NAADP Receptors

Abstract

Of the established Ca²⁺ 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 Ca²⁺ release, and have highlighted a new role of acidic organelles as targets for messenger-evoked Ca²⁺ mobilization. Three distinct roles of TPCs have been identified. The first is to effect local Ca²⁺ release that may play a role in endolysosomal function including vesicular fusion and trafficking. The second is to trigger global calcium release by recruiting Ca²⁺-induced Ca²⁺ release (CICR) channels at lysosomal-ER junctions. The third is to regulate plasma membrane excitability by the targeting of Ca²⁺ release from appropriately positioned subplasma membrane stores to regulate plasma membrane Ca²⁺-activated channels. In this review, I discuss the role of NAADP-mediated Ca²⁺ 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 Ca²⁺ signaling.

NAADP is thought to trigger release of calcium from endolysosomes via TPC channels. This regulates vesicle trafficking, triggers global calcium release, and modulates plasma membrane excitability.

Calcium is the most evolutionarily ubiquitous of intracellular signals and controls cellular mechanisms as diverse as cellular motility, membrane fusion, ion channel function, enzyme activity, and gene expression (Berridge et al. 2003). Free cytoplasmic calcium levels are kept under tight control by pumps, exchangers, and buffering mechanisms including storage by organelles (Pozzan et al. 1994). Ca²⁺ signals may be elicited when these mechanisms are transiently overwhelmed by the opening of calcium permeable channels at the plasma membrane or in membranes of calcium-storing organelles. Chronic activation of such channels may lead to cell death, for example, through the activation of apoptotic signaling cascades (Berridge et al. 1998). Many cell surface receptors are linked to signaling pathways that lead to the mobilization of calcium from intracellular storage organelles through the activation of specific Ca²⁺ release channels (Clapham 1995). Three major intracellular messengers have been established to link cell stimulation with organellar Ca²⁺ release: inositol trisphosphate (IP₃), cyclic adenosine diphosphate ribose (cADPR), and nicotinic acid adenine nucleotide diphosphate (NAADP) (Bootman et al. 2002).

DISCOVERY OF NAADP AS A Ca²⁺-MOBILIZING MOLECULE

NAADP was discovered as a contaminant of commercial batches of β-NADP⁺ by Lee and colleagues while they were investigating the effects of various pyridine nucleotides on calcium release from sea urchin egg homogenates (Clapper et al. 1987). The rationale for this was that at fertilization in sea urchin eggs, dramatic changes in pyridine nucleotide levels occur at a similar time to the generation of the calcium wave. Egg homogenates can be simply prepared from eggs and are remarkably stable, even after freezing, and sequester, and robustly release calcium when challenged with messengers and drugs (Morgan and Galione 2008). Three distinct calcium release mechanisms were shown. These were the early days of IP₃, and IP₃ was found to release calcium from microsomal stores. In addition, two metabolites of pyridine nucleotides, an enzyme-activated metabolite related to NAD⁺, subsequently identified as cADPR (Lee et al. 1989), and alkaline-treated NADP, later shown to be NAADP (Lee and Aarhus 1995), were found to release Ca²⁺ from different subcellular nonmitochondrial fractions from egg homogenate (Fig. 1). A key feature of each mechanism is their display of homologous desensitization underscoring the independence of each of the three mechanisms.

Figure 1.

Structure and function of NAADP. NAADP differs from β -NADP in that the base nicotinic acid is substituted for nicotinamide (upper panel). NAADP, unlike NADP, is a potent Ca²⁺ mobilizing agent and interacts with two-pore channels in the membranes of lysosomes (lower panel).

NAADP AS A Ca²⁺ MOBILIZING MESSENGER

NAADP is the most potent of Ca²⁺ mobilizing messengers described, typically efficacious at pM or low nM concentrations. A growing number of cellular stimuli and activation of cell surface receptors have been found to be coupled to increases in NAADP levels, confirming its role as an intracellular messenger (Churchill et al. 2003; Masgrau et al. 2003; Rutter 2003; Yamasaki et al. 2005; Galione 2006; Gasser et al. 2006; Kim et al. 2008). Mediation of calcium signaling by NAADP has been implicated by two approaches: inhibition of agonist-evoked calcium signals by prior self-inactivation of the NAADP receptor or NAADP receptor pharmacological blocker (Naylor et al. 2009) and measurements of cellular NAADP levels. Measurements of NAADP have been performed using either a radioreceptor assay, based on the high affinity NAADP binding protein of sea urchin eggs (Lewis et al. 2007), or by using a cycling assay of coupled enzyme reactions resulting in fluorescent resorufin production (Graeff and Lee 2002). Although some receptors appear to couple selectively to NAADP production, increasingly it is becoming apparent that several receptors couple to multiple Ca²⁺ mobilizing messengers (Aley et al. 2010), and this may be the norm.

Ca²⁺ STORES TARGETED BY NAADP

Accumulating evidence suggests that the primary Ca²⁺ stores targeted by NAADP are separate from the endoplasmic reticulum and are members of a group known as acidic organelles. The initial evidence for this came from the study of sea urchin eggs and was subsequently extended to mammalian cells.

Sea Urchin Eggs

The initial report of NAADP-evoked Ca²⁺ release using alkaline-activated NADP showed that the subcellular fraction reactive to NAADP in egg homogenates was largely separate from the microsomal/ER fraction sensitive to IP₃ and cADPR (Clapper et al. 1987). Abrogation of Ca²⁺ storage by the ER SERCA inhibitor, thapsigargin, while inhibiting Ca²⁺ release by either IP₃ or cADPR, only partially reduced Ca²⁺ release evoked by NAADP in both sea urchin egg homogenates (Genazzani and Galione 1996) and intact eggs (Churchill and Galione 2001a). Visualization of two separate Ca²⁺ stores was observed in elegant sea urchin egg stratification studies (Lee and Aarhus 2000). Stratification by centrifugation of intact eggs results in eggs forming elongated structures with different organelles separating to different “poles.” Uniform photolysis of caged derivatives of Ca²⁺ mobilizing messengers resulted in IP₃ and cADPR releasing Ca²⁺ from the nuclear pole where ER accumulated, whereas NAADP released Ca²⁺ from the opposite end of the structure. These experiments are consistent with the primary Ca²⁺ store targeted by NAADP as being distinct from the ER.

In a series of important experiments using pharmacological analyses and subcellular fractionation, lysosomal-related organelles were implicated as the primary target organelle for NAADP-evoked Ca²⁺ release in sea urchin eggs (Churchill et al. 2002). Acidic stores, such as lysosomes, have been shown to sequester Ca²⁺ by mechanisms dependent on their low luminal pH (Patel and Docampo 2010). Inhibition of the vacuolar H⁺-ATPase by bafilomycin decreases proton uptake into acidic stores; if their membranes are sufficiently leaky to protons, this leads to the alkalinization of their lumen. Uptake of Ca²⁺ into these stores appears to be dependent on the maintenance of the proton gradient because bafilomycin and protonophores inhibit Ca²⁺ storage by these organelles, although the detailed mechanisms are not well understood. A dense membrane fraction from sea urchin egg homogenates was isolated from a percoll gradient and consisted of “reserve granules.” This fraction was enriched with lysosomal markers and supported ATP-dependent Ca²⁺ sequestration, which was inhibited by preincubation with bafilomycin or the protonophore, nigericin, but not thapsigargin. This fraction was found to contain [³²P]NAADP binding sites, and displayed NAADP but not IP₃/cADPR-evoked Ca²⁺ release. Reserve granules from sea urchin eggs are lysosome-related organelles. In intact sea urchin eggs, treatment with the lysosomotropic agent, glycyl-phenylalanine 2-naphthylamide (GPN), caused the reversible lysis of lysotracker-stained vesicles, resulting in a series of small-amplitude cytoplasmic Ca²⁺ signals, consistent with their role as Ca²⁺ stores. Importantly, GPN treatment in either intact eggs or egg homogenates selectively abolished NAADP-evoked Ca²⁺ release with little effect on Ca²⁺ release by either IP₃ or cADPR. From these data it was proposed that in sea urchin the primary target of NAADP are acidic stores rather than the endoplasmic reticulum. Consistent with this, experiments in sea urchin egg homogenates employing luminal pH indicators such as acridine orange or lysosensor also have shown that NAADP uniquely among Ca²⁺ mobilizing messengers also causes the alkalinization of store lumena, representing another possible signaling mechanism for this molecule (Morgan and Galione 2007b).

Mammalian Cells

Following these studies in sea urchin eggs, it was shown that NAADP also targeted acidic stores in a wide range of mammalian cells and in response to a variety of cellular stimuli (Mitchell et al. 2003; Kinnear et al. 2004; Yamasaki et al. 2004; Galione 2006; Gerasimenko et al. 2006; Menteyne et al. 2006; Zhang et al. 2006; Macgregor et al. 2007; Gambara et al. 2008; Jardin et al. 2008; Kim et al. 2008; Lloyd-Evans et al. 2008; Brailoiu et al. 2009b; Pandey et al. 2009; Thai et al. 2009; Dickinson et al. 2010; Zhang et al. 2010).

DESENSITIZATION OF NAADP-EVOKED Ca²⁺ RELEASE

The Ca²⁺ release mechanism activated by NAADP shows unusual and profound inactivation properties. One major area of confusion in this field is that the inactivation properties of Ca²⁺ release varies markedly between sea urchin egg and mammalian systems, which we have termed type I and type II, respectively (Morgan and Galione 2008) (Fig. 2).

Figure 2.

Differences between desensitization of mammalian and sea urchin NAADP receptors. Left-hand traces depict stylized Ca²⁺ dye fluorescence traces in response to messengers. Right-hand panels represent concentration-response graphs for activation or inactivation of intracellular Ca²⁺ release channels. (A) IP3 and cADPR-mediated Ca²⁺ release. Left-hand traces depict stylized Ca²⁺ 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 diminution of a maximal “test” challenge (orange). (B) Desensitization of sea urchin NAADP receptors (Type I desensitization). The blue left-hand traces show stylized Ca²⁺ dye fluorescence traces in response to increasing concentrations of NAADP, which increases Ca²⁺ 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 Ca²⁺ release desensitizes Ca²⁺ 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 Ca²⁺ release to a maximum (left and center panels). Thereafter, increasing concentrations of NAADP evoke progressively smaller Ca²⁺ release to a point when no Ca²⁺ 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).

Sea urchin eggs: The initial report demonstrating the efficacy of NAADP as a Ca²⁺ mobilizing molecule showed that NAADP released Ca²⁺ by a mechanism independent of IP₃ or ryanodine receptors (RyRs), based on each of these mechanisms showing homologous desensitization (Lee and Aarhus 1995). After NAADP stimulated Ca²⁺ release in egg homogenates, they became refractory to subsequent challenge with NAADP, but still responded to either IP₃ or cADPR. This was the first piece of evidence that NAADP activated a novel Ca²⁺ release channel, distinct from the principal Ca²⁺ release channels on the endoplasmic reticulum.

Further analysis of the phenomenon of self-inactivation of NAADP-evoked Ca²⁺ release in sea urchin eggs and homogenates revealed several profound and unusual features. A surprising finding was that pM concentrations of NAADP, although subthreshold for triggering Ca²⁺ release in egg homogenates, were able to inactivate completely the NAADP Ca²⁺ release mechanism to a subsequent challenge by nM concentration of NAADP that would normally evoke a maximal Ca²⁺ release (Aarhus et al. 1996; Genazzani et al. 1996). The extent of inactivation was dependent on both the concentration and duration of incubation (Genazzani et al. 1996; Genazzani et al. 1997b). Mechanisms of inactivation of the NAADP receptor are not understood, but may be related to the apparent irreversible binding of [³²P]NAADP. The radioligand appears to become occluded on binding in a time-dependent manner (Aarhus et al. 1996). Studies with the selective NAADP receptor antagonist, Ned-19, (Naylor 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 high affinity, whose occupancy leads to slow inactivation of the receptor, and a second lower affinity site that leads to rapid channel opening. Ned-20 blocks inactivation, but not activation of Ca²⁺ release by NAADP (Rosen et al. 2009).

Mammalian cells: There are key differences between desensitization of the NAADP receptor between sea urchin eggs, in which subthreshold concentrations of NAADP can fully inactivate the NAADP-sensitive Ca²⁺ release mechanism; whereas in a mammalian cell, high concentrations of NAADP are needed for full inactivation, which can occur in the apparent absence of receptor activation. The first report of NAADP action as a Ca²⁺-mobilizing agent in a mammalian cell was in the pancreatic acinar cell (Cancela et al. 1999), which was also the system in which IP₃ was first shown to mobilize Ca²⁺ from nonmitochondrial stores (Streb et al. 1983). Using whole cell patch and measuring Ca²⁺-activated currents, we found that a pipet concentration of 10 μM NAADP failed to elicit any responses. However, we noticed that after intracellular application of this concentration of NAADP, cholecystokinin (CCK), which usually increases cytosolic Ca²⁺ at pM concentrations by mobilizing Ca²⁺ from intracellular stores, now failed to evoke any response. We speculated that we may have inactivated the NAADP-evoked Ca²⁺ release mechanism that could be a key component of the CCK signal transduction mechanism. We therefore tried a range of NAADP concentrations and found that concentrations of NAADP as low as 50 nM in the pipet, elicited robust oscillatory responses, similar to those evoked by CCK in untreated cells. The concentration-response relationship for NAADP was found to be “bell-shaped,” with maximal responses occurring at around 100 nM NAADP, whereas with concentrations in excess of 1 μM no effects were seen. Using caged NAADP, we showed that photolysis of this compound also evokes a series of spikes in Ca²⁺-activated currents, which were suppressed in the presence of supramicromolar concentrations of free NAADP in the patch pipet. Bell-shaped concentration-response curves seem to be a major hallmark of mammalian NAADP-induced Ca²⁺ release. A subsequent study in a Jurkat T-cell line showed maximal Ca²⁺ release upon microinjection of around 100 nM NAADP. However, concentrations of >1 μM failed to elicit any response per se whilst inhibiting T-cell receptor activation (Berg et al. 2000). A number of further studies in different cell types used this phenomenon to implicate NAADP in the Ca²⁺ signal transduction pathways activated by various stimuli in the absence of selective NAADP antagonists at that time. These include glucose-evoked Ca²⁺ spiking in MIN6 cells (Masgrau et al. 2003), ET1-evoked Ca²⁺ release in pulmonary vascular smooth myocytes (Kinnear et al. 2004), and β₁ adrenoreceptor enhancement of Ca²⁺ signaling and contractility in ventricular cardiac myocytes (Macgregor et al. 2007).

PHARMACOLOGICAL PROPERTIES OF NAADP RECEPTORS

The pharmacology of NAADP-evoked Ca²⁺ release, initially investigated in sea urchin egg systems, showed major differences with the known Ca²⁺ release mechanisms in the ER. In egg homogenates, NAADP-evoked Ca²⁺ release was unaffected by the competitive IP₃R inhibitor, heparin, or by ryanodine or eight-substituted cADPR analogs that antagonize RyR-mediated Ca²⁺ release. An initial report that thio-NADP was a selective antagonist of NAADP (Chini et al. 1995) was subsequently explained by inactivation of the NAADP-sensitive Ca²⁺ release mechanism by traces of contaminating NAADP (Dickey et al. 1998).

A number of channel blockers were found to inhibit NAADP-evoked Ca²⁺ release selectively in sea urchin egg homogenates with little effect on either IP₃ or cADPR-mediated Ca²⁺ release (Genazzani et al. 1997a). These included voltage-gated Ca²⁺ channel (VGCC) blockers such as diltiazem, nifedipine, and D600 (although greater concentrations were required to block NAADP-evoked Ca²⁺ release than VGCCs), and purinoceptor antagonists such as aspyridoxal-phosphate-6-azophenyl-2’,4’-disulfonate (PPADS) also display a degree of NAADP antagonism (Billington and Genazzani 2007). Because the NAADP receptor effectively discriminates between NAADP and NADP, which differs only by the substitution of a nicotinic acid moiety instead of nicotinamide, nicotinic acid analogs were developed that antagonize NAADP-induced Ca²⁺ release. These include CMA008 (Dowden et al. 2006) and BZ194 (Dammermann et al. 2009), which also have the advantage of being membrane permeant. Recently, a series of novel compounds have been identified by in silico screening strategies based on the three-dimensional shape and electrostatic properties of NAADP that are the most potent of NAADP antagonists developed so far (Naylor et al. 2009; Rosen et al. 2009). Ned-19, the founding member of these analogs, is becoming 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).

Interestingly, Ned-19 analogs have been used to dissect the activation and inactivation effects of NAADP at the sea urchin egg NAADP receptor (Rosen et al. 2009). Ned-20, which differs only from Ned-19 by the para rather than ortho position of a fluorine, blocks the inactivation of NAADP-sensitive Ca²⁺ release mechanism by subthreshold NAADP concentrations for Ca²⁺ release, without affecting NAADP-evoked Ca²⁺ release by higher NAADP concentrations and inhibits high affinity [³²P]NAADP binding to egg membranes (Rosen et al. 2009). These findings are consistent with multiple binding sites for the sea urchin egg NAADP receptor, with high affinity sites leading to inactivation and lower affinity sites leading to activation.

TWO-PORE CHANNELS

A family of novel intracellular channels termed two-pore channels (TPCs) have emerged as the leading candidates for NAADP-gated Ca²⁺ release channels. The founding member of this family, TPC1, was cloned in 2000 from a rat kidney cDNA library in a search for novel members of voltage-gated cation channels (Ishibashi et al. 2000). The putative channel had only a 20% homology with the transmembrane domains of the α subunit of voltage-gated Na⁺ and Ca²⁺ channels, but the highest homology with a deposited sequence of a putative Ca²⁺ channel from the plant Arabidopsis thaliana. Subsequent analysis of the plant clone, AtTPC1, implicated a role for this protein in Ca²⁺ transport and signaling when expressed in yeast and Arabidopsis (Furuichi et al. 2001), and a role in germination and stomatal physiology as a component of the slow vacuolar channel (Peiter et al. 2005). The putative channel, rather than having four repeats of six transmembrane segments, as for voltage-gated Na⁺ and Ca²⁺ channels, only has two. Thus in effect, the protein is the equivalent of half a Na⁺ or Ca²⁺ channel, and may represent an ancestral form that has been duplicated later in evolution to give rise to the four domain channels (Fig. 3).

Figure 3.

Phylogenetic tree for human two-pore channels and their relationship with voltage-gated Ca²⁺ channels and TRP members. It is likely that voltage-gated Ca²⁺ 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 Ca²⁺ channels (24TM).

IDENTIFICATION OF TWO-PORE CHANNELS AS NAADP RECEPTORS

Two clues as to the candidature of TPCs as NAADP receptors emerged in the last few years. Michael Zhu, searching for novel TRP family membranes in around 2000, had cloned a second member of the TPC family, termed TPC2, and found that when heterologously expressed in HEK293 cells, it localized with the lysosomal marker, LAMP1. The second was the further analysis of AtTPC1 function by Sanders and colleagues, showing that AtTPC1 localized to plant vacuoles, the major plant acidic organelle and the functional equivalent of lysosomes in plants (Peiter et al. 2005). The localization of TPCs to acidic stores, and the partial pharmacological overlap of NAADP receptors with voltage-gated Ca²⁺ channels and TRP proteins, which show homologies with TPCs, made these proteins credible candidates as the elusive NAADP receptor. Over four years or so from 2005, we worked extensively with Zhu and collaborators, to test exhaustively the hypothesis that TPCs represented a family of NAADP-gated intracellular channels from several key standpoints. First, we examined the subcellular localization of the human TPC1 and TPC2 isoforms in HEK293 cells. In addition, because the genomes of many species, but not human or rodent, also express a third isoform, TPC3 (Cai and Patel 2010; Zhu et al. 2010), we also expressed the chicken TPC3 to examine its subcellular distribution in HEK293 cells (Calcraft et al. 2009). All three TPCs localize to the endolysosomal system with no apparent expression in Golgi, mitochondria, or ER. Only TPC2 consistently colocalized with the lysosomal marker, LAMP2, but not early or late endosomes. In contrast, TPC1 and TPC3 predominantly were expressed in endosomal and other unidentified compartments, but with only sparse colocalization with lysosomal markers. In HEK293 cells, TPCs are endogenously expressed at low levels, and endogenous TPC2 was also immunolocalized to lysosomes. Overexpression of the human HsTPC2 was associated with increased specific [³²P]NAADP binding to HEK293 cell membranes and immunoprecipitated TPC2 proteins. Both high and low affinity binding sites were manifest in membranes from TPC2-overexpressing cells, with K_d of 5 nM and 7 μM, remarkably similar to endogenous binding in membranes from mouse liver, a tissue which has a particularly high expression of TPCs. Photolysis of caged NAADP in patched wild-type HEK293 cells elicited a small Ca²⁺ response, whereas in cells stably overexpressing TPC2, a large biphasic Ca²⁺ response was evoked on NAADP uncaging or dialysis: an initial pacemaker-like ramp of Ca²⁺ was followed by a larger and faster transient Ca²⁺ release. Bafilomycin treatment abolished both phases of the Ca²⁺ response, whereas the IP₃R antagonist heparin blocked the second phase alone. This finding is consistent with the “trigger” hypothesis for a mode of NAADP action, whereby NAADP evokes a localized Ca²⁺ signal by mobilizing bafilomycin-sensitive acidic stores, which is then globalized by recruiting Ca²⁺-induced Ca²⁺ release (CICR) from nearby ER, in this case by activating IP₃Rs. The concentration-response relationship between NAADP and Ca²⁺ release was of the characteristic bell-shape for NAADP in mammalian cells, with maximal Ca²⁺ release occurring at between 10 nM and 1 μM, whereas 1 mM was without effect. Importantly, shRNA against TPC2 completely abolished the response to NAADP. We also created Tpc2^‐/‐ mice, and found that NAADP-evoked activation of oscillatory Ca²⁺-dependent cation currents in pancreatic β cells seen in wild-type cells, were abolished in cells from the knockout mice.

In contrast to TPC2, we found that HEK cells stably expressed with HsTPC1 evoked only a localized Ca²⁺ release in response to NAADP, which failed to globalize, as was the case for TPC2. One possibility is that the endosomal localization of TPC1 means that there is little close apposition with ER so that coupling with CICR channels is weaker. Two subsequent publications broadly confirmed our findings (Brailoiu et al. 2009a; Zong et al. 2009).

PROPERTIES OF ENDOGENOUS TPCs FROM SEA URCHIN EGGS

The properties of heterologously expressed mammalian TPCs made them strong candidates as NAADP receptors. However, most of the studies of NAADP-mediated Ca²⁺ release and [³²P]NAADP binding sites have been performed in sea urchin egg preparations, where the Ca²⁺ mobilizing effects of NAADP were first discovered. It was important to ascertain whether sea urchin eggs express TPCs and whether they functioned as NAADP receptors. Screening of the genome of the sea urchin Strongylocentrotus purpuratus revealed three TPC isoforms that were cloned from ovaries that displayed around 30% sequence homology between the isoforms (Brailoiu et al. 2010; Ruas et al. 2010). Importantly, immunoprecipitation of TPCs from solubilized egg membranes with polyclonal antibodies raised against each of the three TPC isoforms of TPCs produced immunocomplexes that specifically bound [³²P]NAADP with K_ds of around 1 nM. Binding of [³²P]NAADP to the immunocomplexes mirrored all the key features of binding to intact egg membranes, including K⁺-dependent irreversibility and a similar binding selectivity for NAADP over NADP. These data provided compelling evidence that TPCs form complexes that can explain all the properties of [³²P]NAADP binding sites previously characterized from sea urchin egg preparations. As with their mammalian homologs, heterologous expression of the sea urchin TPC1 and TPC2 isoforms in HEK293 cells enhanced NAADP-evoked Ca²⁺ release from acidic Ca²⁺ stores, which was amplified by recruitment of IP₃Rs, although coupling between TPC1 and IP₃Rs appeared looser. In contrast, TPC3 actually suppressed the small NAADP-evoked response observed in control cells and also abolished the enhancement in cells stably transfected with TPC2 (Fig. 4). This effect of TPC3 is puzzling for several reasons. The effect of TPC3 cannot be accounted by a general dysregulation of acidic Ca²⁺ stores since measurement of both Ca²⁺ storage and luminal pH do not appear to be altered in cells overexpressing TPC3 expressing cells. Another possibility is that TPC3 has a dominant negative effect, perhaps by forming heterodimers, because it is likely, given the proposed structure of TPCs, that functional channels would form dimers. Indeed, homodimerization of human TPC2 has been reported (Zong et al. 2009), but given the differing subcellular localizations of each of the TPCs, at least when heterologously expressed, it is unclear whether heterodimerization can explain TPC3 suppression of NAADP-evoked Ca²⁺ release.

Figure 4.

NAADP-mediated Ca²⁺ release in HEK293 cells expressing each of the three sea urchin TPC isoforms. Representative Ca²⁺ 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 µM) or the IP₃R antagonist, heparin (200 µg/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 Ca²⁺ 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).

SINGLE-CHANNEL PROPERTIES OF HUMAN TPCs

Although TPCs are emerging as promising candidates as NAADP-gated Ca²⁺ release channels in the endolysosomal system, it is important to characterize their biophysical channel properties to show that they do indeed function in this way. However, their localization in organelles presents several problems because they are not readily amenable for electrophysiological analysis as for channels resident at the plasma membrane, and there is no evidence at present that they cycle to the plasma membrane as for other Ca²⁺ release channels (Taylor et al. 2009). The traditional way of studying organellar channels is their reconstitution into artificial bilayers for single channel analysis, as exemplified for IP₃R (Ehrlich and Watras 1988) and RyR (Lai et al. 1988) single-channel studies; although for ER channels, nuclear envelope patching has gained increasing popularity (Mak and Foskett 1997). In a preliminary report, immunopurified human TPC2 was reconstituted into lipid bilayers and shown to form NAADP-gated cation conductances (Pitt et al. 2010). Channels were generally silent until application of NAADP to the cis or cytoplasmic face of the bilayer, and the channels showed a selectivity for cations with conductances of around 300 pS and 15 pS for K⁺ and Ca²⁺ ions as the conducting species. Interestingly, NAADP sensitivity may be regulated by store filling with Ca²⁺, because NAADP sensitivity was markedly dependent on trans or luminal Ca²⁺, with the EC₅₀ for NAADP-evoked enhancement of open probability decreasing from 500 nM to 5 nM as luminal Ca²⁺ increased to 200 μM. This is in the range of reported luminal free Ca²⁺ levels in lysosomes (Christensen et al. 2002; Lloyd-Evans et al. 2008). Thus, fluctuations in luminal Ca²⁺ because of cycles of release and uptake of Ca²⁺ could be important determinants of the effects of NAADP on Ca²⁺ release, and offers one explanation for how constant NAADP levels may elicit trains of Ca²⁺ spikes, as widely observed in various cell types (Cancela et al. 1999). Another variable is luminal pH of acidic stores, since NAADP has also been found to alkalinize acidic stores in sea urchin eggs and homogenates (Morgan and Galione 2007a; Morgan and Galione 2007b), and it is possible that luminal pH has significant effects on TPC2 channel properties. Importantly, the NAADP antagonist was also found to block single channel TPC2 currents (Pitt et al. 2010). However, it should be stressed here that although the immunopurified TPC complexes both form NAADP-gated Ca²⁺ channels (Pitt et al. 2010) and bind [³²P]NAADP (Calcraft et al. 2009; Ruas et al. 2010), the possibility remains that NAADP could interact with an accessory protein associated with TPCs instead of a direct interaction with TPC proteins themselves (Galione et al. 2009).

A single-channel analysis of NAADP-gated channels has also been performed from lysosomal enriched fractions derived from liver (Zhang and Li 2007) and bovine coronary vascular smooth muscle (Zhang et al. 2009). These channels conducted Cs⁺ and were sensitive to NAADP, with open probabilities displaying a bell-shaped concentration dependence, and with maximum P_o occurring at 1 μM NAADP in both preparations. The pharmacology was consistent with previous studies of NAADP-evoked Ca²⁺ release, with block by VGCC antagonists, PPADS, and also amiloride. Interestingly, P_o was increased at acidic pH. In contrast to the situation in most mammalian cells examined so far, pretreatment with concentrations of NAADP as low as 0.5 nM blocked subsequent channel openings by higher NAADP concentrations, as seen for sea urchin egg receptors and in liver (Mandi et al. 2006). The identity of these channels were ascribed to mucolipin-1 (TRPML-1), a lysosomal TRP channel linked to the lysosomal storage disease, mucolipidosis IV, on the basis of a blocking effect of an anti-TPRML1 antibody and reduction of channel activity from cells treated with an siRNA TPRML1 construct. However, the identity of TRPML1 as an NAADP receptor candidate remains controversial (Pryor et al. 2006). In addition, a recent report suggests that NAADP may increase levels of a short variant of a TRPML2 transcript in lymphoid cells (Samie et al. 2009), underscoring the likely complex interactions between lysosomal channels.

INTERACTIONS OF NAADP AND OTHER Ca²⁺ SIGNALING PATHWAYS

NAADP-evoked Ca²⁺ release from lysosomes appears to be small and highly localized. Given the dynamic properties of these organelles, they are ideally suited to be targeted to the vicinity of Ca²⁺-regulated effectors. Three modes of NAADP-mediated Ca²⁺ signaling mechanisms have been highlighted (Fig. 5).

Figure 5.

Three modes of NAADP-mediated Ca²⁺ signaling. (A) NAADP is a local trigger mechanism for detonating global CICR responses from the ER. (B) Local Ca²⁺ release by NAADP from acidic stores positioned under the plasma membrane may regulate membrane excitability (excitable cells) or ion fluxes (nonexcitable cells) by modulating Ca²⁺-activated plasma membrane channels. (C) NAADP regulates local cytoplasmic Ca²⁺/pH and luminal Ca²⁺/pH in endolysosomal compartments that may regulate vesicular fusion of late endosomes/lysosomes. Modified from Galione et al. (2009) and Ruas et al. (2010).

NAADP and Lysosomal-ER Interactions

Organelle interactions in Ca²⁺-signaling is not a new concept. For example, Ca²⁺-microdomains may arise around sites of ER Ca²⁺ release, and neighboring organelles may be profoundly affected physiologically. Indeed ER-mitochondrial interactions have been well studied in the context of IP₃R and RyR-mediated Ca²⁺-release (Rizzuto et al. 1998; Csordas et al. 2001), which impacts on mitochondrial metabolism and apoptotic pathways. The protein mitofusin 2 has been proposed to tether ER and mitochondrial membranes (de Brito and Scorrano 2008).

NAADP-evoked Ca²⁺ release and its effects on Ca²⁺-release channels on the ER/SR was first noted in pancreatic acinar cells (Cancela et al. 1999). This phenomenon, whereby a localized microdomain of Ca²⁺ release from acidic stores triggers a larger release from the ER, is widely observed in both the sea urchin egg and in many types of mammalian cell, and is one of the fundamental principles of NAADP-mediated Ca²⁺ signaling. The trigger hypothesis was formulated by the finding that NAADP-evoked responses in pancreatic acinar cells could be blocked by either heparin or ryanodine, as well as self-inactivation of the NAADP receptor with NAADP itself (Cancela et al. 1999). This was visualized in the larger sea urchin egg by detailed imaging studies (Churchill and Galione 2000; Churchill and Galione 2001b; Churchill and Galione 2001a). NAADP was found to act as a local messenger to form Ca²⁺ gradients across the cell based on NAADP diffusion. These gradients could be amplified and globalized by CICR through the recruitment of IP₃R and RyR-dependent mechanisms on the ER. Because of the distinct self-inactivation properties of NAADP receptors, subsequent NAADP-evoked Ca²⁺-signaling patterns only occur in regions of the cell where NAADP had not previously evoked a response (Churchill and Galione 2001b). This effect lasts for many minutes, representing a basic type of spatiotemporal memory in terms of the generation of Ca²⁺-signal patterning. As well as spatial complexities, NAADP could produce temporal patterns in Ca²⁺ signals by the uptake of Ca²⁺ released from NAADP-sensitive stores into the ER to produce a series of Ca²⁺ spikes dependent on IP₃R and RyRs (Churchill and Galione 2001a).

In pulmonary vascular smooth muscle cells, NAADP and the vasoactive hormone, endothelin-1, evoke a localized Ca²⁺ release from lysosomes at lysosomal-SR junctions, which is then amplified and globalized by a mechanism dependent on recruitment of RyRs on the SR (Kinnear et al. 2004; Kinnear et al. 2008). Similar results have been reported in coronary smooth muscle myocytes (Zhang et al. 2006), and also implicated for early Fas signaling processes, which eventually lead to apoptosis (Zhang et al. 2010).

In Jurkat T cells, NAADP triggers Ca²⁺ release, which can be amplified by RyRs and IP₃Rs, but it has been proposed in this system that RyR1 may be the primary target of NAADP on the ER (Dammermann and Guse 2005; Dammermann et al. 2009). A role for RyR as the direct target for NAADP has also been proposed in pancreatic acinar cell ER/nuclear membranes, although other evidence points to direct activation of acidic stores (Yamasaki et al. 2004; Menteyne et al. 2006) followed by amplification by CICR. Such discrepancies are not surprising given the small release of Ca²⁺ released by lysosomes that TPC studies have revealed (Calcraft et al. 2009; Ruas et al. 2010), with amplification by ER mechanisms providing much larger Ca²⁺ signals. Thus in small cells, dissection of contributory Ca²⁺ release mechanisms can prove difficult (Galione and Petersen 2005), but employment of emerging molecular insights and tools may prove insightful (Galione et al. 2009).

Modulation of Plasma Membrane Excitability

As well as their involvement in organelle communication, NAADP and TPCs appear to play an important role in regulating ion fluxes across the plasma membrane and hence also excitability of excitable cells. NAADP has been shown to stimulate Ca²⁺ influx across the plasma membrane of several cell types including starfish oocytes (Moccia et al. 2003; Moccia et al. 2006), sea urchin eggs (Churchill et al. 2003), where it uniquely among Ca²⁺ mobilizing messengers mediates the polyspermic blocking “cortical flash,” and Jurkat T cells (Langhorst et al. 2004). What is not clear is whether NAADP directly activates plasma membrane channels or whether NAADP first releases Ca²⁺ from intracellular stores, which then leads to activation of plasma membrane conductances. Indeed, at present, there is no evidence for TPC localization at the plasma membrane.

However, local NAADP-evoked Ca²⁺ release from acidic stores in the vicinity of the plasma membrane has been shown in several cell types to open Ca²⁺-activated ion channels. This was first shown in non-excitable pancreatic acinar cells, where activation of such channels is likely to contribute to fluid secretion (Cancela et al. 1999). However, this may be a major mechanism in excitable cells. In pancreatic β cells, NAADP also evokes Ca²⁺-dependent currents, which may contribute to glucose-mediated depolarization of the cells during stimulus-secretion coupling (Naylor et al. 2009), and which are absent in cells derived from Tpc2^‐/‐ mice (Calcraft et al. 2009). In neurons from the rat medulla oblongata (Brailoiu et al. 2009b), NAADP also depolarizes cells through a mechanism dependent on Ca²⁺ release from acidic stores.

NAADP and its Receptors in Endolysosomal Physiology

NAADP may be unique among Ca²⁺ mobilizing messengers in that in contrast to IP₃ or cADPR, it may in most cases directly evoke Ca²⁺ release from the endolysosomal system. NAADP-regulated TPCs are a new group of channels that are targeted to the endolysosomal system, along with mucolipins (Dong et al. 2010), P2X₄ receptors (Qureshi et al. 2007), and TRPM2 (Lange et al. 2009), all of which are likely to influence the ionic environment in acidic organelles. Interestingly, TRPM2 channels have also been proposed to be NAADP receptors (Beck et al. 2006); however, they have much lower affinities for NAADP, in the high μM range. TRPM2 channels could provide local Ca²⁺ signals that may directly impinge on the pleiotropic roles of the endolysosomal system including lysosomal biogenesis, vesicular trafficking and transport, and autophagy. Both local and luminal Ca²⁺ is important for many of these processes including homotypic fusion processes of endosomes and heterotypic fusions of late endosomes with lysosomes, as well as condensation of luminal contents (Piper and Luzio 2004; Luzio et al. 2007); release of Ca²⁺ from endolysosomal stores is thought to be a crucial regulatory mechanism. Overexpression of TPCs in HEK293 causes profound changes in trafficking, lysosomal size, and distribution as observed in certain lysosomal storage diseases (Ruas et al. 2010). These effects can be ameliorated by treatment with the NAADP antagonist, Ned-19. These data are suggestive of a major role for NAADP and TPC proteins in the regulation of luminal Ca²⁺, Ca²⁺ release, and local Ca²⁺ signaling in endolysosomal physiology, and are thus likely to be key regulators of trafficking, autophagy, and other functions of these organelles.

Conclusions: Why Have Multiple Messengers for Ca²⁺ Release?

Over the last decade or so, NAADP has joined IP₃ and cADPR as a major Ca²⁺ mobilizing messenger. A major question in Ca²⁺ signaling research is how ubiquitous Ca²⁺ signals can encode specificity. A general view is that the complex spatial and temporal patterns of Ca²⁺ signals widely observed in cells are key to understanding this problem. The coordination of Ca²⁺ signals by multiple messengers acting at differentially distributed target Ca²⁺ release channels with different properties offers one possible solution. For example, NAADP-evoked Ca²⁺ release leads to neuronal cell differentiation (Brailoiu et al. 2006), whereas cADPR-mediated Ca²⁺ release leads to cell proliferation, but delays differentiation (Yue et al. 2009). On the other hand, activation of certain cell surface receptors may produce different combinations of messengers that are required to mimic the specific Ca²⁺ signaling patterns evoked by the particular receptor agonist (Cancela et al. 2002; Yamasaki et al. 2005), thus increasing the repertoire of cellular responses mediated by Ca²⁺.

The emerging view that NAADP directly targets acidic stores rather than the ER is an important new principle in Ca²⁺ signaling and cellular homeostasis, and allows NAADP to evoke distinct Ca²⁺ signals from those directly mobilizing Ca²⁺ from the ER. This was initially proposed on the basis of pharmacological studies, but the identification of endolysosomal TPC proteins as major targets for NAADP has begun to cement this hypothesis in molecular terms. Three major consequences of NAADP-evoked Ca²⁺ release have been identified. The unifying principle is that NAADP, by mobilizing acidic stores, leads to localized Ca²⁺ signals that may trigger key cellular responses. Depending on the subcellular localization of these stores, there are fundamentally different consequences of NAADP-mediated Ca²⁺ release. For stores proximal to the plasma membrane, Ca²⁺-activated plasma channels may be activated. Such ion fluxes produced in nonexcitable cells may, for example, be important in fluid secretion. In excitable cells, depolarization and changes in membrane excitability may result. For stores apposed to the ER, NAADP-evoked Ca²⁺ release from acidic stores may trigger globalized Ca²⁺ responses by activating IP₃Rs or RyRs by CICR. The third major aspect is the regulation of luminal Ca²⁺ and pH, as well as local Ca²⁺ signals in the endolysosomal system that may have a major impact on the many roles of these organelles in key cellular processes that they control, including vesicular trafficking, autophagy, apoptosis, and autolysis, as well as their role in fighting infection. Cellular stimuli may be selectively coupled to NAADP signaling pathways, or as is commonly observed, to multiple messenger pathways, either providing distinct patterns of Ca²⁺ signals leading to specific responses.

The establishment of a role of the endolysosomal system in Ca²⁺ signaling, the identification of specific Ca²⁺ release channels of acidic organelles as the targets for NAADP, open up new possibilities for a better understanding of the mechanisms of cellular Ca²⁺ signaling and how this goes awry in disease, and its control and pharmacological manipulation.