Targeting Two-Pore Channels: Current Progress and Future Challenges
Authors: Xuhui Jin, Yuxuan Zhang, Abeer Alharbi, Ali Hanbashi, Ali Alhoshani, John Parrington
Journal: Trends in Pharmacological Sciences (2020)

Two-pore channel (TPC)-mediated endolysosomal Ca2+ signalling regulates a variety of processes, including cell proliferation, differentiation, metabolism, viral infection, and cardiac function.Despite the well-established model that TPCs are Ca2+-selective channels indirectly activated by nicotinic acid adenine dinucleotide phosphate (NAADP), it has also been proposed that TPCs as Na+ channels are activated directly by phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2].3D structures of mouse TPC1 and human TPC2 were recently determined, which made it possible for structure-based virtual screening methods to identify pharmacological modulators of TPC.Recent identification by high-throughput screens of pharmacological modulators that target TPCs will help reveal the molecular mechanisms underlying the role of endolysosomal Ca2+ signalling in different pathophysiological processes, and to develop new therapeutics. Two-pore channels (TPCs) are cation-permeable channels located on endolysosomal membranes and important mediators of intracellular Ca2+ signalling. TPCs are involved in various pathophysiological processes, including cell growth and development, metabolism, and cancer progression. Most studies of TPCs have used TPC–/– cell or whole-animal models, or Ned-19, an indirect inhibitor. The TPC activation mechanism remains controversial, which has made it difficult to develop selective modulators. Recent studies of TPC structure and their interactomes are aiding the development of direct pharmacological modulators. This process is still in its infancy, but will facilitate future research and TPC targeting for therapeutical purposes. Here, we review the progress of current research into TPCs, including recent insights into their structures, functional roles, mechanisms of activation, and pharmacological modulators. Two-pore channels (TPCs) are cation-permeable channels located on endolysosomal membranes and important mediators of intracellular Ca2+ signalling. TPCs are involved in various pathophysiological processes, including cell growth and development, metabolism, and cancer progression. Most studies of TPCs have used TPC–/– cell or whole-animal models, or Ned-19, an indirect inhibitor. The TPC activation mechanism remains controversial, which has made it difficult to develop selective modulators. Recent studies of TPC structure and their interactomes are aiding the development of direct pharmacological modulators. This process is still in its infancy, but will facilitate future research and TPC targeting for therapeutical purposes. Here, we review the progress of current research into TPCs, including recent insights into their structures, functional roles, mechanisms of activation, and pharmacological modulators. Changes in intracellular calcium (Ca2+) constitutes a key signalling mechanism that mediates diverse cellular functions [1.Parrington J. Tunn R. Ca 2+ signals, NAADP and two-pore channels: role in cellular differentiation.Acta Physiol. 2014; 211: 285-296Crossref PubMed Scopus (21) Google Scholar]. One source of cytoplasmic Ca2+ is extracellular Ca2+, the entry of which into the cell is regulated by voltage-operated calcium channels (VOCCs), receptor-operated calcium channels (ROCCs), and storage-operated calcium entry (SOCE) (Figure 1) [2.Berridge M.J. et al.Calcium signalling: dynamics, homeostasis and remodelling.Nat. Rev. Mol. Cell Biol. 2003; 4: 517-529Crossref PubMed Scopus (4450) Google Scholar]. Another source is internal stores, which release Ca2+ when their receptors are activated by Ca2+-mobilising messengers, such as inositol 1,4,5-trisphosphate (IP3), cyclic adenosine diphosphate ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP). IP3 and cADPR activate IP3 receptors (IP3Rs) and ryanodine receptors (RyRs), respectively, both of which regulate Ca2+ release from the endo/sarcoplasmic reticulum (ER/SR); NAADP targets TPCs to mediate Ca2+ release from acidic endolysosomal organelles (Figure 1) [3.Lee H.C. Aarhus R. A derivative of NADP mobilizes calcium stores insensitive to inositol trisphosphate and cyclic ADP-ribose.J. Biol. Chem. 1995; 270: 2152-2157Crossref PubMed Scopus (414) Google Scholar,4.Calcraft P.J. et al.NAADP mobilizes calcium from acidic organelles through two-pore channels.Nature. 2009; 459: 596-600Crossref PubMed Scopus (626) Google Scholar]. NAADP is the most potent Ca2+-mobilising messenger, exerting its action even at low nanomolar concentrations [5.Galione A. NAADP receptors.Cold Spring Harb. Perspect. Biol. 2019; 11: a035071Crossref Scopus (42) Google Scholar]. NAADP-evoked Ca2+ signalling can further trigger Ca2+ release from the ER/SR, via Ca2+-induced Ca2+ release (CICR) [6.Davis L.C. et al.NAADP activates two-pore channels on T cell cytolytic granules to stimulate exocytosis and killing.Curr. Biol. 2012; 22: 2331-2337Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar]. TPCs have gained increasing attention as integral components of the NAADP-regulated Ca2+ channel system, although they can also act as sodium (Na+) channels regulated by phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] [7.Jha A. et al.Convergent regulation of the lysosomal two-pore channel-2 by Mg2+, NAADP, PI(3,5)P2 and multiple protein kinases.EMBO J. 2014; 33: 501-511Crossref PubMed Scopus (150) Google Scholar,8.Wang X. et al.TPC proteins are phosphoinositide-activated sodium-selective ion channels in endosomes and lysosomes.Cell. 2012; 151: 372-383Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar]. The exact activation mechanisms mediated by NAADP and PI(3,5)P2 remain controversial, adding further to the TPC enigma. The animal TPC family comprises three subtypes: TPC1, TPC2 and TPC3. Only TPC1 and TPC2 are found in human and mouse cells, and differ in their pattern of distribution: TPC1 is found in a range of endolysosomal organelles, while TPC2 is distributed more predominantly in late endosomes and lysosomes (Figure 1) [9.Ruas M. et al.TPC1 has two variant isoforms, and their removal has different effects on endo-lysosomal functions compared to loss of TPC2.Mol. Cell. Biol. 2014; 34: 3981-3992Crossref PubMed Scopus (70) Google Scholar]. The past decade has witnessed remarkable progress in the development of detector technology and computational algorithms in structural biology study, facilitating the investigation of TPCs at an atomic level. Phylogenetic analysis of TPCs revealed that they share a common evolutionary origin with four-domain voltage-gated Ca2+ and Na+ channels (CaV and NaV), allowing the first predictions of their 3D conformations by homology modelling. This study also identified conserved residues at the channel pore region (see Glossary), suggesting that, similar to NaV and CaV, TPCs also exploit an asparagine-gated filter for cation selectivity [10.Rahman T. et al.Two-pore channels provide insight into the evolution of voltage-gated Ca2+ and Na+ channels.Sci. Signal. 2014; 7ra109Crossref PubMed Scopus (84) Google Scholar]. In 2016, the 3D architecture of TPC1 from Arabidopsis thaliana (AtTPC1) was determined using X-ray crystallography, at a resolution of 3.3 Å and 2.87 Å [11.Guo J. et al.Structure of the voltage-gated two-pore channel TPC1 from Arabidopsis thaliana.Nature. 2016; 531: 196-201Crossref PubMed Scopus (188) Google Scholar,12.Kintzer A.F. Stroud R.M. Structure, inhibition and regulation of two-pore channel TPC1 from Arabidopsis thaliana.Nature. 2016; 531: 258-262Crossref PubMed Scopus (133) Google Scholar]. This was the first structural determination of this homodimer, where each subunit comprises two homologous six-transmembrane (6-TM) domain helices. The two 6-TMs are domain swapped, so that the C-terminal of 6-TM 1 is connected with the N-terminal of 6-TM 2 by an EF hand motif [11.Guo J. et al.Structure of the voltage-gated two-pore channel TPC1 from Arabidopsis thaliana.Nature. 2016; 531: 196-201Crossref PubMed Scopus (188) Google Scholar,12.Kintzer A.F. Stroud R.M. Structure, inhibition and regulation of two-pore channel TPC1 from Arabidopsis thaliana.Nature. 2016; 531: 258-262Crossref PubMed Scopus (133) Google Scholar]. However, the lack of critical glutamate and aspartate residues at the pore region makes AtTPC1 less selective to Ca2+ and Na+ compared with its mammalian counterpart [13.Patel S. et al.Two-pore channels enter the atomic era: structure of plant TPC revealed.Trends Biochem. Sci. 2016; 41: 475-477Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar] (see later). Recently, the 3D structures of mouse TPC1 (MmTPC1) and human TPC2 (HsTPC2) were determined with single-particle electron cryo-microscopy (cryo-EM) [14.She J. et al.Structural insights into the voltage and phospholipid activation of the mammalian TPC1 channel.Nature. 2018; 556: 130-134Crossref PubMed Scopus (140) Google Scholar,15.She J. et al.Structural mechanisms of phospholipid activation of the human TPC2 channel.eLife. 2019; 8e45222Crossref Scopus (86) Google Scholar]. Despite the low sequence similarity with AtTPC1, the overall homodimer architecture is conserved between species. The first four helices in each domain (IS1–IS4 for subunit 1, and IIS1–IIS4 for subunit 2) are voltage-sensing domains (VSDs), while the fifth and sixth helices (IS5 and IS6 for subunit 1, and IIS5 and IIS6 for subunit 2) constitute the pore region (Figure 2). Interestingly, a β-hairpin structure and a prolonged (horseshoe-shaped) C-terminal were identified in MmTPC1 but not in HsTPC2 (Figure 2) [14.She J. et al.Structural insights into the voltage and phospholipid activation of the mammalian TPC1 channel.Nature. 2018; 556: 130-134Crossref PubMed Scopus (140) Google Scholar,15.She J. et al.Structural mechanisms of phospholipid activation of the human TPC2 channel.eLife. 2019; 8e45222Crossref Scopus (86) Google Scholar]. The binding site of PI(3,5)P2 is located at 6-TM 1 for both MmTPC1 and HsTPC2, and this binding is sufficient to open the pore region of HsTPC2. In MmTPC1, the activation is both ligand and voltage dependent; IIS1–IIS4 from VSDs (but not IS1–IS4) are involved in sensing changes in membrane potential to open the channel [14.She J. et al.Structural insights into the voltage and phospholipid activation of the mammalian TPC1 channel.Nature. 2018; 556: 130-134Crossref PubMed Scopus (140) Google Scholar]. VSDs in HsTPC2 are independent from the channel activation process [15.She J. et al.Structural mechanisms of phospholipid activation of the human TPC2 channel.eLife. 2019; 8e45222Crossref Scopus (86) Google Scholar]. Ion selectivity of both MmTPC1 and HsTPC2 is achieved by not only a 'filter', which is an asparagine-gated size sieve (in line with a previous prediction, as mentioned earlier [10.Rahman T. et al.Two-pore channels provide insight into the evolution of voltage-gated Ca2+ and Na+ channels.Sci. Signal. 2014; 7ra109Crossref PubMed Scopus (84) Google Scholar]), but also a 'gate', which opens and closes while also restricting the size and charge of ions [14.She J. et al.Structural insights into the voltage and phospholipid activation of the mammalian TPC1 channel.Nature. 2018; 556: 130-134Crossref PubMed Scopus (140) Google Scholar,15.She J. et al.Structural mechanisms of phospholipid activation of the human TPC2 channel.eLife. 2019; 8e45222Crossref Scopus (86) Google Scholar]. The involvement of certain amino acids is the key in this cation selection process, and can vary between subtypes and species (Table 1).Table 1Comparison of Key Amino Acid Residues that Contribute to Ion Selectivity between AtTPC1, MmTPC1, and HsTPC2TPC subtype'Filter' region amino acids'Gate' region amino acidsIon selectivityRefsAtTPC1: Arabidopsis thalianaThr263, Thr264, Ser265, Asn266, and Asn267 from pore region of subunit 1; Val628, Met629, Gly630, Asn631, and Trp632 from pore region of subunit 2Leu301 and Tyr305 from IS6; Val668, Leu672, and Phe676 from IIS6Limited selectivity, selective to Ca2+, Na+ Ba2+, Li+, and K+[11.Guo J. et al.Structure of the voltage-gated two-pore channel TPC1 from Arabidopsis thaliana.Nature. 2016; 531: 196-201Crossref PubMed Scopus (188) Google Scholar,94.Guo J. et al.Tuning the ion selectivity of two-pore channels.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: 1009-1014Crossref PubMed Scopus (83) Google Scholar]MmTPC1: Mus musculusThr280, Ala281, and Asn282 from pore region of subunit 1; Val647, Asn648, and Asn649 from pore region of subunit 2Leu317, Phe321, and Asp322 from IS6; Val684, Leu688, and Glu689 from IIS6Highly selective to Na+, also selective to Ca2+[14.She J. et al.Structural insights into the voltage and phospholipid activation of the mammalian TPC1 channel.Nature. 2018; 556: 130-134Crossref PubMed Scopus (140) Google Scholar]HsTPC2: Homo sapiensThr271, Ala272, and Asn273 from pore region of subunit 1; Val652, Asn653, and Asn654 from pore region of subunit 2Thr308 and Tyr312 from IS6; Leu690 and Leu694 from IIS6Highly selective to Na+, also selective to Ca2+[15.She J. et al.Structural mechanisms of phospholipid activation of the human TPC2 channel.eLife. 2019; 8e45222Crossref Scopus (86) Google Scholar,94.Guo J. et al.Tuning the ion selectivity of two-pore channels.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: 1009-1014Crossref PubMed Scopus (83) Google Scholar] Open table in a new tab Endolysosomal Ca2+ homeostasis and its underlying mechanisms and pathophysiological relevance were poorly investigated until the discovery of NAADP signalling and its link with TPCs. The use of genetically modified animal models further broadened knowledge in this area. TPCs have been shown to have important roles in various diseases and physiological conditions. Here, we briefly discuss the latest discoveries relating to their roles in growth, differentiation, development, pathophysiological processes in the pancreas, viral infection, and cardiac dysfunction. NAADP-mediated Ca2+ signalling via TPC1 has been found to regulate the acrosome reaction of mouse spermatozoa [16.Arndt L. et al.NAADP and the two-pore channel protein 1 participate in the acrosome reaction in mammalian spermatozoa.Mol. Biol. Cell. 2014; 25: 948-964Crossref Scopus (46) Google Scholar]. Similarly, knockdown of TPCs in sea star oocytes showed defected embryo development due to debilitated Ca2+ signalling [17.Ramos I. et al.Two-pore channels function in calcium regulation in sea star oocytes and embryos.Development. 2014; 141: 4598-4609Crossref Scopus (15) Google Scholar]. Another study reported that TPC2 mediates osteoclastogenesis [18.Notomi T. et al.Role of lysosomal channel protein TPC2 in osteoclast differentiation and bone remodeling under normal and low-magnesium conditions.J. Biol. Chem. 2017; 292: 20998-21010Crossref Scopus (19) Google Scholar]. This finding is consistent with previous studies that identified a role for NAADP/TPC2-induced Ca2+ signals in neuronal and skeletal muscle differentiation [16.Arndt L. et al.NAADP and the two-pore channel protein 1 participate in the acrosome reaction in mammalian spermatozoa.Mol. Biol. 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A recent study found that cell proliferation was hindered by TPC2 gene silencing [24.Sun W. Yue J. TPC2 mediates autophagy progression and extracellular vesicle secretion in cancer cells.Exp. Cell Res. 2018; 370: 478-489Crossref Scopus (31) Google Scholar]. The critical role of NAADP/TPC/Ca2+ signalling in cellular differentiation and development and the role of TPC2 in cancer progression, from tumorigenesis to metastasis, has been reviewed extensively elsewhere [27.Webb S.E. et al.Role of two-pore channels in embryonic development and cellular differentiation.Cold Spring Harb. Perspect. Biol. 2020; 12: a035170Crossref Scopus (12) Google Scholar,28.Alharbi A.F. Parrington J. Endolysosomal Ca2+ signaling in cancer: the role of TPC2, from tumorigenesis to metastasis.Front. Cell Dev. Biol. 2019; 7: 302Crossref Scopus (27) Google Scholar]. Collectively, these studies highlight the critical role of NAADP/TPC/Ca2+ signalling in cellular differentiation, development, and associated pathophysiological processes, such as cancer. TPC2-regulated Ca2+ release from acidic intracellular stores has important roles in both insulin and glucagon secretion by the endocrine pancreas. Studies have highlighted that intracellular Ca2+ signals regulate how adrenaline, a blood glucose-elevating hormone, works as a stimulus of glucagon secretion, and identified a link with the cAMP-protein kinase A (PKA) pathway [29.Hamilton A. et al.Adrenaline stimulates glucagon secretion by Tpc2-dependent Ca2+ mobilization from acidic stores in pancreatic α-cells.Diabetes. 2018; 67: 1128-1139Crossref PubMed Scopus (62) Google Scholar]. cAMP has long been recognised as a key intracellular messenger in insulin release and reduced glucagon secretion because its cytoplasmic concentration regulates actions of many hormones [30.Butcher R.W. et al.The role of cyclic AMP in hormone actions.Adv. Enzym. Regul. 1968; 6: 357-389Crossref PubMed Scopus (79) Google Scholar]. In fact, cAMP is involved in the process by which Ca2+ triggers both glucagon and insulin secretion by α cells and β cells in pancreas islets [31.Arredouani A. et al.Nicotinic acid adenine dinucleotide phosphate (NAADP) and endolysosomal two-pore channels modulate membrane excitability and stimulus-secretion coupling in mouse pancreatic β cells.J. Biol. Chem. 2015; 290: 21376-21392Crossref Scopus (44) Google Scholar]. A study used islets from TPC2-null mice or islets with pharmacologically inhibited TPC2 to test the participation of TPC2 in glucagon secretion and found that the stimulating function of adrenaline in glucagon secretion was dependent on TPC2-evoked Ca2+ mobilisation [29.Hamilton A. et al.Adrenaline stimulates glucagon secretion by Tpc2-dependent Ca2+ mobilization from acidic stores in pancreatic α-cells.Diabetes. 2018; 67: 1128-1139Crossref PubMed Scopus (62) Google Scholar]. This research linking TPC2 to glucostasis enhanced the signalling map of cellular factors maintaining stable glucagon levels and identified TPC2 as a possible drug target in α cells for the treatment of diabetes. In a whole-animal TPC-knockout (KO) mouse study, NAADP-evoked Ca2+, via TPCs, was shown to be positively correlated to glucose- and sulfonylurea-elicited membrane potential, intracellular Ca2+ signals, and insulin secretion [31.Arredouani A. et al.Nicotinic acid adenine dinucleotide phosphate (NAADP) and endolysosomal two-pore channels modulate membrane excitability and stimulus-secretion coupling in mouse pancreatic β cells.J. Biol. Chem. 2015; 290: 21376-21392Crossref Scopus (44) Google Scholar]. However, another study found that pancreatic β cells (the predominant pancreatic cell type and the only source of circulating insulin [32.Rutter G.A. et al.Pancreatic β-cell identity, glucose sensing and the control of insulin secretion.Biochem. 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Moving onto the exocrine pancreas, a 1999 study revealed that NAADP selectively modulates cholecystokinin (CCK)-evoked Ca2+ release in pancreatic acinar cells; this was the first study to indicate NAADP as a potent Ca2+ modulator in mammalian cells, and also established the pancreatic acinar cell as a good model for studies of NAADP-evoked Ca2+ release [34.Cancela J.M. et al.Coordination of agonist-induced Ca2+-signalling patterns by NAADP in pancreatic acinar cells.Nature. 1999; 398: 74-76Crossref PubMed Scopus (333) Google Scholar]. Subsequent research showed that NAADP selectively modulates Ca2+ release from lysosome-related organelles in pancreatic acinar cells [35.Yamasaki M. et al.Organelle selection determines agonist-specific Ca 2+ signals in pancreatic acinar and β cells.J. Biol. Chem. 2004; 279: 7234-7240Crossref PubMed Scopus (186) Google Scholar]. Under normal physiological conditions, Ca2+ released from the ER evokes the exocytosis of zymogens in pancreatic acinar cells, while NAADP releases small but detectable amounts of Ca2+ from endolysosomes, resulting from CICR from zymogen-containing granules and the ER, indicating that, in healthy pancreatic acinar cells, the NAADP-TPC-Ca2+ pathway is an important contributor to the regulation of zymogen secretion [36.Gerasimenko J.V. et al.Both RyRs and TPCs are required for NAADP-induced intracellular Ca2+ release.Cell Calcium. 2015; 58: 237-245Crossref Scopus (47) Google Scholar]. Ebola virus (EBOV) infection causes symptoms that include fever, severe diarrhoea, vomiting, and massive internal and external bleeding, with a high mortality rate [37.Baize Emergence of Zaire Ebola virus disease in Guinea.N. Engl. J. Med. 2014; 371: 1418-1425Crossref PubMed Scopus (1065) Google Scholar]. EBOV is endocytosed into host cells and subsequently transported through the endolysosomal system, where it binds to an intracellular glycoprotein receptor, Niemann-Pick C1 (NPC1) [38.Carette J.E. et al.Ebola virus entry requires the cholesterol transporter Niemann-Pick C1.Nature. 2011; 477: 340-343Crossref PubMed Scopus (986) Google Scholar] and fuses with late lysosomes, following which viral RNA is released into the cytoplasm [39.Sakurai Y. et al.Two-pore channels control Ebola virus host cell entry and are drug targets for disease treatment.Science. 2015; 347: 995-998Crossref PubMed Scopus (417) Google Scholar]. Therefore, blocking membrane fusion within the endolysosomal system could inhibit EBOV infection and, because the maturation and vesicle fusion of endosomes are regulated by the NAADP signalling pathway [40.Ruas M. et al.Purified TPC isoforms form NAADP receptors with distinct roles for Ca2+ signaling and endolysosomal trafficking.Curr. 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TPCs also appear to have a role in the infectious cycle of another virus, Middle East Respiratory Syndrome coronavirus (MERS-CoV), where NAADP-released Ca2+ supports the activity of proprotein convertases, such as furin, which are required for MERS-CoV fusion activity into host cells and its cytoplasmic translocation (including internalisation, trafficking, and release of virus into cytoplasm) [42.Millet J.K. Whittaker G.R. Host cell entry of Middle East respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 15214-15219Crossref PubMed Scopus (496) Google Scholar]. Indeed, knockdown of TPCs depressed MERS-CoV fusion with host cell membranes [43.Gunaratne G.S. et al.NAADP-dependent Ca2+ signaling regulates Middle East respiratory syndrome-coronavirus pseudovirus translocation through the endolysosomal system.Cell Calcium. 2018; 75: 30-41Crossref Scopus (90) Google Scholar]. TPCs are involved in the life cycle of HIV; blocking TPC action by gene knockdown or pharmacological inhibition inhibits the release of HIV Tat protein from endolysosomes and transactivation of the viral long terminal repeat (LTR) gene promoter, which suppresses the efficiency of HIV replication in host cells [44.Khan N. et al.Two-pore channels regulate Tat endolysosome escape and Tat-mediated HIV-1 LTR transactivation.FASEB J. 2020; 34: 4147-4162Crossref Scopus (28) Google Scholar]. Such findings indicate that pharmacological modulators of TPCs could have therapeutic value for the treatment of a range of viral infections. In cardiac ischaemia, a reduced blood supply to the heart can result in shortage of oxygen and nutrients for cardiac myocytes. In addition, restoration of blood supply after ischaemia can also damage myocytes by inducing excessive oxidative stress, known as ischaemia-reperfusion injury (IRI); the involvement of TPCs in IRI was recently shown in TPC1-KO mice, which have alleviated IRI-induced cardiac infarction [45.Davidson S.M. et al.Inhibition of NAADP signalling on reperfusion protects the heart by preventing lethal calcium oscillations via two-pore channel 1 and opening of the mitochondrial permeability transition pore.Cardiovasc. Res. 2015; 108: 357-366Crossref PubMed Scopus (39) Google Scholar]. This was suggested to be due to decreased TPC1-induced CICR, because excessive Ca2+ can be detrimental during IRI by stimulating oxidative stress in mitochondria [45.Davidson S.M. et al.Inhibition of NAADP signalling on reperfusion protects the heart by preventing lethal calcium oscillations via two-pore channel 1 and opening of the mitochondrial permeability transition pore.Cardiovasc. Res. 2015; 108: 357-366Crossref PubMed Scopus (39) Google Scholar]. Apart from IRI, there is also evidence that links TPC2 with arrhythmia and cardiac hypertrophy. The NAADP-TPC2 pathway is involved in this process as a downstream effect of abnormal β-adrenoceptor stimulation [46.Gul R. et al.Nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic ADP-ribose (cADPR) mediate Ca2+ signaling in cardiac hypertrophy induced by β-adrenergic stimulation.PLoS ONE. 2016; 11e0149125Crossref PubMed Scopus (39) Google Scholar]. This concept was also confirmed by an in vivo model, whereby TPC2-KO mice exhibited decreased proneness to both arrhythmia and hypertrophy [47.Capel R.A. et al.Two-pore channels (TPC2s) and nicotinic acid adenine dinucleotide phosphate (NAADP) at lysosomal-sarcoplasmic reticular junctions contribute to acute and chronic β-adrenoceptor signaling in the heart.J. Biol. Chem. 2015; 290: 30087-30098Crossref PubMed Scopus (56) Google Scholar]. 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