Pflugers Arch - Eur J Physiol (2009) 458:869–876 DOI 10.1007/s00424-009-0682-y

ION CHANNELS, RECEPTORS AND TRANSPORTERS

The acid test: the discovery of two-pore channels (TPCs) as NAADP-gated endolysosomal Ca2+ release channels

Antony Galione & A. Mark Evans & Jianjie Ma & John Parrington & Abdelilah Arredouani & Xiaotong Cheng & Michael X. Zhu

Received: 7 May 2009 /Revised: 9 May 2009 /Accepted: 12 May 2009 /Published online: 28 May 2009 # Springer-Verlag 2009

Abstract In this review, we describe the background and NAADP as an intracellular Ca2+ signalling messenger implications of our recent discovery that two-pore channels (TPCs) comprise a novel class of calcium release channels NAADP is the most potent Ca2+ releasing molecule gated by the intracellular messenger nicotinic acid adenine described, discharging Ca2+ from Ca2+ storage organelles dinucleotide phosphate (NAADP). Their localisation to the to elicit cytoplasmic Ca2+ signals at picomolar to low endolysosomal system highlights a new function for these nanomolar concentrations in sea urchin egg preparations organelles as targets for NAADP-mediated Ca2+ mobilisation. [34]. In some mammalian cells, Ca2+ responses are evoked In addition, we describe how TPCs may also trigger further by as low as 10 nM NAADP [4]. Measurement of cell and Ca2+ release by coupling to the endoplasmic reticular stores tissue levels have shown that NAADP is synthesised in through activation of IP3 receptors and ryanodine receptors. response to activation of various cell surface receptors and other stimuli, confirming NAADP’s role as an intracellular Keywords Inositol 1,4,5-trisphosphate . Pancreatic β-cell . messenger [18, 53]. In contrast to the other established Ca2+ 2+ 2+ Endothelin . Cholecystokinin . Ca -induced Ca release . mobilising messengers, inositol 1,4,5-trisphosphate (IP3) . Ca2+ mobilisation . Ca2+ stores . and cyclic ADP ribose (cADPR), that release Ca2+ from the

Calcium signalling . Calcium transient endoplasmic reticulum (ER) by activating IP3 receptors (IP3Rs) and ryanodine receptors (RyRs), respectively, * : : : NAADP is enigmatic since in many cells it appears to act A. Galione ( ) J. Parrington A. Arredouani X. Cheng 2+ Department of , , via a distinct Ca release mechanism, which resides not on Mansfield Road, the ER but on acidic organelles [14]. Oxford OX1 3QT, UK NAADP was discovered as a trace contaminant of e-mail: [email protected] commercially available NADP [35]. This may not be too A. M. Evans surprising since the structure of NAADP is very close to that Centre for Integrative Physiology, of NADP, differing only by the substitution of nicotinic acid College of Medicine and Veterinary Medicine, as the base instead of nicotinamide. This small change, University of Edinburgh, however, has enormous biological effects since whilst NADP Edinburgh, Scotland, UK is inactive, NAADP is very potent at generating Ca2+ J. Ma signals. This discovery was made by Lee and colleagues Department of Physiology and Biophysics, whilst studying calcium release mechanisms in homogenates – UMDNJ Robert Wood Johnson Medical School, prepared from sea urchin eggs [15]. This preparation has Piscataway, NJ, USA done much to advance our understanding of novel Ca2+ M. X. Zhu (*) signalling mechanisms including the first demonstration that Department of Neuroscience, cADPR mobilises Ca2+ through the activation of RyRs [20, Biochemistry and Center for Molecular Neurobiology, 44], and much of the ground work in studying the The Ohio State University, Columbus, OH, USA mechanisms of NAADP action has been gleaned from this e-mail: [email protected] system before being widened to mammalian preparations. 870 Pflugers Arch - Eur J Physiol (2009) 458:869–876

NAADP targets acidic organelles Given the smaller size, and perhaps the lower Ca2+ content, Ca2+ release from acidic stores is often smaller The initial report on the Ca2+ mobilising action of the then than that evoked from the larger reticular ER network. unidentified NADP-related molecule implicated a different However, even the first report of NAADP-evoked Ca2+ organellar target to the ER since it released Ca2+ from a release in a mammalian cell type proposed that NAADP- partially separate subcellular fraction of sea urchin egg induced Ca2+ signals also trigger a subsequent, larger Ca2+ homogenate [15]. Later, NAADP-evoked Ca2+ release was release from the ER through the Ca2+-induced Ca2+ release 2+ found to persist in homogenates and intact eggs even when (CICR) mechanisms of IP3Rs and RyRs [9]. That Ca the ER Ca2+ pool had been abrogated by the SERCA pump release in response to NAADP in turn triggers CICR inhibitor, thapsigargin. Another feature of sea urchin eggs is through both IP3Rs and RyRs has been extensively studied that, when centrifuged, they stratify; that is, they change in sea urchin eggs [11, 12] and demonstrated in a wide from spherical to a more oblong shape with different range of mammalian systems [2, 4, 7, 9, 22, 39]. Indeed, organelles migrating to different poles. Under these tight junctions, likely less than 100 nm across, between the 2+ conditions, IP3 and cADPR mobilise Ca from the nuclear RyR-containing sarcoplasmic reticulum and a subpopula- pole where the ER is, but NAADP releases Ca2+ predom- tion of lysosomes have been observed in vascular smooth inantly from the opposite pole [36]. Taken together, these muscle, and these may constitute a “trigger zone” for the studies favoured a target for NAADP in organelles distinct initiation of propagating Ca2+ signals by NAADP [32, 33]. from the ER, the traditional site of action of Ca2+ Thus, bafilomycin completely abolishes NAADP responses mobilising messengers. whilst thapsigargin and IP3R and RyR blockage may only A major new lead in the physiology of NAADP came reduce the amplitude of the responses [7, 22, 32, 39, 41, from the finding that in sea urchin eggs the major target for 52]. NAADP thus not only has a role in regulating NAADP was acidic organelles such as reserve granules, the endolysosomal luminal Ca2+ content and cytosolic Ca2+ urchin egg’s equivalent of lysosomes [14]. Stratification signals arising from these organelles, but also a second role studies revealed that acidic stores stained with lysotracker in coordinating ER-mediated Ca2+ release. Such interac- localised to the non-ER pole exactly where NAADP was tions have often confounded experiments designed to found to act as described above. Further fractionation of sea elucidate the identity of the NAADP receptor, with some urchin egg homogenates showed that binding sites for [32P] proposing that RyRs are a direct target [16]. As we have NAADP co-localised with lysosomal markers. The isolated stated previously, the identification of NAADP binding NAADP-sensitive stores showed ATP-dependent Ca2+ are required to resolve such controversies [21]. uptake that was insensitive to thapsigargin, but abrogated by protonophores and bafilomycin A1, a vacuolar proton pump inhibitor, suggesting a role for a proton-dependent Distinct pharmacology and properties 2+ 2+ Ca uptake mechanism. The lysosomotropic agent glycyl-L- of the NAADP-regulated Ca release mechanism phenylalanine 2-naphthylamide (GPN) selectively abolished NAADP-induced Ca2+ release in both homogenates and The initial descriptions of NAADP-evoked Ca2+ release intact eggs whilst leaving IP3- and cADPR-sensitive mech- from sea urchin egg homogenates showed that NAADP anisms unaffected. likely activated a novel Ca2+ release mechanism distinct 2+ NAADP-evoked Ca release has now been shown to be from IP3Rs and RyRs. This was based on the individual widespread in mammalian cells, and even in plants [21]. In ability of IP3, cADPR and NAADP to induce homologous most cases where investigated, including neurones, cardiac desensitisation of their target mechanisms without affecting myocytes, smooth muscle, exocrine cells, and platelets, the responsiveness of the other two and regardless of the 2+ NAADP also appears to release Ca from acidic stores order of the additions of each messenger [35]. Log10 rather than from the ER. The study of NAADP thus has concentration–response curves for NAADP-induced Ca2+ highlighted acidic stores as important Ca2+ storage organ- release are quite unlike the familiar sigmoid curves seen for elles that can be discharged by their own intracellular either IP3 or cADPR. In sea urchin eggs, subthreshold messenger. The role of Ca2+ storage in the endolysosomal concentrations of NAADP for Ca2+ release can nevertheless system may be pivotal to endomembrane fusion and fully desensitise NAADP receptors [24], whilst in mam- trafficking and for other physiological functions of these malian cells the concentration–response curves are bell organelles [47, 48]. Recently, it has been proposed that shaped, with micromolar concentrations of NAADP induc- reduced lysosomal Ca2+ storage is a primary defect in ing desensitisation in the absence of apparent Ca2+ release. Niemann–Pick type C1 disease, a lysosomal storage This latter phenomenon has been exploited to identify disease, and this is associated with abolition of NAADP- receptor mechanisms coupled to NAADP-mediated Ca2+ induced Ca2+ release [37]. signalling [9, 18]. Pflugers Arch - Eur J Physiol (2009) 458:869–876 871

In another approach, pharmacological antagonism of the myocytes [55]. However, other experimental data suggest

IP3Rs by heparin or RyRs by ryanodine, or the selective that TRPML1 functions either as a proton channel or an iron cADPR antagonist analogue 8-NH2-cADPR, did not affect release channel at the lysosomes [17, 50]. the ability of NAADP to evoke a Ca2+ release response In a recent study, we demonstrated that two-pore 2+ [19]. Whilst IP3Rs and RyRs are also Ca -activated channels (TPCs or TPCNs for names) function as channels and can propagate Ca2+ release as regenerative NAADP receptors [8]. Based on primary sequences, TPCs waves through CICR, this property is apparently not shared are novel members of the superfamily of voltage-gated by the intrinsic NAADP-sensitive Ca2+ release mechanism cation channels. Hydropathy analyses revealed that the full- either in egg homogenates [10, 23] or in intact cells [11]. length contains 12 putative transmembrane (TM) As outlined above, rather NAADP evokes local Ca2+ segments that are segregated into two homologous domains signals that may only be propagated and globalised by of six TM segments each with an intervening hydrophobic subsequent recruitment of IP3Rs and RyRs. In addition, loop between the fifth and the sixth segments (Fig. 1). This NAADP-induced Ca2+ release is markedly less pH sensitive 6-TM organisation resembles that of all 6-TM voltage- than IP3Rs and RyRs [11] allowing the messenger to gated cation channel family members including the four efficiently act at acidic stores, where large pH variation is repeated domains found in the pore-forming subunits of inevitable because protons are co-released with Ca2+ [43]. voltage-gated Ca2+ and Na+ channels [54]. The sequence Some pharmacological agents have been reported to homology of the TPC TM domains is also closer to selectively block NAADP-induced Ca2+ release without voltage-gated Ca2+ and Na+ channels than to K+ channels. affecting either IP3Rs or RyRs. These include voltage-gated In fact, among the single domain 6-TM channels, CatSpers (VGCC) blockers such as dihydropyri- and some TRP channels are more closely related to TPCs dines [25] and the store-operated channel and TRPC than Kv channels are, but nevertheless their homologies to channel blocker SKF96365 [42]. Recently, a potent and TPCs are slightly lower than that of Cav and Nav channels. selective membrane permeant NAADP antagonist, NED19, This has led to the hypothesis that TPCs may represent has been described, which will be invaluable in helping evolutional intermediates of the two round duplications of dissect the contribution of NAADP during Ca2+ signalling single 6-TM domain channels that gave rise to the four 6-TM

[45]. Taken together, these data support the hypothesis that domain Cav and Nav channels [1]. The fact that related TPCs NAADP activates a unique Ca2+ release mechanism are widely expressed in plants suggests that this is an ancient involving channels that are perhaps more similar to VGCC ion channel family that had been in existence before the or TRP families than the two established ER Ca2+ release divergence of plants and animals. On the other hand, TPCN channel families, the IP3Rs and RyRs. are completely missing in some model animal species, such as flies and nematodes, indicating that these channels may not be essential for survival. With the report of the first mammalian TPC (TPC1) Identification of TPCs as NAADP receptors sequence some years ago [28], so was the name given but this should not be confused with two-pore potassium The search for the molecular identity of NAADP receptors channels, which have two repeats of a 2-TM architecture. had been a bumpy ride to say the least. Initially, multi-step However, functional data had been completely lacking, at fractionations of sea urchin egg homogenates were attempted least in animal cells, for this unique group of ion channels and a protein target that bound to [32P]NAADP, but was that apparently have an important position in the evolution substantially smaller than IP3Rs and RyRs, was demonstrat- of voltage-gated cation channels. By contrast, active ed [3]. However, the NAADP binding proteins were not research on plant cells revealed that plant TPC1, which is identified at the molecular level. Since the demonstration that not equivalent to animal TPC1 but rather an equally distant NAADP elicited Ca2+ release from lysosome-related acidic homologue of all animal TPCs, forms a Ca2+ release stores, lysosomal localised cation channels have been channel in intracellular vacuoles [46]. Indeed, even before considered as candidates of NAADP receptors. For example, the work on the plant TPC1 was reported, we had noticed mucolipin 1, or TRPML1, was tested and failed to show that human TPC1 and TPC2 expressed in HEK293 cells [32P]NAADP binding or an NAADP-evoked Ca2+ response were not expressed on the plasma membrane but rather on in heterologous systems [49]. Despite these findings, another membranes of endosomes and lysosomes. Given that group showed that a TRPML1 antibody blocked NAADP- NAADP acts at acidic endolysosomal stores [14] and its evoked currents in planar lipid bilayers incorporated with action is inhibited by Ca2+ channel antagonists [25], the lysosomal membranes prepared from rat liver and coronary specific subcellular locations and the homology to Cav and arterial myocytes [55, 56] and siRNA silencing of TRPML1 TRP channels prompted us to examine whether TPCs form expression attenuated lysosome-associated Ca2+ release in NAADP receptors. 872 Pflugers Arch - Eur J Physiol (2009) 458:869–876

Four pore a Two pore b Domain I Domain II

Lysosome lumen P loop P loop

.2 3 .1 1 . 1

. 1 1 3 v v v 2 a a 3 v a TPC N C C a C TP C2 C C N TP S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6 G CNG B3 er3 B1 CatSp NH2 COOH C er2 NG CatSp A4 cytoplasmic CNG atSper1 A1 C 4 H CatSper CN1 c Lysosome lumen TRPP2 Kv10

Kv12 TRPML1

Kv11

TRPM8 TRP Kv7 M4 T 6 RPM Kv T 6 RP Kv9 M 8 1 K v TR cytoplasmic 5 T P K v R C 2 T P 1 K v R C 4 T P 4 K v 3 R C v 1 P NAADP K v T 2 K T TR C R TR R 3 P P P P V Single pore A V V 1 1 6 4

Fig. 1 Two-pore channels: relationships with other voltage-gated subunit contains 12 putative TM segments that can be divided into channels and structure organisation. a A phylogenetic tree generated two 6-TM homologous domains, each containing also a pore loop (P by ClustalW (http://align.genome.jp) and plotted using Neighbour- loop). Based on orientation of voltage-gated channels, the amino and Joining algorithm for TPCs and selected members of the superfamily carboxyl termini, as well as the large loop in between the two of voltage-gated channels. The second transmembrane (TM) domain domains, are on the cytoplasmic side of the endolysosomal mem- of TPCs and the fourth TM domain of Cav and Nav were aligned with branes. Red branches indicate a putative glycosylation site in human the TM domains (including all six TM segments and the pore loop) of TPC2. c 3-D projection model of an assembled TPC channel, which is representative Kv, CNG, TRP and CatSper channels. b A single TPC most likely a dimer

Because of its prominent localisation on lysosomal through CICR, resulting, originally, from the NAADP- membranes, we focused initially on TPC2 and obtained induced Ca2+ release from acidic stores. This biphasic several lines of evidence that TPC2 is involved in NAADP phenomenon and the dependence of the secondary phase on binding and NAADP-mediated Ca2+ release from acidic the ER stores are entirely consistent with NAADP-elicited stores [8]. First, NAADP binds to membranes from cells Ca2+ responses in native cells as described above. Interest- over-expressing TPC2 with a high affinity of 5–10 nM. ingly, the wild-type HEK293 cells lacked the biphasic Second, NAADP, at concentrations as low as 10 nM, response and exhibited only small and short-lived Ca2+ elicited intracellular Ca2+ concentration increases in transients upon stimulation by higher concentrations of HEK293 cells that overexpress TPC2. Interestingly, the NAADP, suggesting that the relatively low endogenous rise in intracellular Ca2+ exhibited two phases, an initial level of TPC expression in this cell type does not favour pacemaker phase with low to moderate Ca2+ transients akin globalisation of the NAADP-evoked Ca2+ signal and/or that to a “Ca2+ burst” [4], followed by a secondary rapid and lysosomes form “loose junctions” with the ER in HEK293 much larger Ca2+ increase. The initial phase represents cells and thereby fail to adequately mediate, via endoge- lysosomal Ca2+ release evoked by NAADP, as it was nous TPC2, sufficient Ca2+ release to breach the threshold 2+ unaffected by blocking ER Ca release with thapsigargin, for CICR via IP3Rs. Third, shRNA specifically targeted heparin or ryanodine, or all of these together, but against TPC2 abolished NAADP-evoked Ca2+ signals in completely abolished after treating cells with bafilomycin. HEK293 cells over-expressing TPC2 and the native The secondary phase, however, was eliminated by thapsi- NAADP response in a human hepatoblastoma cell line. gargin or heparin, or both of these together with ryanodine, Finally, NAADP-evoked Ca2+ signals activated non- and therefore represents globalisation of the Ca2+ signal selective cation currents in pancreatic β cells prepared Pflugers Arch - Eur J Physiol (2009) 458:869–876 873 from wild-type mice, but no such current was detected in Physiology of TPC-mediated calcium signalling cells prepared from Tpc2 knockout mice. Taken together, these data strongly support a role for TPC2 in NAADP- There is still much to learn about TPC-mediated Ca2+ evoked Ca2+ release in mammalian cells. signalling. The NAADP-induced Ca2+ responses in sea We further noticed that, although overexpression of urchin and starfish eggs are pivotal events at fertilisation [13, TPC1 did not result in globalisation of the Ca2+ signal in 42]. In mammalian systems, this ligand appears to play key response to 10 nM NAADP, the localised Ca2+ transients roles in digestive enzyme and fluid secretion of pancreatic did become more pronounced at this low ligand concentra- acinar cells [52], glucose-induced insulin secretion in β cells tion than in wild-type cells. This, perhaps, reflects the more [40], smooth muscle contraction in the vasculature [4, 32], T discrete intracellular distribution of TPC1 than TPC2, the lymphocyte activation [2], neurotransmitter release and lower Ca2+ content in the TPC1-expressing endosomes than neurite outgrowth [5, 6], and platelet activation [38]. In lysosomes and/or the fact that endosomal TPC1 does not some cases, NAADP evokes specific Ca2+ responses distinct form true junctions with the ER in HEK293 cells and from either IP3 or cADPR such as the cortical flash at marine therefore fails to elicit CICR via IP3Rs. Nevertheless, this invertebrate egg fertilisation [13, 42], and differentiation of finding does suggest that TPC1 may also act as an PC12 cells [5]. In addition, NAADP modulates luminal pH NAADP receptor. Therefore, the TPCs represent a family of its target organelles suggesting another distinct signalling of endolysosomal cation channels targeted by NAADP for event evoked by this messenger [43]. Notwithstanding, the regulated Ca2+ release from acidic stores. Ca2+ release study of NAADP signalling had been greatly hindered by the from this dynamic and mobile source contributes to the lack of knowledge about its molecular target(s) and lack of diversity and versatility of Ca2+ signalling not only specific blockers. Now two recent reports have revealed not because of its unique origin but also because of its ability only a potent and specific NAADP antagonist [45], but also to couple to the ER Ca2+ release under certain but not all the molecular identity of the NAADP receptor [8]. It is conditions (Fig. 2). expected that these findings will greatly accelerate functional

Fig. 2 NAADP regulation of TPCs. Model outlining how NAADP- stores produces a local Ca2+ signal that activates plasma membrane regulated TPCs expressed at acidic Ca2+ stores might modulate the channels [7–9]. In turn, this may drive changes in the plasma membrane activity of closely apposed Ca2+-sensitive channels in a the plasma potential. b A two-pool model of Ca2+ spiking where a highly localised membrane or b endoplasmic reticulum via local Ca2+ (represented by trigger Ca2+ released via TPCs recruits CICR channels on the ER [8]. the green fill). a Ca2+-sensitive ion channels in the plasma membrane Abbreviations: CHX,Ca2+/H+ exchanger; SERCA, sarco(endo)plasmic 2+ are arbitrarily depicted with six transmembrane domains and might be reticulum Ca -ATPase; IP3R,IP3 receptor; ER, endoplasmic reticulum; cation- or anion-selective, inward or outward currents (double-headed CICR,Ca2+-induced Ca2+ release; X+, cations; Y−, anions; ΔΨ, arrow). NAADP-evoked Ca2+ release from sub-plasmalemmal acidic membrane potential. Drawn by Dr. Anthony Morgan 874 Pflugers Arch - Eur J Physiol (2009) 458:869–876 characterisation of NAADP-induced Ca2+ signalling in many NAADP-regulated Ca2+ release channels from acidic different cell types and biological systems (Fig. 2). organelles, we cannot rule out the possibility that other Human genetic studies have revealed some physiological proteins tightly associate with these channels and modulate and pathophysiological functions that may be attributed to their activities. It may also be too early to rule out the direct TPC2. The polymorphic variants of the TPCN2 gene at two involvement of other candidate channels, e.g. RyRs and locations are associated with hair colour in Northern TRPML1, in NAADP-evoked Ca2+ mobilisation because Europeans [51], indicative of an important role for this this will require additional experimentation under specific channel in pigmentation. This would be consistent with the conditions in appropriate cell types. lysosomal function of TPC2 in melanosomes, lysosomal- related organelles of the melanocytes, which produce and Acknowledgments The authors thank American Association, release melanin to keratinocytes for pigmentation. The British Heart Foundation, US National Institutes of Health, and the TPCN2 locus is also within the chromosomal region that is for financial support of their research. linked to autosomal recessive nonsyndromic hearing im- pairment in at least five families [29, 31], and for one of them, the TPCN2 gene has been sequenced to show five References exonic and four intronic variants [29]. Given that progres- sive hearing loss has been linked to several lysosomal 1. Anderson PA, Greenberg RM (2001) Phylogeny of ion channels: storage diseases [30], it is possible that dysfunction of the clues to structure and function. 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