NAADP mobilizes calcium from acidic organelles through two-pore channels.

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

NAADP was first identified as a potent intracellular Ca2+ mobilizing agent in sea urchin eggs 5 and later confirmed as such in various mammalian preparations 6-8. The Ca2+ stores mobilized by NAADP appear to be distinct from S/ER 9-11 and accumulating evidence from a variety of preparations now suggests that NAADP targets lysosome-like acidic compartments 3,4,12-14. However, it remains debated whether NAADP can also release the S/ER Ca2+ stores in certain cell types, perhaps by directly acting on RyRs 15-17. Furthermore, cross-talk between NAADP signaling and that mediated by InsP3, Ca2+, and cADPR exists in many cell types 11,18 (Supplementary Fig. S1), complicating the interpretation of experimental results.

TPCs (also known as TPCNs) are novel members of the superfamily of voltage-gated ion channels 19,20. Their predicted structures indicate 2-fold symmetry with a total of 12 putative transmembrane (TM) α-helices (Fig. 1a). Three non-allelic TPCN genes are present in sea urchins and most vertebrate species, with TPC3 absent in primates and some rodent species (supplementary Fig. S2). The three TPCs are equally distant from each other, and from plant “TPC1”, with <30% amino acid identity in the conserved TM regions (Fig. 1b and supplementary Fig. S2). While Arabidopsis “TPC1” has been shown to mediate Ca2+ release from plant intracellular vacuoles 21, functional data are lacking for animal TPCs.

Figure 1

Tissue and subcellular expression of mammalian TPC2

a, Hydropathy plot of human TPC2. Window size = 9 a.a. b, Evolutionary relationships of TPCs with single-pore and four-pore domain channels. h, human; d, dog; r, rat; at, Arabidopsis thaliana. c, Northern blot analysis of TPC2 expression in human tissues. d, Colocalization of endogenous TPC2 with LAMP-2 in untransfected HEK293 cells. e and f, Colocalization of HA-hTPC2 with LAMP-2 (e) and LysoTracker (f) in hTPC2 stable cell line. Right panel in f shows stacked image of overall LysoTracker accumulation relative to expressed HA-hTPC2 (3D projections, supplementary Movie 1). Scale bars = 10 μm.

Similar to the reported widespread expression of TPC1 mRNA 19, Northern analysis shows that human TPC2 (hTPC2) mRNA is expressed in most human tissues with higher levels in liver and kidney (Fig. 1c). Immunofluorescence labeling of HEK293 cells using an anti-hTPC2 antibody (see supplementary Fig. S3) revealed punctate staining in the cytoplasm, which was blocked by the antigenic peptides (not shown) and overlaps with that of lysosome-membrane associated protein 2 (LAMP-2; Fig. 1d, Pearson’s coefficient = 0.92). Similarly, in a stable cell line expressing hemagglutinin (HA) tagged-hTPC2 (hTPC2 cells), the HA-tagged protein colocalizes with LAMP-2 (Fig. 1e; Supplementary Movie 1, Table, and Fig. S4) but not with markers for early and late endosomes or that for ER, Golgi, or mitochondria (Supplementary Figs. S5a-e). Moreover, LysoTracker (Fig. 1f), but not MitoTracker or fluorescent transferrin (not shown), accumulated in intracellular vesicles surrounded by HA-hTPC2. Similar results were obtained for heterologously expressed mouse TPC2 (Supplementary Fig. S6).

In contrast, heterologously expressed TPC1 and TPC3 display only sparse co-localization with TPC2 or LAMP-2, but instead are predominantly expressed in endosomes and other unidentified intracellular compartments (Supplementary Figs. S5f, S5g, S7, and Table). Therefore, all mammalian TPCs are expressed intracellularly on endo-lysosomes with TPC2 being specifically targeted to lysosomal membranes.

The lysosomal localization and homology to Ca2+ channels prompted us to test whether TPC2 forms a binding site for NAADP. Membranes from the hTPC2 and wild type HEK293 cells were incubated with 0.2 nM [32P]NAADP in the absence and presence of unlabeled NAADP (100 μM). hTPC2 membranes showed more than a three-fold increase in specific binding compared to wild type membranes (Fig. 2a). To confirm that the binding is associated with expressed TPC2 proteins, we depleted HA-hTPC2 from the membranes with an anti-HA antibody and tested [32P]NAADP binding to the resulting supernatant and pellet. With anti-HA the binding was mainly associated with the pellet whereas with a control antibody it remained in the supernatant (Fig. 2b).

Figure 2

[32P]NAADP binding to TPC2

a, Specific binding for membranes from wild type HEK293 and hTPC2 cells (n = 3). b, Depletion of hTPC2 by immunoprecipitation abolished [32P]NAADP binding. The supernatant was depleted of hTPC2 (i) and [32P]NAADP binding (ii) by anti-HA but not by rat IgG (control) antibody; hTPC2 (i) and [32P]NAADP binding (iii) appeared in the pellet with anti-HA only. c and d, Representative ligand competition assay for membranes prepared from hTPC2 cells (c) and mouse liver (d). The maximal specific binding for hTPC2 membranes ranged from 167.6 to 300 dpm and for mouse liver from 1,000 to 1,600 dpm.

A ligand competition assay showed that the hTPC2-containing membranes displayed two affinities to NAADP with Kd values of 5.0 ± 4.2 nM and 7.2 ± 0.8 μM (n = 3) (Fig. 2c). This binding curve closely resembles that of mouse liver membranes (Fig. 2d), which displayed affinity values of 6.6 ± 3.5 nM and 4.6 ± 2.4 μM (n = 4), and these Kd for the high affinity binding site compare well with results reported for other mammalian preparations 22-24.

As expected 22,25, NADP, the precursor of NAADP that is unable to mobilize Ca2+, only showed low affinity binding to hTPC2 and mouse liver membranes, with Kd of 10.3 ± 3.1 μM (n = 3) and 4.5 ± 2.3 μM (n = 4), respectively. This could also arise from contamination by trace amounts of NAADP in NADP preparations 5. Moreover, although wild type membranes displayed specific binding to NAADP, the ligand competition assay could only reveal low affinity binding, indicating that the fraction for high affinity binding must be very low. This is supported by quantitative RT-PCR which showed a >250 fold increase in TPC2 mRNA in hTPC2 compared to wild type cells (Supplementary information). Therefore, TPC2 expression confers the high affinity NAADP binding. Although we cannot exclude that interactions with accessory proteins may be necessary for NAADP binding to TPC2, such proteins would have to associate with TPC2 tightly in order to explain these binding results.

To test if TPC2 mediates Ca2+ release from lysosomes, we studied the effect of flash photolysis of caged-NAADP on intracellular Ca2+ concentration ([Ca2+]i) in wild type HEK293 and hTPC2 cells by Fluo3 fluorescence. As shown in Fig. 3a, all hTPC2 cells responded to photorelease of NAADP with a biphasic [Ca2+]i transient comprising an initial slow pacemaker-like ramp (10-180 sec) and a subsequent large Ca2+ transient. No fluorescence increase occurred after UV flashes if caged-NAADP was not included (n=6, not shown). Furthermore, wild type cells displayed only small and short-lived [Ca2+]i rises and lacked both the ramp-like phase and the secondary transient (Fig. 3b).

Figure 3

TPC2 expression and NAADP-evoked Ca2+ signaling

a-c, Effect of photoreleased NAADP on Fluo3 fluorescence in hTPC2 (a) and wild type HEK293 cells (b). c, Means ± SEM of peak response. H: heparin, R: ryanodine. * p < 0.05 different from hTPC2 only. d-f, Effect of intracellular dialysis of NAADP on Fura2 ratio in hTPC2 (d) and wild type (e, the cell within dashed lines) cells: upper panels pseudocolour images, lower panels ratio against time. Time scale bars = 20 s. f, Means ± SEM (except n = 2) of peak response. * p < 0.05 different from hTPC2, 10 nM NAADP. Baf: bafilomycin, scrbld: scrambled. Number of cells in parentheses.

Consistent with a role for lysosomes in this process, the vacuolar H+-ATPase inhibitor bafilomycin A1 (Baf, 1 μM) abolished both phases of the response to NAADP (Fig. 3a), but failed to affect the [Ca2+]i rise induced by extracellular application of 100 μM ATP, which activates ER Ca2+ release (not shown). By contrast, inclusion of heparin (200 μg/ml), a competitive inhibitor of InsP3Rs, in the patch pipette only blocked the secondary phase of the Ca2+ transient and thereby revealed in its entirety the initial [Ca2+]i signal triggered by NAADP (Fig. 3a). Consistent with the lack of RyR expression in HEK293 cells 26, both phases of the response to photorelease of NAADP persisted in hTPC2 cells preincubated with 10 μM ryanodine (not shown). Furthermore, the combined effects of depleting the ER store by pretreatment with thapsigargin (TG, 1 μM), blocking InsP3Rs with heparin and RyRs with ryanodine caused no further inhibition of the NAADP-induced response than did blocking InsP3Rs with heparin alone (Fig. 3a). Therefore, the initial [Ca2+]i rise is dependent on acidic organelles but independent of the ER, whereas the secondary phase is due to ER Ca2+ release via InsP3Rs, presumably through Ca2+-induced Ca2+ release in concert with resting InsP3 levels.

To determine the concentration-response relationship for NAADP, we dialyzed known concentrations of NAADP into single cells via patch pipettes and monitored [Ca2+]i changes using Fura2 13,18. In hTPC2 cells, while 100 pM NAADP did not cause any appreciable [Ca2+]i rise, 10 nM NAADP elicited a biphasic response reminiscent of those evoked by photolysis of caged-NAADP (Fig. 3d). Pretreatment with TG abolished the second but not the first phase (Fig. 3d). The response was also seen with 1 μM but not with much higher NAADP concentrations (1 mM, Fig. 3f), consistent with the notion that NAADP-induced Ca2+ release desensitizes at high ligand concentrations 6. By contrast, 10 nM NAADP was without effect in wild type cells, while 1 μM only induced a small, perhaps more localized, Ca2+ transient in 3 out of 5 cells (Figs. 3e and 3f).

Again, the Ca2+ transient induced by intracellular dialysis of 10 nM NAADP in hTPC2 cells was blocked by Baf. More importantly, the response was abolished by transfection into hTPC2 cells of an shRNA against TPC2 (Supplementary Fig. S8), but not that of a scrambled control shRNA (Fig. 3f), demonstrating the essential role of TPC2 in mediating NAADP-induced Ca2+ release. All cells responded to extracellularly applied carbachol, which triggers ER Ca2+ release, indicating that cells were viable 27.

To examine the role of endogenous TPC2 in NAADP signaling in a native system, we generated TPC2 knockout mice using a gene trap strategy (Figs. 4a-c and supplementary information) 29 and isolated pancreatic β cells in which previous studies have established that NAADP-dependent Ca2+ mobilization from a TG-insensitive acidic store 12,24 underpins the gating of a Ca2+-activated plasma membrane cation current. Fig. 4d shows that under the whole-cell configuration, intracellular dialysis of 100 nM NAADP elicited oscillatory inward currents in wild type β-cells held at -70 mV. No such currents were detected if NAADP was omitted from the pipette solution, if intracellular Ca2+ was strongly buffered by 10 mM BAPTA, or if the extracellular cations were replaced by N-methyl-D-glucamine (not shown). Strikingly, NAADP failed to activate the cation currents in β-cells from the TPC2 knockout mice (Fig. 4e), strongly suggesting that TPC2 plays a critical role in native NAADP-evoked Ca2+ signaling in β-cells.

Figure 4

Pancreatic β cells from TPC2 knockout mice are NAADP insensitive

a, Approximate position of the gene trap vector in Tpcn2 gene. b, PCR analysis of genomic DNA. +/+, wild type; +/-, heterozygote; -/-, homozygote. c, RT-PCR products from wild type and mutant Tpcn2 mRNAs with approximate positions of amplicons indicated by red bars; numbers indicate exons (see supplementary information for details). d and e, Cation currents at -70 mV evoked by intracellular dialysis of 100 nM NAADP in pancreatic β-cells isolated from wild type (d) and TPC2 knockout (e) mice. Left, representative traces from single cells; right, overlaid traces for 5 cells.

The above results are best explained if TPC2 is a lysosomal Ca2+ release channel targeted by NAADP. Although we have focused on TPC2 because of its predominant lysosomal localization, TPC1 and TPC3 may also mediate NAADP-induced Ca2+ release from distinct subsets of acidic organelles, such as the distinguished endosome populations suggested by their subcellular distributions. Indeed, we have observed significant but highly localized Ca2+ transients in response to 10 nM NAADP in cells that overexpress human TPC1 as opposed to the global [Ca2+]i changes seen in hTPC2 cells (Supplementary Fig. S9 and Movie 2). This distinction is consistent with the more restricted subcellular distribution of TPC1 compared to TPC2 (Supplementary Fig. S7).

The biphasic Ca2+ response to NAADP in hTPC2 overexpressing cells and the dependence of the later phase on InsP3Rs and the ER are consistent with the idea that NAADP-induced Ca2+ signals are small, and perhaps localized, but able to act, at least via TPC2, as discrete triggers for large global [Ca2+]i changes through coupling to InsP3R/RyR-S/ER systems. This adds an intriguing possibility for signal diversification, given that the pure NAADP-evoked Ca2+ signal is small and highly localized once the ER Ca2+ store is depleted by TG, as shown in Figs 3a for heterologously expressed TPC2 as well as the endogenous channels in human hepatoblastoma cell line, HepG2 (Supplementary Fig. S10). However, the localized Ca2+ signals will likely reach high levels, particularly at lysosome-ER junctions with unheralded versatility supplied by the fact that TPC-containing vesicles undergo rapid movement within the cytosoplasm (Supplementary Movie 3). In this respect it is important to note that NAADP-sensitive Ca2+ signals can have multiple coupling targets. In sea urchin eggs and pancreatic acinar cells, NAADP-induced Ca2+ signals are coupled to ER Ca2+ release through InsP3Rs and RyRs 6,11,28; in pulmonary arterial smooth muscle cells, they appear to selectively target RyRs 13,18; in pancreatic β-cells, they are coupled to Ca2+-activated cation channels. Thus, the graded local and mobile endo-lysosome-derived Ca2+ signals released via TPCs, through coupling to other systems, are dynamic and versatile. Future investigations on the role of TPCs as NAADP receptors will therefore provide important advances in our understanding of the mechanisms of regulation, spatial organization and diverse functional roles of Ca2+ signals in mammalian cells.

Method summary

The cDNA for hTPC2 was cloned from HEK293 cells by RACE-PCR. Northern hybridization was performed using a multi-tissue human mRNA blot (BD Biosciences). For stable expression, the N-terminal HA-tagged hTPC2 was placed in pIRESneo (BD Biosciences), transfected in HEK293 cells, and stable clones selected and maintained using G418. [32P]NAADP synthesis, membrane purification and radioligand binding studies were carried out as previously described 23,25. Caged-NAADP was synthesized as described 30. TPC2 knockout mice were developed from an ES cell line (YHD437) containing a gene trap mutation in the Tpcn2 gene. Details for flash photolysis of caged-NAADP, intracellular NAADP dialysis, Ca2+ imaging, and measurement of Ca2+-activated cation currents are described in additional methods.