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) Ion channel . 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 Pharmacology, University of Oxford, 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+ proteins 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 gene 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].