NAADP as an intracellular messenger regulating lysosomal calcium-release channels

Recent studies into the mechanisms of action of the Ca(2+)-mobilizing messenger NAADP (nicotinic acid-adenine dinucleotide phosphate) have demonstrated that a novel family of intracellular Ca(2+)-release channels termed TPCs (two-pore channels) are components of the NAADP receptor. TPCs appear to be exclusively localized to the endolysosomal system. These findings confirm previous pharmacological and biochemical studies suggesting that NAADP targets acidic Ca(2+) stores rather than the endoplasmic reticulum, the major site of action of the other two principal Ca(2+)-mobilizing messengers, InsP(3) and cADPR (cADP-ribose). Studies of the messenger roles of NAADP and the function of TPCs highlight the novel role of lysosomes and other organelles of the endocytic pathway as messenger-regulated Ca(2+) stores which also affects the regulation of the endolysosomal system.     

Triggering of Ca2+ signals by NAADP-gated two-pore channels: a role for membrane contact sites?

NAADP (nicotinic acid-adenine dinucleotide phosphate) is a potent Ca2+-mobilizing messenger implicated in many Ca2+-dependent cellular processes. It is highly unusual in that it appears to trigger Ca2+ release from acidic organelles such as lysosomes. These signals are often amplified by archetypal Ca2+ channels located in the endoplasmic reticulum. Recent studies have converged on the TPCs (two-pore channels) which localize to the endolysosomal system as the likely primary targets through which NAADP mediates its effects. 'Chatter' between TPCs and endoplasmic reticulum Ca2+ channels is disrupted when TPCs are directed away from the endolysosomal system. This suggests that intracellular Ca2+ release channels may be closely apposed, possibly at specific membrane contact sites between acidic organelles and the endoplasmic reticulum.     

An NAADP-gated two-pore channel targeted to the plasma membrane uncouples triggering from amplifying Ca2+ signals

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.     

Photoaffinity labeling of nicotinic acid adenine dinucleotide phosphate (NAADP) targets in mammalian cells

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.     

Molecular mechanisms of endolysosomal Ca2+ signalling in health and disease

Endosomes, lysosomes and lysosome-related organelles are emerging as important Ca2+ storage cellular compartments with a central role in intracellular Ca2+ signalling. Endocytosis at the plasma membrane forms endosomal vesicles which mature to late endosomes and culminate in lysosomal biogenesis. During this process, acquisition of different ion channels and transporters progressively changes the endolysosomal luminal ionic environment (e.g. pH and Ca2+) to regulate enzyme activities, membrane fusion/fission and organellar ion fluxes, and defects in these can result in disease. In the present review we focus on the physiology of the inter-related transport mechanisms of Ca2+ and H+ across endolysosomal membranes. In particular, we discuss the role of the Ca2+-mobilizing messenger NAADP (nicotinic acid adenine dinucleotide phosphate) as a major regulator of Ca2+ release from endolysosomes, and the recent discovery of an endolysosomal channel family, the TPCs (two-pore channels), as its principal intracellular targets. Recent molecular studies of endolysosomal Ca2+ physiology and its regulation by NAADP-gated TPCs are providing exciting new insights into the mechanisms of Ca2+-signal initiation that control a wide range of cellular processes and play a role in disease. These developments underscore a new central role for the endolysosomal system in cellular Ca2+ regulation and signalling.     

Transient receptor potential mucolipin 1 (TRPML1) and two-pore channels are functionally independent organellar ion channels

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.     

Cyclic adenosine diphosphate ribose activates ryanodine receptors, whereas NAADP activates two-pore domain channels

The mechanism by which cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) mobilize intracellular Ca(2+) stores remains controversial. It is open to question whether cADPR regulates ryanodine receptors (RyRs) directly, as originally proposed, or indirectly by promoting Ca(2+) uptake into the sarco/endoplasmic reticulum by sarco/endoplasmic reticulum Ca(2+)-ATPases. Conversely, although we have proposed that NAADP mobilizes endolysosomal Ca(2+) stores by activating two-pore domain channels (TPCs), others suggest that NAADP directly activates RyRs. We therefore assessed Ca(2+) signals evoked by intracellular dialysis from a patch pipette of cADPR and NAADP into HEK293 cells that stably overexpress either TPC1, TPC2, RyR1, or RyR3. No change in intracellular Ca(2+) concentration was triggered by cADPR in either wild-type HEK293 cells (which are devoid of RyRs) or in cells that stably overexpress TPC1 and TPC2, respectively. By contrast, a marked Ca(2+) transient was triggered by cADPR in HEK293 cells that stably expressed RyR1 and RyR3. The Ca(2+) transient was abolished following depletion of endoplasmic reticulum stores by thapsigargin and block of RyRs by dantrolene but not following depletion of acidic Ca(2+) stores by bafilomycin. By contrast, NAADP failed to evoke a Ca(2+) transient in HEK293 cells that expressed RyR1 or RyR3, but it induced robust Ca(2+) transients in cells that stably overexpressed TPC1 or TPC2 and in a manner that was blocked following depletion of acidic stores by bafilomycin. We conclude that cADPR triggers Ca(2+) release by activating RyRs but not TPCs, whereas NAADP activates TPCs but not RyRs.     

Nicotinic acid adenine dinucleotide phosphate (NAADP) regulates autophagy in cultured astrocytes

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca(2+)-mobilizing messenger that in many cells releases Ca(2+) from the endolysosomal system. Recent studies have shown that NAADP-induced Ca(2+) mobilization is mediated by the two-pore channels (TPCs). Whether NAADP acts as a messenger in astrocytes is unclear, and downstream functional consequences have yet to be defined. Here, we show that intracellular delivery of NAADP evokes Ca(2+) signals from acidic organelles in rat astrocytes and that these signals are potentiated upon overexpression of TPCs. We also show that NAADP increases acidic vesicular organelle formation and levels of the autophagic markers, LC3II and beclin-1. NAADP-mediated increases in LC3II levels were reduced in cells expressing a dominant-negative TPC2 construct. Our data provide evidence that NAADP-evoked Ca(2+) signals mediated by TPCs regulate autophagy.     

Interactions between calcium release pathways: multiple messengers and multiple stores

The discovery of cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) as Ca(2+) releasing messengers has provided additional insight into how complex Ca(2+) signalling patterns are generated. There is mounting evidence that these molecules along with the more established messenger, myo-inositol 1,4,5-trisphosphate (IP(3)), have a widespread messenger role in shaping Ca(2+) signals in many cell types. These molecules have distinct structures and act on specific Ca(2+) release mechanisms. Emerging principles are that cADPR enhances the Ca(2+) sensitivity of ryanodine receptors (RYRs) to produce prolonged Ca(2+) signals through Ca(2+)-induced Ca(2+) release (CICR), while NAADP acts on a novel Ca(2+) release mechanism to produce a local trigger Ca(2+) signal which can be amplified by CICR by recruiting other Ca(2+) release mechanisms. Whilst IP(3) and cADPR mobilise Ca(2+) from the endoplasmic reticulum (ER), recent evidence from the sea urchin egg suggests that the major NAADP-sensitive Ca(2+) stores are reserve granules, acidic lysosomal-related organelles.In this review we summarise the role of multiple Ca(2+) mobilising messengers, Ca(2+) release channels and Ca(2+) stores, and the interplay between them, in the generation of specific Ca(2+) signals. Focusing upon cADPR and NAADP, we discuss how cellular stimuli may draw upon different combinations of these messengers to produce distinct Ca(2+) signalling signatures.     

NAADP controls cross-talk between distinct Ca2+ stores in the heart

In cardiac muscle the sarcoplasmic reticulum (SR) plays a key role in the control of contraction, releasing Ca(2+) in response to Ca(2+) influx across the sarcolemma via voltage-gated Ca(2+) channels. Here we report evidence for an additional distinct Ca(2+) store and for actions of nicotinic acid adenine dinucleotide phosphate (NAADP) to mobilize Ca(2+) from this store, leading in turn to enhanced Ca(2+) loading of the SR. Photoreleased NAADP increased Ca(2+) transients accompanying stimulated action potentials in ventricular myocytes. The effects were prevented by bafilomycin A (an H(+)-ATPase inhibitor acting on acidic Ca(2+) stores), by desensitizing concentrations of NAADP, and by ryanodine and thapsigargin to suppress SR function. Bafilomycin A also suppressed staining of acidic stores with Lysotracker Red without affecting SR integrity. Cytosolic application of NAADP by means of its membrane permeant acetoxymethyl ester increased myocyte contraction and the frequency and amplitude of Ca(2+) sparks, and these effects were inhibited by bafilomycin A. Effects of NAADP were associated with an increase in SR Ca(2+) load and appeared to be regulated by beta-adrenoreceptor stimulation. The observations are consistent with a novel role for NAADP in cardiac muscle mediated by Ca(2+) release from bafilomycin-sensitive acidic stores, which in turn enhances SR Ca(2+) release by increasing SR Ca(2+) load.     

Recent breakthroughs in the molecular mechanism of capacitative calcium entry (with thoughts on how we got here)

Activation of phospholipase C by G-protein-coupled receptors results in release of intracellular Ca(2+) and activation of Ca(2+) channels in the plasma membrane. The intracellular release of Ca(2+) is signaled by the second messenger, inositol 1,4,5-trisphosphate. Ca(2+) entry involves signaling from depleted intracellular stores to plasma membrane Ca(2+) channels, a process referred to as capacitative calcium entry or store-operated calcium entry. The electrophysiological current associated with capacitative calcium entry is the calcium-release-activated calcium current, or I(crac). In the 20 years since the inception of the concept of capacitative calcium entry, a variety of activation mechanisms have been proposed, and there has been considerable interest in the possibility of transient receptor potential channels functioning as store-operated channels. However, in the past 2 years, two major players in both the signaling and permeation mechanisms for store-operated channels have been discovered: Stim1 (and possibly Stim2) and the Orai proteins. Activation of store-operated channels involves an endoplasmic reticulum Ca(2+) sensor called Stim1. Stim1 acts by redistributing within a small component of the endoplasmic reticulum, approaching the plasma membrane, but does not appear to translocate into the plasma membrane. Stim1, either directly or indirectly, signals to plasma membrane Orai proteins which constitute pore-forming subunits of store-operated channels.     

Nicotinic acid adenine dinucleotide phosphate potentiates neurite outgrowth

Ca(2+) regulates a spectrum of cellular processes including many aspects of neuronal function. Ca(2+)-sensitive events such as neurite extension and axonal guidance are driven by Ca(2+) signals that are precisely organized in both time and space. These complex cues result from both Ca(2+) influx across the plasma membrane and the mobilization of intracellular Ca(2+) stores. In the present study, using rat cortical neurons, we have examined the effects of the novel intracellular Ca(2+)-mobilizing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) on neurite length and cytosolic Ca(2+) levels. We show that NAADP potentiates neurite extension in response to serum and nerve growth factor and stimulates increases in cytosolic Ca(2+) from bafilomycin-sensitive Ca(2+) stores. Simultaneous blockade of inositol trisphosphate and ryanodine receptors abolished the effects of NAADP on neurite length and reduced the magnitude of NAADP-mediated Ca(2+) signals. This is the first report demonstrating functional NAADP receptors in a mammalian neuron. Interplay between NAADP receptors and more established intracellular Ca(2+) channels may therefore play important signaling roles in the nervous system.     

双孔通道家族与钙信号调节

钙离子（Ca2+）在细胞各项生理活动中发挥着重要作用. 胞浆游离Ca2+浓度（［Ca2+］i）的变化与细胞功能、信号转导及细胞损伤和凋亡都有密切联系．研究证实,烟酸腺嘌呤二核苷磷酸(nicotinic acid adenine dinucleotide phosphate, NAADP)是一种有效的胞内Ca2+释放活化剂,但具体作用机制尚不明确．有研究表明,双孔通道家族(two pore channels,TPCs)可能与此有关．本文对TPCs的结构与功能及其生理病理等相关性的研究进展作一综述,从而为进一步研究TPCs生理功能提供依据．     

Membrane topology of NAADP-sensitive two-pore channels and their regulation by N-linked glycosylation

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.     

Photoaffinity labeling of high affinity nicotinic acid adenine dinucleotide phosphate (NAADP)-binding proteins in sea urchin egg

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a messenger that regulates calcium release from intracellular acidic stores. Recent studies have identified two-pore channels (TPCs) as endolysosomal channels that are regulated by NAADP; however, the nature of the NAADP receptor binding site is unknown. To further study NAADP binding sites, we have synthesized and characterized [(32)P-5-azido]nicotinic acid adenine dinucleotide phosphate ([(32)P-5N(3)]NAADP) as a photoaffinity probe. Photolysis of sea urchin egg homogenates preincubated with [(32)P-5N(3)]NAADP resulted in specific labeling of 45-, 40-, and 30-kDa proteins, which was prevented by inclusion of nanomolar concentrations of unlabeled NAADP or 5N(3)-NAADP, but not by micromolar concentrations of structurally related nucleotides such as NAD, nicotinic acid adenine dinucleotide, nicotinamide mononucleotide, nicotinic acid, or nicotinamide. [(32)P-5N(3)]NAADP binding was saturable and displayed high affinity (K(d) ～10 nM) in both binding and photolabeling experiments. [(32)P-5N(3)]NAADP photolabeling was irreversible in a high K(+) buffer, a hallmark feature of NAADP binding in the egg system. The proteins photolabeled by [(32)P-5N(3)]NAADP have molecular masses smaller than the sea urchin TPCs, and antibodies to TPCs do not detect any immunoreactivity that comigrates with either the 45-kDa or the 40-kDa photolabeled proteins. Interestingly, antibodies to TPC1 and TPC3 were able to immunoprecipitate a small fraction of the 45- and 40-kDa photolabeled proteins, suggesting that these proteins associate with TPCs. These data suggest that high affinity NAADP binding sites are distinct from TPCs.     

NAADP-dependent Ca2+ signaling regulates Middle East respiratory syndrome-coronavirus pseudovirus translocation through the endolysosomal system

Middle East Respiratory Syndrome coronavirus (MERS-CoV) infections are associated with a significant mortality rate, and existing drugs show poor efficacy. Identifying novel targets/pathways required for MERS infectivity is therefore important for developing novel therapeutics. As an enveloped virus, translocation through the endolysosomal system provides one pathway for cellular entry of MERS-CoV. In this context, Ca²⁺-permeable channels within the endolysosomal system regulate both the luminal environment and trafficking events, meriting investigation of their role in regulating processing and trafficking of MERS-CoV. Knockdown of endogenous two-pore channels (TPCs), targets for the Ca²⁺ mobilizing second messenger NAADP, impaired infectivity in a MERS-CoV spike pseudovirus particle translocation assay. This effect was selective as knockdown of the lysosomal cation channel mucolipin-1 (TRPML1) was without effect. Pharmacological inhibition of NAADP-evoked Ca²⁺ release using several bisbenzylisoquinoline alkaloids also blocked MERS pseudovirus translocation. Knockdown of TPC1 (biased endosomally) or TPC2 (biased lysosomally) decreased the activity of furin, a protease which facilitates MERS fusion with cellular membranes. Pharmacological or genetic inhibition of TPC1 activity also inhibited endosomal motility impairing pseudovirus progression through the endolysosomal system. Overall, these data support a selective, spatially autonomous role for TPCs within acidic organelles to support MERS-CoV translocation.     

Physiological roles of NAADP-mediated Ca<Superscript>2+</Superscript> signaling

Nicotinic acid dinucleotide phosphate (NAADP) is unique amongst Ca²⁺ mobilizing messengers in that its principal function is to mobilize Ca²⁺ from acidic organelles. Early studies indicated that it was likely that NAADP activates a novel Ca²⁺ release channel distinct from the well characterized Ca²⁺ release channels on the (sarco)-endoplasmic reticulum (ER), inositol trisphosphate and ryanodine receptors. In this review, we discuss the emergence of a novel family of endolysosomal channels, the two-pore channels (TPCs), as likely targets for NAADP, and how molecular and pharmacological manipulation of these channels is enhancing our understanding of the physiological roles of NAADP as an intracellular Ca²⁺ mobilizing messenger.     

TPCs: Endolysosomal channels for Ca mobilization from acidic organelles triggered by NAADP

Two-pore channels (TPCs or TPCNs) are novel members of the large superfamily of voltage-gated cation channels with slightly higher sequence homology to the pore-forming subunits of voltage-gated Ca²⁺ and Na⁺ channels than most other members. Recent studies demonstrate that TPCs locate to endosomes and lysosomes and form Ca²⁺ release channels that respond to activation by the Ca²⁺ mobilizing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). With multiple endolysosomal targeted NAADP receptors now identified, important new insights into the regulation of endolysosomal function in health and disease will therefore be unveiled.     

Involvement of PI3K in HCV-related lymphoproliferative disorders

We have investigated the role of NAADP-mediated Ca(2+) mobilization in endothelin (ET) signaling via endothelin receptor subtype A (ETA) and endothelin receptor subtype B (ETB) in rat peritubular smooth muscle cells. Microinjection and extracellular application of NAADP were both able to elicit Ca(2+) release which was blocked by inhibitory concentrations of NAADP, by impairing Ca(2+) uptake in acidic stores with bafilomycin, and by thapsigargin. Ca(2+) release in response to selective ETB stimulation was abolished by inhibition of NAADP signaling through the same strategies, while these treatments only partially impaired ETA-dependent Ca(2+) signaling, showing that transduction of the ETB signal is dependent on NAADP. In addition, we show that lipid rafts/caveolae contain ETA, ETB, and NAADP/cADPR generating enzyme CD38 and that stimulation of ETB receptors results in increased CD38 activity; interestingly, ETB- (but not ETA-) mediated Ca(2+) responses were antagonized by disruption of lipid rafts/caveolae with methyl-beta-cyclodextrin. These data demonstrate a primary role of NAADP in ETB-mediated Ca(2+) signaling and strongly suggest a novel role of lipid rafts/caveolae in triggering ET-induced NAADP signaling.