NAADP Receptors

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NAADP Receptors

Antony Galione

Department of Pharmacology, University of Oxford, Mansﬁeld Road, Oxford OX1 3QT, United Kingdom Correspondence: [email protected]

Of the established Ca2þ mobilizing messengers, NAADP is arguably the most tantalizing. It is the most potent, often efficacious at low nanomolar concentrations. Recent studies have identified a new class of calcium release channel, the two-pore channels (TPCs), as the likely targets for NAADP. These channels are endolysosomal in localization where they mediate local Ca2þ release, and have highlighted a new role of acidic organelles as targets for messenger-evoked Ca2þ mobilization. Three distinct roles of TPCs have been identified. The first is to effect local Ca2þ release that may play a role in endolysosomal function including vesicular fusion and trafficking. The second is to trigger global calcium release by recruiting Ca2þ-induced Ca2þ release (CICR) channels at lysosomal-ER junctions. The third is to regulate plasma membrane excitability by the targeting of Ca2þ release from ap- propriately positioned subplasma membrane stores to regulate plasma membrane Ca2þ- activated channels. In this review, I discuss the role of NAADP-mediated Ca2þ release from endolysosomal stores as a widespread trigger for intracellular calcium signaling mechanisms, and how studies of TPCs are beginning to enhance our understanding of the central role of lysosomes in Ca2þ signaling.

alcium is the most evolutionarily ubiqui- such channels may lead to cell death, for exam- Ctous of intracellular signals and controls ple, through the activation of apoptotic signal- cellular mechanisms as diverse as cellular mo- ing cascades (Berridge et al. 1998). Many cell tility, membrane fusion, ion channel function, surface receptors are linked to signaling path- enzyme activity, and gene expression (Berridge ways that lead to the mobilization of calcium et al. 2003). Free cytoplasmic calcium levels from intracellular storage organelles through are kept under tight control by pumps, ex- the activation of speciﬁc Ca2þ release channels changers, and buffering mechanisms includ- (Clapham 1995). Three major intracellular ing storage by organelles (Pozzan et al. 1994). messengers have been established to link cell stim- Ca2þ signals may be elicited when these mech- ulation with organellar Ca2þ release: inositol tri- anisms are transiently overwhelmed by the sphosphate (IP3), cyclic adenosine diphosphate opening of calcium permeable channels at the ribose (cADPR), and nicotinic acid adenine plasma membrane or in membranes of cal- nucleotide diphosphate (NAADP) (Bootman cium-storing organelles. Chronic activation of et al. 2002).

Editors: Martin D. Bootman, Michael J. Berridge, James W. Putney, and H. Llewelyn Roderick Additional Perspectives on Calcium Signaling available at www.cshperspectives.org Copyright # 2011 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a004036 Cite this article as Cold Spring Harb Perspect Biol 2011;3:a004036

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A. Galione

DISCOVERY OF NAADP AS A NADP,later shown to be NAADP (Lee and Aar- Ca2þ-MOBILIZING MOLECULE hus 1995), were found to release Ca2þ from different subcellular nonmitochondrial fractions NAADP was discovered as a contaminant of þ from egg homogenate (Fig. 1). A key feature commercial batches of b-NADP by Lee and of each mechanism is their display of homolo- colleagues while they were investigating the ef- gous desensitization underscoring the inde- fects of various pyridine nucleotides on calci- pendence of each of the three mechanisms. um release from sea urchin egg homogenates (Clapper et al. 1987). The rationale for this was that at fertilization in sea urchin eggs, dramatic NAADP AS A Ca2þ MOBILIZING MESSENGER changes in pyridine nucleotide levels occur at a similar time to the generation of the calcium NAADP is the most potent of Ca2þ mobilizing wave. Egg homogenates can be simply prepared messengers described, typically efficacious at from eggs and are remarkably stable, even after pM or low nM concentrations. A growing num- freezing, and sequester, and robustly release cal- ber of cellular stimuli and activation of cell sur- cium when challenged with messengers and face receptors have been found to be coupled to drugs (Morgan and Galione 2008). Three dis- increases in NAADP levels, confirming its role tinct calcium release mechanisms were shown. as an intracellular messenger (Churchill et al. These were the early days of IP3, and IP3 was 2003; Masgrau et al. 2003; Rutter 2003; Yama- found to release calcium from microsomal saki et al. 2005; Galione 2006; Gasser et al. stores. In addition, two metabolites of pyridine 2006; Kim et al. 2008). Mediation of calcium nucleotides, an enzyme-activated metabolite signaling by NAADP has been implicated by related to NADþ, subsequently identified as two approaches: inhibition of agonist-evoked cADPR (Lee et al. 1989), and alkaline-treated calcium signals by prior self-inactivation of the

NH HO OH 2

N N O N PP N N+ O

HO HO P

Lysosome Cytosol

TPC2 Ca2+ Ca2+

NAADP

Figure 1. Structure and function of NAADP. NAADP differs from b -NADP in that the base nicotinic acid is substituted for nicotinamide (upper panel). NAADP,unlike NADP,is a potent Ca2þ mobilizing agent and inter- acts with two-pore channels in the membranes of lysosomes (lower panel).

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NAADP Receptors

NAADP receptor or NAADP receptor phar- pole where ER accumulated, whereas NAADP macological blocker (Naylor et al. 2009) and released Ca2þ from the opposite end of the struc- measurements of cellular NAADP levels. Mea- ture. These experiments are consistent with the surements of NAADP have been performed primary Ca2þ store targeted by NAADPas being using either a radioreceptor assay, based on the distinct from the ER. high affinity NAADP binding protein of sea In a series of important experiments using urchin eggs (Lewis et al. 2007), or by using a pharmacological analyses and subcellular frac- cycling assay of coupled enzyme reactions tionation, lysosomal-related organelles were resulting in fluorescent resorufin production implicated as the primary target organelle for (Graeff and Lee 2002). Although some recep- NAADP-evoked Ca2þ release in sea urchin tors appear to couple selectively to NAADP pro- eggs (Churchill et al. 2002). Acidic stores, such duction, increasingly it is becoming apparent as lysosomes, have been shown to sequester that several receptors couple to multiple Ca2þ Ca2þ by mechanisms dependent on their low mobilizing messengers (Aley et al. 2010), and luminal pH (Patel and Docampo 2010). Inhib- this may be the norm. ition of the vacuolar Hþ-ATPase by bafilomycin decreases proton uptake into acidic stores; if

2þ their membranes are sufficiently leaky to pro- Ca STORES TARGETED BY NAADP tons, this leads to the alkalinization of their lu- 2þ Accumulating evidence suggests that the pri- men. Uptake of Ca into these stores appears mary Ca2þ stores targeted by NAADP are sepa- to be dependent on the maintenance of the pro- rate from the endoplasmic reticulum and are ton gradient because bafilomycin and protono- 2þ members of a group known as acidic organelles. phores inhibit Ca storage by these organelles, The initial evidence for this came from the although the detailed mechanisms are not well study of sea urchin eggs and was subsequently understood. A dense membrane fraction from extended to mammalian cells. sea urchin egg homogenates was isolated from a percoll gradient and consisted of “reserve granules.” This fraction was enriched with lyso- Sea Urchin Eggs somal markers and supported ATP-dependent The initial report of NAADP-evoked Ca2þ Ca2þ sequestration, which was inhibited by release using alkaline-activated NADP showed preincubation with bafilomycin or the proto- that the subcellular fraction reactive to NAADP nophore, nigericin, but not thapsigargin. This in egg homogenates was largely separate from fraction was found to contain [32P]NAADP the microsomal/ER fraction sensitive to IP3 binding sites, and displayed NAADP but not 2þ and cADPR (Clapper et al. 1987). Abrogation IP3/cADPR-evoked Ca release. Reserve gran- of Ca2þ storage by the ER SERCA inhibi- ules from sea urchin eggs are lysosome-related tor, thapsigargin, while inhibiting Ca2þ release organelles. In intact sea urchin eggs, treatment by either IP3 or cADPR, only partially re- with the lysosomotropic agent, glycyl-phenyl- duced Ca2þ release evoked by NAADP in both alanine 2-naphthylamide (GPN), caused the sea urchin egg homogenates (Genazzani and reversible lysis of lysotracker-stained vesicles, re- Galione 1996) and intact eggs (Churchill and sulting in a series of small-amplitude cytoplas- Galione 2001a). Visualization of two separate mic Ca2þ signals, consistent with their role as Ca2þ stores was observed in elegant sea urchin Ca2þ stores. Importantly, GPN treatment in ei- egg stratification studies (Lee and Aarhus 2000). ther intact eggs or egg homogenates selectively Stratification by centrifugation of intact eggs abolished NAADP-evoked Ca2þ release with lit- 2þ results in eggs forming elongated structures tle effect on Ca release byeither IP3 orcADPR. with different organelles separating to different From these data it was proposed that in sea “poles.” Uniform photolysis of caged derivatives urchin the primary target of NAADP are acidic 2þ of Ca mobilizing messengers resulted in IP3 stores rather than the endoplasmic reticulum. and cADPR releasing Ca2þ from the nuclear Consistent with this, experiments in sea urchin

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A. Galione

egg homogenates employing luminal pH in- sea urchin eggs and homogenates revealed sev- dicators such as acridine orange or lysosensor eral profound and unusual features. A sur- also have shown that NAADP uniquely among prising finding was that pM concentrations of Ca2þ mobilizing messengers also causes the NAADP, although subthreshold for triggering alkalinization of store lumena, representing an- Ca2þ release in egg homogenates, were able to other possible signaling mechanism for this inactivate completely the NAADP Ca2þ release molecule (Morgan and Galione 2007b). mechanism to a subsequent challenge by nM concentration of NAADP that would normally Mammalian Cells evoke a maximal Ca2þ release (Aarhus et al. Following these studies in sea urchin eggs, it 1996; Genazzani et al. 1996). The extent of inac- was shown that NAADP also targeted acidic tivation was dependent on both the concentra- stores in a wide range of mammalian cells and tion and duration of incubation (Genazzani in response to a variety of cellular stimuli et al. 1996; Genazzani et al. 1997b). Mecha- (Mitchell et al. 2003; Kinnear et al. 2004; Yama- nisms of inactivation of the NAADP receptor are not understood, but may be related to the saki et al. 2004; Galione 2006; Gerasimenko 32 et al. 2006; Menteyne et al. 2006; Zhang et al. apparent irreversible binding of [ P]NAADP. 2006; Macgregor et al. 2007; Gambara et al. The radioligand appears to become occluded 2008; Jardin et al. 2008; Kim et al. 2008; on binding in a time-dependent manner (Aar- Lloyd-Evans et al. 2008; Brailoiu et al. 2009b; hus et al. 1996). Studies with the selective Pandey et al. 2009; Thai et al. 2009; Dickinson NAADP receptor antagonist, Ned-19, (Naylor et al. 2010; Zhang et al. 2010). et al. 2009) and its analogs (Rosen et al. 2009) have led to the proposal that there are two distinct binding sites for NAADP. The first is DESENSITIZATION OF NAADP-EVOKED high affinity, whose occupancy leads to slow Ca2þ RELEASE inactivation of the receptor, and a second lower The Ca2þ release mechanism activated by affinity site that leads to rapid channel opening. NAADP shows unusual and profound inactiva- Ned-20 blocks inactivation, but not activation tion properties. One major area of confusion in of Ca2þ release by NAADP (Rosen et al. 2009). this field is that the inactivation properties of Mammalian cells: There are key differences Ca2þ release varies markedly between sea urchin between desensitization of the NAADP receptor egg and mammalian systems, which we have between sea urchin eggs, in which subthreshold termed type I and type II, respectively (Morgan concentrations of NAADP can fully inactivate and Galione 2008) (Fig. 2). the NAADP-sensitive Ca2þ release mechanism; Sea urchin eggs: The initial report demon- whereas in a mammalian cell, high concentra- strating the efficacy of NAADP as a Ca2þ mobi- tions of NAADPare needed for full inactivation, lizing molecule showed that NAADP released which can occur in the apparent absence of 2þ Ca by a mechanism independent of IP3 or receptor activation. The first report of NAADP ryanodine receptors (RyRs), based on each of action as a Ca2þ-mobilizing agent in a mam- these mechanisms showing homologous desen- malian cell was in the pancreatic acinar cell sitization (Lee and Aarhus 1995). After NAADP (Cancela et al. 1999), which was also the system 2þ 2þ stimulated Ca release in egg homogenates, in which IP3 was first shown to mobilize Ca they became refractory to subsequent challenge from nonmitochondrial stores (Streb et al. with NAADP, but still responded to either IP3 1983). Using whole cell patch and measuring or cADPR. This was the first piece of evidence Ca2þ-activated currents, we found that a pi- that NAADP activated a novel Ca2þ release pet concentration of 10 mM NAADP failed to channel, distinct from the principal Ca2þ re- elicit any responses. However, we noticed that lease channels on the endoplasmic reticulum. after intracellular application of this concen- Further analysis of the phenomenon of self- tration of NAADP, cholecystokinin (CCK), inactivation of NAADP-evoked Ca2þ release in which usually increases cytosolic Ca2þ at pM

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NAADP Receptors

A Activation Inactivation Test Activation Increasing d a Conc. +

Ca c b release

Bme 2+

b c Ca Inactivation (test) a d IP3/cADPR Sub-max Max log [IP3 or cADPR] Concs B Activation Inactivation Test

Increasing a Type 1 Conc.

b release 2+ Inactivation Activation c Ca (test) a-d d NAADP Sub-threshold Max log [NAADP] Concs C Activation Inactivation Test Activation a Increasing Type 2 Inactivation Conc. b b release (test) 2+

c c Ca

a,d d NAADP High Optimal log [NAADP] Concs

Figure 2. Differences between desensitization of mammalian and sea urchin NAADP receptors. Left-hand traces depict stylized Ca2þ dye fluorescence traces in response to messengers. Right-hand panels represent concentration-response graphs for activation or inactivation of intracellular Ca2þ release channels. (A) IP3 and cADPR-mediated Ca2þ release. Left-hand traces depict stylized Ca2þ dye fluorescence traces in response to various conditions. Right-hand panels represent concentration-response graphs for activation or inactivation. IP3 and cADPR demonstrate simple, monophasic concentration-response curves for activation (blue). Inactivation by ever-increasing submaximal concentrations (grey period) is revealed by the reciprocal diminu- tion of a maximal “test” challenge (orange). (B) Desensitization of sea urchin NAADP receptors (Type I desensitization). The blue left-hand traces show stylized Ca2þ dye fluorescence traces in response to increasing concentrations of NAADP, which increases Ca2þ release, represented by a classical sigmoid log concentration-response curve (blue line, right-hand panel). However, preincubation with subthreshold concentrations of NAADP that do not evoke Ca2þ release desensitizes Ca2þ release in a time and concentration manner by subsequent challenge by a normally maximal NAADP (test) concentration (central panel, and orange curve, right panel). (C) Desensitization of mammalian NAADP receptors (Type 2 desensitization). Increasing concentrations of NAADP enhances Ca2þ release to a maximum (left and center panels). Thereafter, increasing concentrations of NAADP evoke progressively smaller Ca2þ release to a point when no Ca2þ release is evoked at high NAADP concentrations. This “bell-shaped” or hormetic log concentration-response curve is shown in the right-hand panel (blue curve). Modified from Morgan and Galione (2008).

concentrations by mobilizing Ca2þ from intra- transduction mechanism. We therefore tried a cellular stores, now failed to evoke any response. range of NAADP concentrations and found that We speculated that we may have inactivated the concentrations of NAADP as low as 50 nM in NAADP-evoked Ca2þ release mechanism that the pipet, elicited robust oscillatory responses, could be a key component of the CCK signal similar to those evoked by CCK in untreated

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A. Galione

cells. The concentration-response relationship A number of channel blockers were found for NAADP was found to be “bell-shaped,” with to inhibit NAADP-evoked Ca2þ release selective- maximal responses occurring at around 100 nM ly in sea urchin egg homogenates with little 2þ NAADP, whereas with concentrations in excess effect on either IP3 or cADPR-mediated Ca of 1 mM no effects were seen. Using caged release (Genazzani et al. 1997a). These included NAADP,we showed that photolysis of this com- voltage-gated Ca2þ channel (VGCC) blockers pound also evokes a series of spikes in Ca2þ- such as diltiazem, nifedipine, and D600 (al- activated currents, which were suppressed in the though greater concentrations were required to presence of supramicromolar concentrations blockNAADP-evoked Ca2þ release than VGCCs), of free NAADP in the patch pipet. Bell-shaped and purinoceptor antagonists such as aspyri- concentration-response curves seem to be a doxal-phosphate-6-azophenyl-2’,4’-disulfonate major hallmark of mammalian NAADP-induced (PPADS) also display a degree of NAADP an- Ca2þ release. A subsequent study in a Jurkat tagonism (Billington and Genazzani 2007). T-cell line showed maximal Ca2þ release upon Because the NAADP receptor effectively dis- microinjection of around 100 nM NAADP. criminates between NAADP and NADP, which However, concentrations of .1 mM failed to differs only by the substitution of a nicotinic elicit any response per se whilst inhibiting acid moiety instead of nicotinamide, nicotinic T-cell receptor activation (Berg et al. 2000). A acid analogs were developed that antagonize number of further studies in different cell types NAADP-induced Ca2þ release. These include used this phenomenon to implicate NAADP in CMA008 (Dowden et al. 2006) and BZ194 the Ca2þ signal transduction pathways activated (Dammermann et al. 2009), which also have by various stimuli in the absence of selective the advantage of being membrane permeant. NAADP antagonists at that time. These include Recently, a series of novel compounds have been glucose-evoked Ca2þ spiking in MIN6 cells identified by in silico screening strategies based (Masgrau et al. 2003), ET1-evoked Ca2þ release on the three-dimensional shape and electro- in pulmonary vascular smooth myocytes (Kin- static properties of NAADP that are the most near et al. 2004), and b1 adrenoreceptor en- potent of NAADP antagonists developed so far hancement of Ca2þ signaling and contractility (Naylor et al. 2009; Rosen et al. 2009). Ned-19, in ventricular cardiac myocytes (Macgregor the founding member of these analogs, is be- et al. 2007). coming the most widely used antagonist on account of its potency, membrane permeability, and selectivity (Naylor et al. 2009; Rosen et al. 2009; Thai et al. 2009; Aley et al. 2010). PHARMACOLOGICAL PROPERTIES Interestingly, Ned-19 analogs have been used OF NAADP RECEPTORS to dissect the activation and inactivation effects The pharmacology of NAADP-evoked Ca2þ of NAADP at the sea urchin egg NAADP recep- release, initially investigated in sea urchin egg tor (Rosen et al. 2009). Ned-20, which differs systems, showed major differences with the only from Ned-19 by the para rather than ortho known Ca2þ release mechanisms in the ER. In position of a fluorine, blocks the inactivation of egg homogenates, NAADP-evoked Ca2þ release NAADP-sensitive Ca2þ release mechanism by 2þ was unaffected by the competitive IP3R in- subthreshold NAADP concentrations for Ca hibitor, heparin, or by ryanodine or eight- release, without affecting NAADP-evoked Ca2þ substituted cADPR analogs that antagonize release by higher NAADP concentrations and RyR-mediated Ca2þ release. An initial report inhibits high affinity [32P]NAADP binding to that thio-NADP was a selective antagonist of egg membranes (Rosen et al. 2009). These find- NAADP (Chini et al. 1995) was subsequently ings are consistent with multiple binding sites explained by inactivation of the NAADP- for the sea urchin egg NAADP receptor, with sensitive Ca2þ release mechanism by traces of high affinity sites leading to inactivation and contaminating NAADP (Dickey et al. 1998). lower affinity sites leading to activation.

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NAADP Receptors

TWO-PORE CHANNELS represent an ancestral form that has been du- plicated later in evolution to give rise to the A family of novel intracellular channels termed four domain channels (Fig. 3). two-pore channels (TPCs) have emerged as the leading candidates for NAADP-gated Ca2þ release channels. The founding member of this family, TPC1, was cloned in 2000 from a rat kid- IDENTIFICATION OF TWO-PORE CHANNELS AS NAADP RECEPTORS ney cDNA library in a search for novel members of voltage-gated cation channels (Ishibashi et al. Two clues as to the candidature of TPCs as 2000). The putative channel had only a 20% NAADP receptors emerged in the last few years. homology with the transmembrane domains of Michael Zhu, searching for novel TRP family the a subunit of voltage-gated Naþ and Ca2þ membranes in around 2000, had cloned a second channels, but the highest homology with a member of the TPC family, termed TPC2, and deposited sequence of a putative Ca2þ channel found that when heterologously expressed in from the plant Arabidopsis thaliana. Subsequent HEK293 cells, it localized with the lysosomal analysis of the plant clone, AtTPC1, implicated marker, LAMP1. The second was the further a role for this protein in Ca2þ transport and sig- analysis of AtTPC1 function by Sanders and naling when expressed in yeast and Arabidopsis colleagues, showing that AtTPC1 localized to (Furuichi et al. 2001), and a role in germination plant vacuoles, the major plant acidic organelle and stomatal physiology as a component of and the functional equivalent of lysosomes in the slow vacuolar channel (Peiter et al. 2005). plants (Peiter et al. 2005). The localization of The putative channel, rather than having four TPCs to acidic stores, and the partial phar- repeats of six transmembrane segments, as for macological overlap of NAADP receptors with voltage-gated Naþ and Ca2þ channels, only voltage-gated Ca2þ channels and TRP proteins, has two. Thus in effect, the protein is the equiv- which show homologies with TPCs, made these alent of half a Naþ or Ca2þ channel, and may proteins credible candidates as the elusive

Cav2.1 Four-pore channels Cav2.2 Cav2.3 Cav1.2 (L) Cav3.1 (T) Two-pore channels TPC1 TPC2

TRPP2 One-pore channels TRPM1 TRPV1b TRPC1 TRPA1 TRPML1 CatSper3 CatSper4 CatSper1 CatSper2

Figure 3. Phylogenetic tree for human two-pore channels and their relationship with voltage-gated Ca2þ channels and TRP members. It is likely that voltage-gated Ca2þ channels have arisen from two rounds of tandem duplication in evolution. Thus, TPCs having 12 transmembrane domains (12TM) may be considered ancient intermediate proteins between TRP channels (6TM), such as CatSpers in sperm or mucolipins or polycystins, and voltage-gated Ca2þ channels (24TM).

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A. Galione

NAADP receptor. Over four years or so from release (CICR) from nearby ER, in this case by 2005, we worked extensively with Zhu and col- activating IP3Rs. The concentration-response laborators, to test exhaustively the hypothesis relationship between NAADP and Ca2þ release that TPCs represented a family of NAADP- was of the characteristic bell-shape for NAADP gated intracellular channels from several key in mammalian cells, with maximal Ca2þ release standpoints. First, we examined the subcellular occurring at between 10 nM and 1 mM, whereas localization of the human TPC1 and TPC2 iso- 1 mM was without effect. Importantly, shRNA forms in HEK293 cells. In addition, because the against TPC2 completely abolished the res- genomes of many species, but not human or ponse to NAADP. We also created Tpc2-/- mice, rodent, also express a third isoform, TPC3 and found that NAADP-evoked activation of (Cai and Patel 2010; Zhu et al. 2010), we also oscillatory Ca2þ-dependent cation currents in expressed the chicken TPC3 to examine its sub- pancreatic b cells seen in wild-type cells, were cellular distribution in HEK293 cells (Calcraft abolished in cells from the knockout mice. et al. 2009). All three TPCs localize to the endo- In contrast to TPC2, we found that HEK lysosomal system with no apparent expression cells stably expressed with HsTPC1 evoked in Golgi, mitochondria, or ER. Only TPC2 con- only a localized Ca2þ release in response to sistently colocalized with the lysosomal marker, NAADP, which failed to globalize, as was the LAMP2, but not early or late endosomes. In case for TPC2. One possibility is that the endo- contrast, TPC1 and TPC3 predominantly were somal localization of TPC1 means that there is expressed in endosomal and other unidentified little close apposition with ER so that coupling compartments, but with only sparse colocaliza- with CICR channels is weaker. Two subsequent tion with lysosomal markers. In HEK293 cells, publications broadly confirmed our findings TPCs are endogenously expressed at low levels, (Brailoiu et al. 2009a; Zong et al. 2009). and endogenous TPC2 was also immunolocal- ized to lysosomes. Overexpression of the human PROPERTIES OF ENDOGENOUS TPCs HsTPC2 was associated with increased specific FROM SEA URCHIN EGGS [32P]NAADP binding to HEK293 cell membranes and immunoprecipitated TPC2 pro- The properties of heterologously expressed teins. Both high and low affinity binding sites mammalian TPCs made them strong candi- were manifest in membranes from TPC2-over- dates as NAADP receptors. However, most of 2þ expressing cells, with Kd of 5 nM and 7 mM, the studies of NAADP-mediated Ca release remarkably similar to endogenous binding in and [32P]NAADP binding sites have been per- membranes from mouse liver, a tissue which formed in sea urchin egg preparations, where has a particularly high expression of TPCs. Pho- the Ca2þ mobilizing effects of NAADP were tolysis of caged NAADP in patched wild-type first discovered. It was important to ascertain HEK293 cells elicited a small Ca2þ response, whether sea urchin eggs express TPCs and whereas in cells stably overexpressing TPC2, a whether they functioned as NAADP receptors. large biphasic Ca2þ response was evoked on Screening of the genome of the sea urchin NAADP uncaging or dialysis: an initial pace- Strongylocentrotus purpuratus revealed three maker-like ramp of Ca2þ was followed by a larger TPC isoforms that were cloned from ovaries and faster transient Ca2þ release. Bafilomycin that displayed around 30% sequence homology treatment abolished both phases of the Ca2þ between the isoforms (Brailoiu et al. 2010; Ruas response, whereas the IP3R antagonist heparin et al. 2010). Importantly, immunoprecipitation blocked the second phase alone. This finding is of TPCs from solubilized egg membranes with consistent with the “trigger” hypothesis for a polyclonal antibodies raised against each of mode of NAADP action, whereby NAADP the three TPC isoforms of TPCs produced evokes a localized Ca2þ signal by mobilizing bafi- immunocomplexes that specifically bound 32 lomycin-sensitive acidic stores, which is then [ P]NAADP with Kds of around 1 nM. Binding globalized by recruiting Ca2þ-induced Ca2þ of [32P]NAADP to the immunocomplexes

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NAADP Receptors

mirrored all the key features of binding to in- subcellular localizations of each of the TPCs, tact egg membranes, including Kþ-dependent at least when heterologously expressed, it is irreversibility and a similar binding selectivity unclear whether heterodimerization can ex- for NAADP over NADP. These data provided plain TPC3 suppression of NAADP-evoked compelling evidence that TPCs form com- Ca2þ release. plexes that can explain all the properties of [32P]NAADP binding sites previously charac- SINGLE-CHANNEL PROPERTIES terized from sea urchin egg preparations. As OF HUMAN TPCs with their mammalian homologs, heterologous expression of the sea urchin TPC1 and TPC2 Although TPCs are emerging as promising can- isoforms in HEK293 cells enhanced NAADP- didates as NAADP-gated Ca2þ release channels evoked Ca2þ release from acidic Ca2þ stores, in the endolysosomal system, it is important to which was amplified by recruitment of IP3Rs, characterize their biophysical channel proper- although coupling between TPC1 and IP3Rs ties to show that they do indeed function in appeared looser. In contrast, TPC3 actually sup- this way. However, their localization in organ- pressed the small NAADP-evoked response elles presents several problems because they observed in control cells and also abolished are not readily amenable for electrophysiolog- the enhancement in cells stably transfected ical analysis as for channels resident at the with TPC2 (Fig. 4). This effect of TPC3 is plasma membrane, and there is no evidence at puzzling for several reasons. The effect of TPC3 present that they cycle to the plasma membrane cannot be accounted by a general dysregulation as for other Ca2þ release channels (Taylor et al. of acidic Ca2þ stores since measurement of both 2009). The traditional way of studying organel- Ca2þ storage and luminal pH do not appear to lar channels is their reconstitution into artificial be altered in cells overexpressing TPC3 express- bilayers for single channel analysis, as exempli- ing cells. Another possibility is that TPC3 has a fied for IP3R (Ehrlich and Watras 1988) and dominant negative effect, perhaps by forming RyR (Lai et al. 1988) single-channel studies; heterodimers, because it is likely, given the although for ER channels, nuclear envelope proposed structure of TPCs, that functional patching has gained increasing popularity channels would form dimers. Indeed, homodi- (Mak and Foskett 1997). In a preliminary re- merization of human TPC2 has been reported port, immunopurified human TPC2 was recon- (Zong et al. 2009), but given the differing stituted into lipid bilayers and shown to form

4 +NAADP +NAADP+bafilomycin 3

0 +NAADP+heparin F/F 2

1 mCherry mCherry.SpTPC1 mCherry.SpTPC2 mCherry.SpTPC3 1 min

Figure 4. NAADP-mediated Ca2þ release in HEK293 cells expressing each of the three sea urchin TPC isoforms. Representative Ca2þ traces of cells dialyzed with NAADP (100 nM) and fura-2 via patch pipette in whole-cell configuration, in absence or presence of bafilomycin A1 (1 mM) or the IP3R antagonist, heparin (200 mg/ ml). Arrows indicate break-in. In wild-type cells, only a small endogenous response to NAADP was seen. In SpTPC1 and SpTPC2 cells, NAADP-evoked biphasic responses. The first component was from acidic stores (bafilomycin-sensitive), whereas the second phase, which requires the first to trigger it, is caused by recruitment of IP3Rs (heparin-sensitive). TPC3 expression suppresses the endogenous response. Models for NAADP- triggered Ca2þ responses, based on interaction between different organelles (circle, lysosome and network, ER) are also shown above each series of traces. Modified from Ruas et al. (2010).

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A. Galione

NAADP-gated cation conductances (Pitt et al. in both preparations. The pharmacology was 2010). Channels were generally silent until consistent with previous studies of NAADP- application of NAADP to the cis or cytoplasmic evoked Ca2þ release, with block by VGCC antag- face of the bilayer, and the channels showed a onists, PPADS, and also amiloride. Interestingly, selectivity for cations with conductances of Po was increased at acidic pH. In contrast to around 300 pS and 15 pS for Kþ and Ca2þ the situation in most mammalian cells exam- ions as the conducting species. Interestingly, ined so far, pretreatment with concentrations of NAADP sensitivity may be regulated by store NAADP as low as 0.5 nM blocked subsequent filling with Ca2þ, because NAADP sensitivity channel openings by higher NAADP concentra- was markedly dependent on trans or luminal tions, as seen for sea urchin egg receptors and 2þ Ca , with the EC50 for NAADP-evoked en- in liver (Mandi et al. 2006). The identity of hancement of open probability decreasing from these channels were ascribed to mucolipin-1 500 nM to 5 nM as luminal Ca2þ increased to (TRPML-1), a lysosomal TRP channel linked 200 mM. This is in the range of reported lu- to the lysosomal storage disease, mucolipidosis minal free Ca2þ levels in lysosomes (Christen- IV, on the basis of a blocking effect of an anti- sen et al. 2002; Lloyd-Evans et al. 2008). Thus, TPRML1 antibody and reduction of channel fluctuations in luminal Ca2þ because of cycles activity from cells treated with an siRNA of release and uptake of Ca2þ could be impor- TPRML1 construct. However, the identity of tant determinants of the effects of NAADP TRPML1 as an NAADP receptor candidate on Ca2þ release, and offers one explanation remains controversial (Pryor et al. 2006). In for how constant NAADP levels may elicit trains addition, a recent report suggests that NAADP of Ca2þ spikes, as widely observed in various cell may increase levels of a short variant of a types (Cancela et al. 1999). Another variable is TRPML2 transcript in lymphoid cells (Samie luminal pH of acidic stores, since NAADP has et al. 2009), underscoring the likely complex also been found to alkalinize acidic stores in interactions between lysosomal channels. sea urchin eggs and homogenates (Morgan and Galione 2007a; Morgan and Galione INTERACTIONS OF NAADP AND OTHER 2007b), and it is possible that luminal pH has Ca2þ SIGNALING PATHWAYS significant effects on TPC2 channel properties. Importantly, the NAADP antagonist was also NAADP-evoked Ca2þ release from lysosomes found to block single channel TPC2 currents appears to be small and highly localized. Given (Pitt et al. 2010). However, it should be stressed the dynamic properties of these organelles, they here that although the immunopurified TPC are ideally suited to be targeted to the vicinity complexes both form NAADP-gated Ca2þ chan- of Ca2þ-regulated effectors. Three modes of nels (Pitt et al. 2010) and bind [32P]NAADP NAADP-mediated Ca2þ signaling mechanisms (Calcraft et al. 2009; Ruas et al. 2010), the possi- have been highlighted (Fig. 5). bility remains that NAADP could interact with an accessory protein associated with TPCs NAADP and Lysosomal-ER Interactions instead of a direct interaction with TPC proteins themselves (Galione et al. 2009). Organelle interactions in Ca2þ-signaling is A single-channel analysis of NAADP-gated not a new concept. For example, Ca2þ-micro- channels has also been performed from lyso- domains may arise around sites of ER Ca2þ somal enriched fractions derived from liver release, and neighboring organelles may be (Zhang and Li 2007) and bovine coronary vas- profoundly affected physiologically. Indeed cular smooth muscle (Zhang et al. 2009). These ER-mitochondrial interactions have been well þ channels conducted Cs and were sensitive to studied in the context of IP3R and RyR- NAADP, with open probabilities displaying a mediated Ca2þ-release (Rizzuto et al. 1998; bell-shaped concentration dependence, and Csordas et al. 2001), which impacts on mito- with maximum Po occurring at 1 mM NAADP chondrial metabolism and apoptotic pathways.

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NAADP Receptors

A Ca2+ SERCA

IP3R 2+ ER TPC Ca + H + Acidic store H -ATPase NAADP CHX ? ? ? Ca2+ CICR amplification and Resting Trigger globalization

BC1. Docking 2. Trigger Resting Local Cross Talk Ca2+ X+ or Y– TPC

ΔΨ Endosome NAADP Plasma membrane Ca2+ Lysosome Acidic store

H+ CHX + NAADP 3. Fusion ? H -ATPase ? Ca2+

Figure 5. Three modes of NAADP-mediated Ca2þ signaling. (A) NAADP is a local trigger mechanism for det- onating global CICR responses from the ER. (B) Local Ca2þ release by NAADP from acidic stores positioned under the plasma membrane may regulate membrane excitability (excitable cells) or ion ﬂuxes (nonexcitable cells) by modulating Ca2þ-activated plasma membrane channels. (C) NAADP regulates local cytoplasmic Ca2þ/pH and luminal Ca2þ/pH in endolysosomal compartments that may regulate vesicular fusion of late endosomes/lysosomes. Modiﬁed from Galione et al. (2009) and Ruas et al. (2010).

The protein mitofusin 2 has been proposed to found to act as a local messenger to form tether ER and mitochondrial membranes (de Ca2þ gradients across the cell based on NAADP Brito and Scorrano 2008). diffusion. These gradients could be amplified NAADP-evoked Ca2þ release and its effects and globalized by CICR through the recruit- 2þ on Ca -release channels on the ER/SR was first ment of IP3R and RyR-dependent mechanisms noted in pancreatic acinar cells (Cancela et al. on the ER. Because of the distinct self- 1999). This phenomenon, whereby a localized inactivation properties of NAADP receptors, microdomain of Ca2þ release from acidic stores subsequent NAADP-evoked Ca2þ-signaling pat- triggers a larger release from the ER, is widely ob- terns only occur in regions of the cell where served in both the sea urchin egg and in many NAADP had not previously evoked a response types of mammalian cell, and is one of the (Churchill and Galione 2001b). This effect lasts fundamental principles of NAADP-mediated for many minutes, representing a basic type of Ca2þ signaling. The trigger hypothesis was for- spatiotemporal memory in terms of the genera- mulated by the finding that NAADP-evoked tion of Ca2þ-signal patterning. As well as spatial responses in pancreatic acinar cells could be complexities, NAADP could produce temporal blocked by either heparin or ryanodine, as patterns in Ca2þ signals by the uptake of Ca2þ well as self-inactivation of the NAADP receptor released from NAADP-sensitive stores into the with NAADP itself (Cancela et al. 1999). This ER to produce a series of Ca2þ spikes dependent was visualized in the larger sea urchin egg by on IP3R and RyRs (Churchill and Galione detailed imaging studies (Churchill and 2001a). Galione 2000; Churchill and Galione 2001b; In pulmonary vascular smooth muscle Churchill and Galione 2001a). NAADP was cells, NAADP and the vasoactive hormone,

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A. Galione

endothelin-1, evoke a localized Ca2þ release whether NAADP first releases Ca2þ from intra- from lysosomes at lysosomal-SR junctions, cellular stores, which then leads to activation which is then amplified and globalized by a of plasma membrane conductances. Indeed, at mechanism dependent on recruitment of present, there is no evidence for TPC localiza- RyRs on the SR (Kinnear et al. 2004; Kinnear tion at the plasma membrane. et al. 2008). Similar results have been reported However, local NAADP-evoked Ca2þ release in coronary smooth muscle myocytes (Zhang from acidic stores in the vicinity of the plasma et al. 2006), and also implicated for early Fas sig- membrane has been shown in several cell types naling processes, which eventually lead to apop- to open Ca2þ-activated ion channels. This was tosis (Zhang et al. 2010). first shown in non-excitable pancreatic acinar In Jurkat T cells, NAADP triggers Ca2þ cells, where activation of such channels is likely release, which can be amplified by RyRs and to contribute to fluid secretion (Cancela et al. IP3Rs, but it has been proposed in this system 1999). However, this may be a major mechanism that RyR1 may be the primary target of NAADP in excitable cells. In pancreatic b cells, NAADP on the ER (Dammermann and Guse 2005; also evokes Ca2þ-dependent currents, which Dammermann et al. 2009). A role for RyR as may contribute to glucose-mediated depolariza- the direct target for NAADP has also been tion of the cells during stimulus-secretion cou- proposed in pancreatic acinar cell ER/nuclear pling (Naylor et al. 2009), and which are absent membranes, although other evidence points in cells derived from Tpc2-/- mice (Calcraft to direct activation of acidic stores (Yamasaki et al. 2009). In neurons from the rat medulla et al. 2004; Menteyne et al. 2006) followed by oblongata (Brailoiu et al. 2009b), NAADP also amplification by CICR. Such discrepancies are depolarizes cells through a mechanism depend- not surprising given the small release of Ca2þ ent on Ca2þ release from acidic stores. released by lysosomes that TPC studies have revealed (Calcraft et al. 2009; Ruas et al. 2010), NAADP and its Receptors in Endolysosomal with amplification by ER mechanisms pro- Physiology viding much larger Ca2þ signals. Thus in small cells, dissection of contributory Ca2þ release NAADP may be unique among Ca2þ mobiliz- mechanisms can prove difficult (Galione and ing messengers in that in contrast to IP3 or Petersen 2005), but employment of emerging cADPR, it may in most cases directly evoke molecular insights and tools may prove insight- Ca2þ release from the endolysosomal system. ful (Galione et al. 2009). NAADP-regulated TPCs are a new group of channels that are targeted to the endolysosomal system, along with mucolipins (Dong et al. Modulation of Plasma Membrane Excitability 2010), P2X4 receptors (Qureshi et al. 2007), As well as their involvement in organelle com- and TRPM2 (Lange et al. 2009), all of which munication, NAADP and TPCs appear to play are likely to influence the ionic environment an important role in regulating ion fluxes in acidic organelles. Interestingly, TRPM2 chan- across the plasma membrane and hence also nels have also been proposed to be NAADP excitability of excitable cells. NAADP has been receptors (Beck et al. 2006); however, they shown to stimulate Ca2þ influx across the have much lower affinities for NAADP, in the plasma membrane of several cell types including high mM range. TRPM2 channels could provide starfish oocytes (Moccia et al. 2003; Moccia et al. local Ca2þ signals that may directly impinge 2006), sea urchin eggs (Churchill et al. 2003), on the pleiotropic roles of the endolysosomal where it uniquely among Ca2þ mobilizing mes- system including lysosomal biogenesis, vesicu- sengers mediates the polyspermic blocking lar trafficking and transport, and autophagy. “cortical flash,” and Jurkat T cells (Langhorst Both local and luminal Ca2þ is important for et al. 2004). What is not clear is whether NAADP many of these processes including homotypic directly activates plasma membrane channels or fusion processes of endosomes and heterotypic

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NAADP Receptors

fusions of late endosomes with lysosomes, as from the ER. This was initially proposed on the well as condensation of luminal contents (Piper basis of pharmacological studies, but the identifi- and Luzio 2004; Luzio et al. 2007); release of cation of endolysosomal TPC proteins as major Ca2þ from endolysosomal stores is thought to targets for NAADP has begun to cement this be a crucial regulatory mechanism. Overexpres- hypothesis in molecular terms. Three major con- sion of TPCs in HEK293 causes profound sequences of NAADP-evoked Ca2þ release have changes in trafficking, lysosomal size, and dis- been identified. The unifying principle is that tribution as observed in certain lysosomal stor- NAADP, by mobilizing acidic stores, leads to lo- age diseases (Ruas et al. 2010). These effects can calized Ca2þ signals that may trigger key cellular be ameliorated by treatment with the NAADP responses. Depending on the subcellular locali- antagonist, Ned-19. These data are suggestive zation of these stores, there are fundamentally dif- of a major role for NAADP and TPC proteins ferent consequences of NAADP-mediated Ca2þ in the regulation of luminal Ca2þ,Ca2þ release, release. For stores proximal to the plasma mem- and local Ca2þ signaling in endolysosomal brane, Ca2þ-activated plasma channels may be physiology, and are thus likely to be key regula- activated. Such ion fluxes produced in nonexcit- tors of trafficking, autophagy, and other func- able cells may, for example, be important in fluid tions of these organelles. secretion. In excitable cells, depolarization and changesinmembraneexcitabilitymayresult. For stores apposed to the ER, NAADP-evoked Conclusions: Why Have Multiple Messengers 2þ 2þ Ca release from acidic stores may trigger for Ca Release? 2þ globalized Ca responses by activating IP3Rs or Over the last decade or so, NAADPhasjoined IP3 RyRs by CICR. The third major aspect is the reg- and cADPR as a major Ca2þ mobilizing messen- ulation of luminal Ca2þ and pH, as well as local ger. A major question in Ca2þ signaling research Ca2þ signals in the endolysosomal system that is how ubiquitous Ca2þ signals can encode spe- may have a major impact on the many roles of cificity. A general view is that the complex spatial these organelles in key cellular processes that and temporal patterns of Ca2þ signals widely they control, including vesicular trafficking, observed in cells are key to understanding this autophagy, apoptosis, and autolysis, as well as problem. The coordination of Ca2þ signals by their role in fighting infection. Cellular stimuli multiple messengers acting at differentially dis- may be selectively coupled to NAADP signaling tributed target Ca2þ release channels with dif- pathways, or as is commonly observed, to mul- ferent properties offers one possible solution. tiple messenger pathways, either providing dis- For example, NAADP-evoked Ca2þ release leads tinct patterns of Ca2þ signals leading to specific to neuronal cell differentiation (Brailoiu et al. responses. 2006), whereas cADPR-mediated Ca2þ release The establishment of a role of the endolyso- leads to cell proliferation, but delays differen- somal system in Ca2þ signaling, the identifica- tiation (Yue et al. 2009). On the other hand, acti- tion of specific Ca2þ release channels of acidic vation of certain cell surface receptors may organelles as the targets for NAADP, open up produce different combinations of messengers new possibilities for a better understanding of that are required to mimic the specific Ca2þ sig- the mechanisms of cellular Ca2þ signaling and naling patterns evoked by the particular recep- how this goes awry in disease, and its control tor agonist (Cancela et al. 2002; Yamasaki et al. and pharmacological manipulation. 2005), thus increasing the repertoire of cellular responses mediated by Ca2þ. ACKNOWLEDGMENTS The emerging view that NAADP directly targets acidic stores rather than the ER is an impor- This work in AG’s laboratory is funded by the tant new principle in Ca2þ signaling and cellular Wellcome Trust, and AG is a Principal Investiga- homeostasis, and allows NAADP to evoke distinct tor of the British Heart Foundation Centre of Ca2þ signals from those directly mobilizing Ca2þ Research Excellence at the University of Oxford.

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A. Galione

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NAADP Receptors

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NAADP Receptors

Antony Galione

Cold Spring Harb Perspect Biol 2011; doi: 10.1101/cshperspect.a004036 originally published online November 3, 2010

Subject Collection Calcium Signaling

The Endoplasmic Reticulum−Plasma Membrane Primary Active Ca2+ Transport Systems in Health Junction: A Hub for Agonist Regulation of Ca 2+ and Disease Entry Jialin Chen, Aljona Sitsel, Veronick Benoy, et al. Hwei Ling Ong and Indu Suresh Ambudkar Calcium-Handling Defects and Neurodegenerative Signaling through Ca2+ Microdomains from Disease Store-Operated CRAC Channels Sean Schrank, Nikki Barrington and Grace E. Pradeep Barak and Anant B. Parekh Stutzmann Lysosomal Ca2+ Homeostasis and Signaling in Structural Insights into the Regulation of Ca2+ Health and Disease /Calmodulin-Dependent Protein Kinase II (CaMKII) Emyr Lloyd-Evans and Helen Waller-Evans Moitrayee Bhattacharyya, Deepti Karandur and John Kuriyan Ca2+ Signaling in Exocrine Cells Store-Operated Calcium Channels: From Function Malini Ahuja, Woo Young Chung, Wei-Yin Lin, et al. to Structure and Back Again Richard S. Lewis Functional Consequences of Calcium-Dependent Bcl-2-Protein Family as Modulators of IP3 Synapse-to-Nucleus Communication: Focus on Receptors and Other Organellar Ca 2+ Channels Transcription-Dependent Metabolic Plasticity Hristina Ivanova, Tim Vervliet, Giovanni Monaco, et Anna M. Hagenston, Hilmar Bading and Carlos al. Bas-Orth Identifying New Substrates and Functions for an Calcium Signaling in Cardiomyocyte Function Old Enzyme: Calcineurin Guillaume Gilbert, Kateryna Demydenko, Eef Dries, Jagoree Roy and Martha S. Cyert et al. Fundamentals of Cellular Calcium Signaling: A Cytosolic Ca2+ Buffers Are Inherently Ca2+ Signal Primer Modulators Martin D. Bootman and Geert Bultynck Beat Schwaller

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Role of Two-Pore Channels in Embryonic Organellar Calcium Handling in the Cellular Development and Cellular Differentiation Reticular Network Sarah E. Webb, Jeffrey J. Kelu and Andrew L. Wen-An Wang, Luis B. Agellon and Marek Michalak Miller

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