Questioning Regulation of Two-Pore Channels by NAADP

MESSENGER Article

Jonathan S. Marchant1 ∗ and Sandip Patel2 ∗ MESSENGER

1Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA 2Department of Cell and Developmental Biology, University College London, London, WC1E 6BT, UK

NAADP is a potent Ca2+ mobilizing messenger. Since its discovery in 1995 a considerable volume of literature has shown that NAADP couples cell stimulation to endolysosomal Ca2+ release and thereby the regulation of many cellular functions. However deﬁnition of its molecular mechanism of action has proved far from easy. Since 2009, a consensus emerged as several independent groups coalesced upon the two-pore channel (TPC) family as NAADP-activated channels essential for Ca2+ release from endolysosomal Ca2+ stores. However this view has been recently challenged by data clearly showing that TPCs function as Na+-selective channels apparently insensitive

to NAADP. Given the two fundamental characteristics deﬁning an ion channel comprise the opening stimulus and MESSENGER the nature of the permeant ions, scrutiny of these seeming irreconcilable viewpoints is essential. The purpose of this commentary is to distil the remaining consensus while interrogating these divergent viewpoints. From this analysis, critical experimental needs are identiﬁed. Keywords: NAADP, Two-Pore Channels, TPCN1, TPCN2, Lysosomes, Endosomes, Ca2+.

Three independent studies published in 2009 first intro- A1-sensitive manner in this cell type was recently con- duced TPCs as the elusive targetIP: channels 192.168.39.210 for NAADP On: Fri,firmed 01 Oct by 2021 Wang 01:37:16 et al. (2012). Second, it is clear that (Brailoiu et al., 2009; Calcraft et al.,Copyright: 2009; Zong American et al., ScientificTPCs function Publishers as organellar ion channels. TPCs were 2009). Wang et al. (2012) and Cang et al. (2013)Delivered dis- bycloned Ingenta from plants and animals MESSENGER at the turn of the cen- MESSENGER pute this view. For non-aficionados interfacing with these tury (Furuichi et al., 2001; Ishibashi et al., 2000). In plants, recent developments in the rapidly growing TPC literature, TPCs emerged as the molecular correlate of the slow vac- + + let’s start by outlining a baseline consensus that investi- uolar (SV) current–a Ca2 -sensitive Ca2 release current + gators agree upon. In our opinion, there are three broad localized to the plant vacuole, in essence an acidic Ca2 areas of agreement. First, considerable evidence indicates store (Peiter et al., 2005; Peiter, 2011). Animal biolo- that NAADP evokes cytosolic Ca2+ signals by mobiliz- gists subsequently resolved an endolysosomal location for ing ‘acidic’ Ca2+ stores (Patel and Docampo, 2010). This TPCs in several studies published in 2009, again confirmed evidence builds from early work in the sea urchin egg recently (Wang et al., 2012). Third, several aspects of the demonstrating pharmacological and physical separation of molecular architecture of these channels also seem clear- 2+ 2+ cut namely: (i) TPCs are composed from glycosylated sub- Ca stores sensitive to NAADP from Ca stores within MESSENGER the ER (Lee and Aarhus, 1995; Genazzani and Galione, units that oligomerize (Zong et al., 2009; Hooper et al., 1996; Lee and Aarhus, 2000). The identity of these ‘acidic’ 2011; Rietdorf et al., 2011; Churamani et al., 2012). (ii) Ca2+ stores was revealed in 2002 to comprise lysosome- mutagenesis of the putative pore regions of TPCs results like organelles (yolk platelets) in the egg (Churchill et al., in loss of channel activity (Brailoiu et al., 2009; Wang 2002). This discovery paved the way for subsequent stud- et al., 2012; Brailoiu et al., 2010b; Schieder et al., 2010; ies focussing on endolysosomal Ca2+ stores within mam- Rybalchenko et al., 2012) and (iii) the N-terminus is crit- malian cell types, which showed that NAADP responses ical for faithful subcellular targeting of both animal and could be inhibited by interfering with acidic organelles plant TPCs (Wang et al., 2012; Brailoiu et al., 2010b; using agents such as GPN and bafilomycin (Galione et al., Yamaguchi et al., 2011; Larisch et al., 2012). Beyond these commonalities, experimental consensus 2010). These data encompassed several studies using pan- diverges. Counterbalancing the consensus derived from creatic beta cells and cell lines (Patel, 2003). The find- + several independent groups over the last four years that ing that NAADP-evokes Ca2 signals in a bafilomycin TPCs exhibit properties consistent with NAADP-activated Ca2+ release channels (see left hand side of Table I), ∗Authors to whom correspondence should be addressed. recent studies evidence a completely different viewpoint E-mails: [email protected], [email protected] namely that TPCs are PI(3,5)P2-activated, ATP-inhibited

MESSENGER 2013, Vol. 2, No. 2 2167-955X/2013/2/113/007 doi:10.1166/msr.2013.1027 113 Questioning Regulation of Two-Pore Channels by NAADP Marchant and Patel

Table I. Divergent views of TPCs: a comparison. Summary of contrasting data reporting the effects of TPC overexpression and inhibition on Ca2+ signalling and channel properties. Abbreviations: Hs, Homo sapiens; Sp Stronglylocentrotus purpuratus (sea urchin); Mm, Mus musculus (mouse). *Ba2+ used as charge carrier.

NAADP model PI(3,5)P2 model Ca2+signalling TPC over-expression Enhances NAADP-mediated Ca2+ release. Not tested HsTPC1 (Brailoiu et al., 2009; Calcraft et al., 2009; Yamaguchi et al., 2011; Ogunbayo et al., 2011; Dionisio et al., 2011; Pereira et al., 2011), HsTPC2 (Calcraft et al., 2009; Brailoiu et al., 2010b; Yamaguchi et al., 2011; Ogunbayo et al., 2011; Dionisio et al., 2011; Pereira et al., 2011; Brailoiu et al., 2010a), MmTPC2 (Zong et al., 2009), SpTPC1 (Ruas et al., 2010; Brailoiu et al., 2010a), SpTPC2 (Ruas et al., 2010; Brailoiu et al., 2010a), SpTPC3 (Brailoiu et al., 2010a) TPC inhibition + MESSENGER Knockout mice Inhibits NAADP-mediated Ca2 -dependent inward Not tested cation currents in pancreatic beta cells from TPC2 KO mouse (Calcraft et al., 2009) Not tested No effect on NAADP-mediated Ca2+ signals in pancreatic islets fromTPC1/2 DKO mouse (Wang et al., 2012) siRNA Inhibits NAADP-mediated Ca2+ release. SKBR3 Not tested cells (TPC1) (Brailoiu et al., 2009), HepG2 cells (TPC2) (Calcraft et al., 2009), MEG01 cells (TPC1 or 2) (Dionisio et al., 2011), T lymphocytes (TPC1 or 2) (Davis et al., 2012). Dominant negative TPC Inhibits NAADP-mediated Ca2+ release. SKBR3 Not tested constructs cells (TPC1 L273P) (Brailoiu et al., 2009), MESSENGER MESSENGER astrocytesIP: 192.168.39.210 (TPC2 L265P) On: (Pereira Fri, et01 al., Oct 2011) 2021 01:37:16 Channel properties Copyright: American Scientific Publishers Delivered by Ingenta TPC over-expression Channel activity stimulated by NAADP Channel activity insensitive to NAADP Regulation HsTPC1 (Rybalchenko et al., 2012), HsTPC1 (Wang et al., 2012), HsTPC2 (Brailoiu et al., 2010b; Yamaguchi HsTPC2 (Wang et al., 2012) et al., 2011; Pitt et al., 2010), MmTPC2 (Schieder et al., 2010).

Not tested Channel activity stimulated by PI(3,5)P2. HsTPC1, HsTPC2 (Wang et al., 2012) Not tested Channel activity inhibited by phosphorylation consequent to ATP activation of mTORC1 HsTPC1, HsTPC2 (Cang et al., 2013) MESSENGER Permeability Ca2+-permeable Weakly Ca2+-permeable HsTPC1 (Rybalchenko et al., 2012)*, HsTPC2 (Wang et al., 2012) HsTPC2 (Brailoiu et al., 2010b; Pitt et al., 2010), MmTPC2 (Schieder et al., 2010). Not tested Na+-permeable HsTPC2 (Wang et al., 2012; Cang et al., 2013) Pharmacology Inhibited by Ned-19 Partially Inhibited by Ned-19 HsTPC2 (Brailoiu et al., 2010b; Pitt et al., 2010) HsTPC2 (Wang et al., 2012) Not tested Inhibited by verapamil HsTPC2 (Wang et al., 2012)

TPC inhibition Not tested Inhibits endogenous PI(3,5)P2-mediated channel Knockout mice activity in macrophages (TPC1/2

MESSENGER DKO) (Wang et al., 2012) siRNA Inhibits endogenous NAADP-mediated channel Endogenous NAADP-mediated channel activity activity not detectable. HEK cells (TPC1) (Rybalchenko et al., 2012) COS-1, INS1, MIN6 cells (Wang et al., 2012) Dominant negative TPC Inhibits endogenous NAADP-mediated channel Endogenous NAADP-mediated channel constructs activity. activity not detectable COS-1, INS1, MIN6 HEK cells (TPC1 L273P) (Rybalchenko et al., cells (Wang et al., 2012) 2012)

114 MESSENGER 2, 113–119, 2013 MESSENGER MESSENGER Marchant and Patel Questioning Regulation of Two-Pore Channels by NAADP

Na+ channels, insensitive to NAADP (see right hand side confirming NAADP-mediated Ca2+ release from acidic of Table I). This new work clearly questions whether TPCs organelles in intact cells (Wang et al., 2012). An explana- are relevant either as targets for NAADP, or for the process tion that currents are too small to measure seems unlikely, of NAADP-evoked Ca2+ release from acidic Ca2+ stores. as endogenous NAADP currents have been described in In this context, reported actions of NAADP on the ER bilayer studies using vesicular preparations (Rybalchenko and PM, and candidate NAADP-sensitive channels distinct et al., 2012; Zhang and Li, 2007). Clearly, effort in defin- from TPCs (although in the minority) should not be over- ing recording conditions that maintain NAADP sensitivity looked (reviewed in (Guse, 2009; Patel, 2010)). But are the is warranted. divergent viewpoints as irreconcilable as they first appear? One key avenue for exploration is the recent proposal MESSENGER Let us consider in turn the implications for TPC activa- that NAADP may not bind TPCs directly but rather target tion (via unique ligands, NAADP vs. PI(3,5)P2 and TPC small molecular weight accessory proteins within a larger permeation (cation selectivity). TPC complex (Lin-Moshier et al., 2012; Walseth et al., 2012). This evidence was based on use of a novel photoaffinity labelling (PAL) probe which labelled proteins TPC ACTIVATION significantly smaller than the molecular weight of TPCs By direct patch clamp of vacuolin-1 enlarged endolyso- but with diagnostic NAADP pharmacology (Lin-Moshier somes, Wang et al.. demonstrate that TPCs mediate an et al., 2012; Walseth et al., 2012). These proteins per- endolysosomal conductance that is selectively activated sist in TPC transgenic mice (Lin-Moshier et al., 2012),

and the labelled targets co-immunoprecipitated with sea MESSENGER by PI(3,5)P2 (Wang et al., 2012). TPCs displayed com- plete insensitivity to NAADP in their hands, irrespective of urchin TPCs (Walseth et al., 2012). If NAADP does medi- whether activity was probed via this technically demand- ate its actions indirectly through an intermediate TPC ing method, or by more conventional whole cell/excised accessory protein then the presence of these proteins (the patch recording of a mutant TPC2 channel retargeted to the true ‘NAADP’ receptors) must be considered in evaluat- cell surface (Wang et al., 2012). These data clearly contrast ing NAADP sensitivity. The presence or absence of these with the results from other groups demonstrating NAADP proteins might then correlate with observable NAADP sensitivity using in some cases similar methods (for exam- sensitivity across experimental systems, or in the same ple, vacuolin-induced organellar swelling (Schieder et al., preparation studied by different techniques that may differ- IP: 192.168.39.210 On: Fri,entially 01 Oct preserve 2021 01:37:16 this interaction. In this context, the intra- 2010) or an identical mutant TPC2Copyright: construct (Brailoiu American Scientific Publishers et al., 2010b)). When considering these disparateDelivered obser- bycellular Ingenta whole organelle patch MESSENGER clamping method, where MESSENGER vations, the experimental capacity to record endogenous individual organelles are enlarged 10-fold in diameter NAADP responses coupled with the ability to interro- and perfused may not be optimal for retaining accessory gate such responses via loss of function approaches is protein interactions (and thereby NAADP-sensitivity), as paramount. However, unifying these experimental needs is noted by Cang et al. (2013). This line of investigation cer- not trivial for several technical reasons as we will now tainly has appeal as a unifying hypothesis for the broader discuss. NAADP literature (Marchant et al., 2012; Guse, 2012). The Ca2+-mobilizing ability of NAADP has proved For example, if these proteins display promiscuous asso- + most easily demonstrated in specific experimental prepa- ciation with a variety of intracellular Ca2 channels then 2+ rations. Unlike the ubiquitous efficacy of IP3 asaCa - reports of NAADP activation of unique channels could mobilizing messenger, many experimental systems exhibit be resolved. However this proposal remains in the realms an apparent insensitivity to NAADP. For example, reports of speculation until the putative NAADP-binding proteins MESSENGER of NAADP action in broken preparations outside of the are identified and their cellular biology resolved. A helpful “gold” standard sea urchin egg homogenate are limited, first step, would simply be to compare the abundance of clearly contrasting with the actions of IP3 which are NAADP-binding proteins in NAADP-responsive and unre- readily demonstrable upon cell disruption. Even in the sponsive preparations. robust sea urchin egg, NAADP-insensitive preparations Beyond the mercurial demonstration of endogenous are occasionally encountered (Patel et al., 2000). The NAADP sensitivity, it is worthwhile highlighting the con- restricted efficacy of NAADP to particular systems has trasting data obtained by different groups employing TPC no obvious molecular explanation at present, but is note- knockout mice. In the transgenic TPC2 mice derived worthy when negative data concerning NAADP activity by Calcraft et al. (2009) NAADP-stimulated responses is obtained. Is insensitivity of certain cells/preparations (Ca2+-dependent ionic currents at the plasma membrane) to NAADP due to the physiological absence of core were abolished in pancreatic beta cells whereas in the dou- NAADP-regulated machinery, or more of a technical issue ble TPC1/2 transgenic mice used by Wang et al. (2012), relating to loss of essential cofactors? It is thus perhaps NAADP responses (cytosolic Ca2+ elevations) persisted. significant that, Wang et al. could not detect endogenous These experimental outcomes are clearly difficult to rec- NAADP-mediated currents in broken preparations despite oncile. To generate these models, Calcraft et al. used the

MESSENGER 2, 113–119, 2013 115 Questioning Regulation of Two-Pore Channels by NAADP Marchant and Patel

Genetrap approach for inactivation, where the gene of considerably more uncertainty about the composition and interest is effectively replaced by a reporter under con- magnitude of ionic gradients across intracellular mem- trol of the endogenous promoter (Calcraft et al., 2009). branes and their dynamic range during cellular activ- The targeting strategy used by Wang et al. relied on dele- ity. Therefore, experimental data defining the permeation tion of only the first exon of TPC1 and TPC2 gene (Wang signature of organellar channels has consistently yielded et al., 2012). As acknowledged by the authors, this raises more variability. For example, electrophysiological anal- the possibility that N-terminally truncated proteins lacking yses of TRPML1 (mucolipin 1) variants (which are also the first 69 and 49 amino acids could be expressed (Wang expressed on acidic organelles) struggled to reach a con- et al., 2012). In this context, it is notable that deletion sensus, over the direction of current flow or cation selectiv- of the first 24 amino acids of human TPC2 (or point ity; at times Ca2+ or monovalent selective, a H+ channel, mutations of an endo-lysosomal targeting motif within or most recently a channel permeable to Ca2+,Na+,K+, 2+ 2+ the NH2 terminus) redirects TPC2 to the plasma mem- and Fe /Mn (Puertollano and Kiselyov, 2009; Cheng brane (Brailoiu et al., 2010b)–findings confirmed by Wang et al., 2010). Wang et al. demonstrate that TPCs are Na+- et al. (2012). Similarly, a recent study showed that deletion selective channels (Wang et al., 2012), rather than Ca2+ of the first 85 amino acids of human TPC1 re-directs it to channels as first implied (Brailoiu et al., 2010b; Schieder the ER (Churamani et al., 2013). In the case of re-directed et al., 2010; Rybalchenko et al., 2012; Pitt et al., 2010). TPC2, both channel activity and/or NAADP–evoked Ca2+ While the initial cohort of electrophysiological studies that MESSENGER influx were clearly resolvable in two independent stud- characterized NAADP-mediated TPC activity all resolved ies (Brailoiu et al., 2010b; Yamaguchi et al., 2011) despite permeability to Ca2+ (or the surrogate Ba2+ despite the rerouting of TPC2 to the cell surface. Thus, as argued by quite different preparations employed, none of these stud- Morgan and Galione (2013) it is possible that the trans- ies examined Na+ permeability (Brailoiu et al., 2010b; genic animals generated by Wang et al. might not be truly Schieder et al., 2010; Rybalchenko et al., 2012; Pitt “null” for TPCs but rather harbour potentially active chan- et al., 2010). Further investigation of TPC permeation is nels in non-lysosomal locations. Although currents were required. not detectable for recombinant TPCs corresponding to the But the key point here would seem to be whether truncated constructs, these measurements relied on vacuo- a limited permeability of TPCs to Ca2+ (Wang et al. lar preparations from which TPC channels may have been resolve a Ca2+/Na+ permeability ratio of ∼0.1) (Wang 2+ MESSENGERre-routed (Wang et al., 2012). MESSENGER AIP: more 192.168.39.210 expansive charac- On: Fri,et 01 al., Oct 2012) 2021 is 01:37:16 sufficient to evoke cytosolic Ca sig- terization of TPC expression and targetingCopyright: in the currently American Scientificnals. NAADP Publishers is thought to provide a small release of available transgenic models would be informativeDelivered. byCa Ingenta2+ which is subsequently amplified by neighbouring A final cautionary note regarding preservation of ER Ca2+ release channels through the process of Ca2+- NAADP sensitivity for recombinant proteins relates to the induced Ca2+-release (Patel et al., 2001; Guse and Lee,

use of N-terminally tagged TPC constructs. NH2-terminal 2008). In some cell types, such as the pancreatic acinar tagging of TPC1 has recently been shown to abolish sen- cell (Cancela et al., 1999), the putative “trigger” events are sitivity of human TPC1 to NAADP (Churamani et al., not resolvable following removal of the contributions of 2+ 2013). Indeed, previous studies using NH2-terminally ER Ca channels by pharmacological blockade. This sug- tagged mouse TPC1 also noted a lack of sensitivity to gests that these signals are small, transient and/or highly NAADP (Zong et al., 2009). Beyond the immediate impli- localized, features rendering them inaccessible to con- MESSENGER cations for TPC functional analyses (Wang et al., 2012), ventional light microscopy (spatial resolution ∼200 nm.

the broader implications of NH2 terminal structure to Clandestine channel “chatter” might then occur at spe- the mechanism of both trafficking and activation of TPCs cialized junctions between acidic organelles and the ER requires further study. through surreptitious Ca2+ fluxes supported by TPC activity or perhaps even protein-protein interactions with ER 2+ TPC PERMEATION Ca release channels (Patel, 2010; Patel and Brailoiu, 2012). Certainly, non-amplified NAADP-mediated Ca2+ Electrophysiological studies of cell surface ion channels signals can be imaged upon overexpression of TPC2 re- have provided exquisite biophysical insight into the gat- routed to the PM (Brailoiu et al., 2010b), evidencing ing and permeation of numerous ionotropic families. The the existence of Ca2+ fluxes likely masked within the

MESSENGER presentation of channels at the cell surface facilitates specialized morphology of interorganellar membrane con- experimental accessibility and the different ionic environ- tact sites. The morphological correlates of ER-lysosomal ments segregated by the plasma membrane are clearly junctions (<20 nm) have recently been identified at the defined. In contrast, analysis of intracellular ion channels electron microscopic level in human fibroblasts with evi- holds more challenges. Recording channel activity within dence for inter-organelle tethers akin to those between the native lipid composition of organellar environments, the ER and PM/mitochondria (Kilpatrick et al., 2013). likely critical for faithfully reproducing endogenous chan- Light microscopy also provides evidence for close con- nel behaviour is much more difficult. Further there remains tacts between the ER and lysosomes in COS-7 cells and

116 MESSENGER 2, 113–119, 2013 MESSENGER MESSENGER Marchant and Patel Questioning Regulation of Two-Pore Channels by NAADP sea urchin eggs (Lopez-Sanjurjo et al., 2013; Morgan, ligand and permeability clearly differ, future work sug- 2013). Indeed, in the former case, live cell TIRF imag- gested here may prompt convergence of these opinions ing of organelle mobility showed that lysosomes and the when viewed in an appropriate experimental and physi- ER are also likely physically coupled (Lopez-Sanjurjo ological context. Indeed, it is intriguing that Ned-19 and et al., 2013). These restricted spaces might serve as a plat- verapamil, both antagonists of NAADP action (Genazzani form for translation of modest changes in cytosolic Ca2+ et al., 1996; Naylor et al., 2009), also partially antagonize originating from acidic organelles to larger ones depen- PI(3,5)P2-stimulated TPC currents (Wang et al., 2012). dent on the ER. Flux of Ca2+ ions through NAADP- TPCs may regulate endolysosomal cation homeostasis via regulated channels in a cellular setting therefore need not integration of multiple cytoplasmic cues. MESSENGER be large to be physiologically impactful. Further definition and manipulation of lysosome-ER contacts is important to Acknowledgments: We thank Anthony Morgan and interpret the significance of biophysical permeation sig- Shmuel Muallem for helpful comments. Work in the natures of TPC proteins recorded in vitro with respect to authors laboratories are supported by grants from the NAADP action in a physiological context. NIH (JSM), NSF (JSM), BBSRC (SP) and Parkinson’s Final points relate to the cell biological significance UK (SP). of TPC permeability in the context of the ionic composition of the lysosomal lumen. Trafficking events within REFERENCES the endolysosomal system are regulated by local Ca2+

fluxes (Pryor et al., 2000; Luzio et al., 2007) as elegantly Brailoiu, E., Churamani, D., Cai, X., Schrlau, M. G., Brailoiu, G. C., Gao, MESSENGER demonstrated by the authors of the recent TPC studies in X., Hooper, R., Boulware, M. J., Dun, N. J., Marchant, J. S., and Patel, S. (2009). Essential requirement for two-pore channel 1 in NAADP- independent work (Shen et al., 2012). TPC activity also mediated calcium signaling. J. Cell Biol. 186, 201–209. has been shown to perturb endomembrane dynamics (Ruas Brailoiu, E., Hooper, R., Cai, X., Brailoiu, G. C., Keebler, M. V., Dun, et al., 2010), but these phenomena are now suggested to N. J., Marchant, J. S., and Patel, S. (2010a). An ancestral deuteros- + be supported by TPC-dependent Na fluxes (Wang et al., tome family of two-pore channels mediate nicotinic acid adenine dinu- 2012). This new insight, coupled with experimental revi- cleotide phosphate-dependent calcium release from acidic organelles. J. sion of the ionic composition of the lysosomal lumen indi- Biol. Chem. 285, 2897–2901. cating that Na+ (∼140–150 mM) not K+ (Morgan, 2011) Brailoiu, E., Rahman, T., Churamani, D., Prole, D. L., Brailoiu, G. C., as the predominant cation necessitatesIP: 192.168.39.210 proposition of On: a Fri,Hooper, 01 Oct R., 2021 Taylor, 01:37:16 C. W., and Patel, S. (2010b). An NAADP-gated two- + Copyright: American Scientificpore channel Publishers targeted to the plasma membrane uncouples triggering from + novel mechanism for how local Na currents impact mem- MESSENGER MESSENGER Delivered byamplifying Ingenta Ca2 signals. J. Biol. Chem. 285, 38511–38516. brane fusion. It is suggested that depolarization due to + Calcraft, P. J., Ruas, M., Pan, Z., Cheng, X., Arredouani, A., Hao, X., Na fluxes as a result of local production of PI(3,5)P2 Tang, J., Rietdorf, K., Teboul, L., Chuang, K. T., Lin, P., Xiao, R., Wang, production facilitates fusion through electrostatic inter- C., Zhu, Y., Lin, Y., Wyatt, C. N., Parrington, J., Ma, J., Evans, A. M., actions (Wang et al., 2012). This is an attractive idea, Galione, A., and Zhu, M. X. (2009). NAADP mobilizes calcium from but whether such a mechanism is physiologically relevant acidic organelles through two-pore channels. Nature 459, 596–600. remains to be established particularly given parallel evi- Cancela, J. M., Churchill, G. C., and Galione, A. (1999). Coordination of + dence by the authors that TPCs are tonically inhibited by agonist-induced Ca2 -signalling patterns by NAADP in pancreatic acinar physiological ATP concentrations (Cang et al., 2013). Fur- cells. Nature 398, 74–76. ther work is required in dissecting the role of specific ion Cang, C., Zhou, Y., Navarro, B., Seo, Y. J., Aranda, K., Shi, L., Battaglia- fluxes through TPCs in regulating fusion events within the Hsu, S., Nissim, I., Clapham, D. E., and Ren, D. (2013). mTOR regulates lysosomal ATP-sensitive two-pore Na(+) channels to adapt to metabolic MESSENGER endolysosomal system. state. Cell 152, 778–790. Cheng, X., Shen, D., Samie, M., and Xu, H. (2010). Mucolipins: Intra- CONCLUSION cellular TRPML1-3 channels. FEBS Lett. 584, 2013–2021. Churamani, D., Hooper, R., Brailoiu, E., and Patel, S. (2012). Domain In light of the recent work, the field has transitioned assembly of NAADP-gated two-pore channels. Biochem. J. 441, 317– to distinct routes for TPC regulation (NAADP activation 323. versus PI(3,5)P2 activation/ATP inhibition). None of these Churamani, D., Hooper, R., Rahman, T., Brailoiu, E., and Patel, S. mechanisms are currently associated with a defined bind- (2013). The N-terminal region of two-pore channel 1 regulates trafficking ing/regulatory site on the TPC complex. For understanding and activation by NAADP. Biochem. J. 453, 147–151. NAADP efficacy, a key solution may come from identifi- Churchill, G. C., Okada, Y., Thomas, J. M., Genazzani, A. A., Patel, S., + cation of the recently proposed NAADP receptor distinct and Galione, A. (2002). NAADP mobilizes Ca2 from reserve granules, Cell from the TPCs themselves, and this is now arguably the lysosome-related organelles, in sea urchin eggs. 111, 703–708. key molecular unknown in the broader field of Ca2+ sig- Davis, L. C., Morgan, A. J., Chen, J. L., Snead, C. M., Bloor-Young, D., Shenderov, E., Stanton-Humphreys, M. N., Conway, S. J., Churchill, nalling. Equally, there is onus to map sites for PI(3,5)P2 G. C., Parrington, J., Cerundolo, V., and Galione, A. (2012). NAADP binding and regulatory phosphorylation. Although current activates two-pore channels on T cell cytolytic granules to stimulate exo- perspectives of the TPCs with respect to their activating cytosis and killing. Curr. Biol. 22, 2331–2337.

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Received: 29 May 2013. Accepted: 5 June 2013. MESSENGER

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