affinity NAADP binding sites was preserved in pancreatic samples from TPC1 and TPC2 knock-out mice. These photolabeling data suggest that an accessory component within a larger TPC complex is responsible for binding NAADP that is unique from the core channel itself. This observation necessitates critical evaluation of current models of NAADP-triggered activation of the TPC family.

TPCs (two-pore channels) have recently been identified as targets for the Ca2+-mobilizing messenger NAADP (nicotinic acid-adenine dinucleotide phosphate). TPCs have a unique structure consisting of cytosolic termini, two hydrophobic domains (I and II) each comprising six transmembrane regions and a pore, and a connecting cytosolic loop; however, little is known concerning how these channels are assembled. In the present paper, we report that both domain I and II of human TPCs are capable of independent insertion into membranes, whereas the loop linking the domains fails to insert. Pairs of transmembrane regions within domain I of TPC1 are also capable of insertion, consistent with sequential translational integration of hydrophobic regions. Insertion of the first two transmembrane regions, however, was inefficient, indicating possible interaction between transmembrane regions during translation. Both domains, and each pair of transmembrane regions within domain I, were capable of forming oligomers, highlighting marked redundancy in the molecular determinants driving oligomer formation. Each hydrophobic domain formed dimers upon cross-linking. The first four transmembrane regions of TPC1 also formed dimers, whereas transmembrane regions 5 and 6, encompassing the pore loop, formed both dimers and tetramers. TPCs thus probably assemble as dimers through differential interactions between transmembrane regions. The present study provides new molecular insight into the membrane insertion and oligomerization of TPCs.

NAADP is a potent second messenger that mobilizes Ca(2+) from acidic organelles such as endosomes and lysosomes. The molecular basis for Ca(2+) release by NAADP, however, is uncertain. TRP mucolipins (TRPMLs) and two-pore channels (TPCs) are Ca(2+)-permeable ion channels present within the endolysosomal system. Both have been proposed as targets for NAADP. In the present study, we probed possible physical and functional association of these ion channels. Exogenously expressed TRPML1 showed near complete colocalization with TPC2 and partial colocalization with TPC1. TRPML3 overlap with TPC2 was more modest. TRPML1 and to some extent TRPML3 co-immunoprecipitated with TPC2 but less so with TPC1. Current recording, however, showed that TPC1 and TPC2 did not affect the activity of wild-type TRPML1 or constitutively active TRPML1(V432P). N-terminally truncated TPC2 (TPC2delN), which is targeted to the plasma membrane, also failed to affect TRPML1 and TRPML1(V432P) channel function or TRPML1(V432P)-mediated Ca(2+) influx. Whereas overexpression of TPCs enhanced NAADP-mediated Ca(2+) signals, overexpression of TRPML1 did not, and the dominant negative TRPML1(D471K) was without affect on endogenous NAADP-mediated Ca(2+) signals. Furthermore, the single channel properties of NAADP-activated TPC2delN were not affected by TRPML1. Finally, NAADP-evoked Ca(2+) oscillations in pancreatic acinar cells were identical in wild-type and TRPML1(-/-) cells. We conclude that although TRPML1 and TPCs are present in the same complex, they function as two independent organellar ion channels and that TPCs, not TRPMLs, are the targets for NAADP.

Two-pore channels (TPCs) localize to the endolysosomal system and have recently emerged as targets for the Ca(2+)-mobilizing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). However, their membrane topology is unknown. Using fluorescence protease protection assays, we show that human TPC1 and TPC2 possess cytosolic N and C termini and therefore an even number of transmembrane regions. Fluorophores placed at position 225 or 347 in TPC1, or 339 in TPC2 were also cytosolic, whereas a fluorophore at position 628 in TPC1 was luminal. These data together with sequence similarity to voltage-gated Ca(2+) and Na(+) channels, and unbiased in silico predictions are consistent with a topology in which two homologous domains are present, each comprising 6 transmembrane regions and a re-entrant pore loop. Immunocytochemical analysis of selectively permeabilized cells using antipeptide antibodies confirmed that the C-terminal tails of recombinant TPCs are cytosolic and that residues 240-254 of TPC2 prior to putative pore 1 are luminal. Both TPC1 and TPC2 are N-glycosylated with residues 599, 611, and 616 contributing to glycosylation of TPC1. This confirms the luminal position of these residues, which immediately precede the putative pore loop of the second domain. Mutation of all three glycosylation sites in TPC1 enhances NAADP-evoked cytosolic Ca(2+) signals. Our data establish essential features of the topology of two-pore channels.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a ubiquitous messenger proposed to stimulate Ca(2+) release from acidic organelles via two-pore channels (TPCs). It has been difficult to resolve this trigger event from its amplification via endoplasmic reticulum Ca(2+) stores, fuelling speculation that archetypal intracellular Ca(2+) channels are the primary targets of NAADP. Here, we redirect TPC2 from lysosomes to the plasma membrane and show that NAADP evokes Ca(2+) influx independent of ryanodine receptors and that it activates a Ca(2+)-permeable channel whose conductance is reduced by mutation of a residue within a putative pore. We therefore uncouple TPC2 from amplification pathways and prove that it is a pore-forming subunit of an NAADP-gated Ca(2+) channel.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a widespread and potent calcium-mobilizing messenger that is highly unusual in activating calcium channels located on acidic stores. However, the molecular identity of the target protein is unclear. In this study, we show that the previously uncharacterized human two-pore channels (TPC1 and TPC2) are endolysosomal proteins, that NAADP-mediated calcium signals are enhanced by overexpression of TPC1 and attenuated after knockdown of TPC1, and that mutation of a single highly conserved residue within a putative pore region abrogated calcium release by NAADP. Thus, TPC1 is critical for NAADP action and is likely the long sought after target channel for NAADP.

The sea urchin is an extensively used model system for the study of calcium signalling by the messenger molecules NAADP and cyclic ADP-ribose. Both are synthesized by ADP-ribosyl cyclases but our molecular understanding of these enzymes in the sea urchin is limited. We have recently reported the cloning of an extended family of sea urchin ADP-ribosyl cyclases and shown that one of these enzymes (SpARC1) is active within the endoplasmic reticulum lumen. These studies suggest that production of messengers is compartmentalized. Here we characterize the properties of SpARC2. SpARC2 catalyzed both NAADP and cyclic ADP-ribose production. Unusually, the NAD surrogate, NGD was a poor substrate. In contrast to SpARC1, heterologously expressed SpARC2 localized to the plasma membrane via a glycosylphosphatidylinositol (GPI)-anchor. Transcripts for SpARC2 were readily detectable in sea urchin eggs and a majority of the endogenous membrane bound activity was found to be GPI-anchored. Our data reveal striking differences in the properties of sea urchin ADP-ribosyl cyclases and provide further evidence that messenger production may occur outside of the cytosol.

Background: ADP-ribosyl cyclases are remarkable enzymes capable of catalyzing multiple reactions including the synthesis of the novel and potent intracellular calcium mobilizing messengers, cyclic ADP-ribose and NAADP. Not all ADP-ribosyl cyclases however have been characterized at the molecular level. Moreover, those that have are located predominately at the outer cell surface and thus away from their cytosolic substrates., Methodology/principal Findings: Here we report the molecular cloning of a novel expanded family of ADP-ribosyl cyclases from the sea urchin, an extensively used model organism for the study of inositol trisphosphate-independent calcium mobilization. We provide evidence that one of the isoforms (SpARC1) is a soluble protein that is targeted exclusively to the endoplasmic reticulum lumen when heterologously expressed. Catalytic activity of the recombinant protein was readily demonstrable in crude cell homogenates, even under conditions where luminal continuity was maintained., Conclusions/significance: Our data reveal a new intracellular location for ADP-ribosyl cyclases and suggest that production of calcium mobilizing messengers may be compartmentalized.

NAADP (nicotinic acid-adenine dinucleotide phosphate) is a newly described intracellular messenger molecule that mediates Ca2+ increases in a variety of cells. However, little is known of the mechanism whereby ligand binding regulates the target protein. We report in the present paper that NAADP receptors from sea urchin eggs undergo an unusual stabilization process that appears to be dependent upon the time during which receptors are exposed to their ligand. We demonstrate that receptors 'tagged' with NAADP for short periods were more readily dissociated following subsequent delipidation than those labelled for longer. Stabilization of NAADP receptors by their ligand was delayed relative to ligand association taking on the order of minutes to develop at picomolar concentrations. The stabilizing effects of NAADP did not require cytosolic factors or the continued presence of NAADP and persisted upon solubilization. NAADP receptors, however, failed to stabilize at reduced temperature. We conclude that NAADP receptors possess a simple molecular memory endowing them with the remarkable ability to detect the duration of their activation.

Cells possess several Ca2+-mobilizing messengers, which couple stimulation at the cell surface by a multitude of extracellular cues to the regulation of intracellular Ca2+-sensitive targets. Recent studies suggest that agonists differentially select from this molecular palette to generate their characteristic Ca2+ signals but it is still unclear whether different messengers mediate different functions or whether they act in a redundant fashion. In this study, we compared the effects of nicotinic acid adenine dinucleotide phosphate (NAADP), a novel Ca2+-mobilizing messenger, with that of the prototypical messenger inositol trisphosphate on cytosolic Ca2+ levels and differentiation status of PC12 cells. We demonstrate that liposomal delivery of NAADP mediated release of Ca2+ from acidic Ca2+ stores and that this stimulus was sufficient to drive differentiation of the cells to a neuronal-like phenotype. In sharp contrast, cell fate was unaffected by more transient Ca2+ signals generated by inositol trisphosphate-evoked release of endoplasmic reticulum Ca2+ stores. Our data establish for the first time (i) the presence of novel NAADP-sensitive Ca2+ stores in PC12 cells, (ii) a role for NAADP in differentiation, and (iii) that Ca2+-dependent function can be messenger-specific. Thus, differential recruitment of intracellular Ca2+-mobilizing messengers and their target Ca2+ stores may represent a robust means of maintaining stimulus fidelity in the control of Ca2+-dependent cell function.

The mechanisms through which pancreatic beta cells recognize and respond to changes in circulating glucose are well understood. Evidence is accumulating that a subpopulation of neurons in the ventromedial hypothalamus (VMH) use similar cellular mechanisms to sense changes in extracellular glucose. In the present study, we used PCR and single-cell calcium imaging techniques to investigate whether glucose-sensing cells in the pancreas and hypothalamus employ a similar set of stimulus-response elements. Dispersed cells from mouse pancreata and hypothalamus were used in conjunction with the insulin-secreting cell line MIN6. We present functional data suggesting that both pancreatic and a subpopulation of hypothalamic cells exhibit glucose- and tolbutamide-evoked changes in cytosolic calcium and consider some clinical implications of different glucose sensors using the same mechanisms.

Mobilization of intracellular Ca2+ pools by NAADP (nicotinic acid-adenine dinucleotide phosphate) is becoming increasingly recognized as an important determinant of complex Ca2+ signals. However, the properties of the putative Ca2+ channel activated by NAADP are poorly defined. In the present study, we provide evidence that binding of NAADP to its target protein in sea urchin eggs requires phospholipids. Decreasing the level of protein-bound lipid in detergent extracts by either dilution of the preparation at a fixed detergent concentration or increasing the detergent concentration at a fixed protein concentration inhibited [32P]NAADP binding. These effects were prevented by the addition of phospholipids, but not other related molecules, were reversible and were associated with a marked decrease in the apparent affinity of the target protein for its ligand. Additionally, we show that the extent of dissociation of NAADP-receptor ligand complexes during gel filtration in the presence of detergent correlates well with the extent of delipidation. Our data highlight the importance of the lipid environment for interaction of NAADP with its target protein.

Nicotinic acid-adenine dinucleotide phosphate (NAADP) is fast emerging as a new intracellular Ca2+-mobilizing messenger. In sea urchin egg homogenates, binding of NAADP to its receptor is not readily reversible; hence, prior incubation with low concentrations of NAADP is more effective in inhibiting subsequent binding of radiolabelled NAADP than incubating the preparation with the two ligands simultaneously [Patel, Churchill and Galione (2000) Biochem. J. 352, 725-729]. We extend this finding to show that NAADP is more effective still in inhibiting the subsequent radioligand binding at lower homogenate concentrations, an effect again quite probably due to the non-reversible nature of the receptor-ligand interaction. Enhanced sensitivity of the preparation to NAADP afforded by simple manipulation of the experimental conditions has been applied to determine low levels of NAADP in acid extracts from human red blood cells, rat hepatocytes and Escherichia coli without interference from NADP breakdown. Our improved method for the quantification of NAADP should prove useful in the further assessment of its signalling role within cells.