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Sandip Patel - One of the best experts on this subject based on the ideXlab platform.
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the two pore Channel tpc interactome unmasks isoform specific roles for tpcs in endolysosomal morphology and cell pigmentation
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Yaping Linmoshier, Dev Churamani, Robert Hooper, Eugen Brailoiu, Michael J Boulware, Michael V Keebler, Xiaolong Liu, Mary E Abood, Timothy F Walseth, Sandip PatelAbstract:The Two-Pore Channels (TPC1 and TPC2) belong to an ancient family of intracellular ion Channels expressed in the endolysosomal system. Little is known about how regulatory inputs converge to modulate TPC activity, and proposed activation mechanisms are controversial. Here, we compiled a proteomic characterization of the human TPC interactome, which revealed that TPCs complex with many proteins involved in Ca2+ homeostasis, trafficking, and membrane organization. Among these interactors, TPCs were resolved to scaffold Rab GTPases and regulate endomembrane dynamics in an isoform-specific manner. TPC2, but not TPC1, caused a proliferation of endolysosomal structures, dysregulating intracellular trafficking, and cellular pigmentation. These outcomes required both TPC2 and Rab activity, as well as their interactivity, because TPC2 mutants that were inactive, or rerouted away from their endogenous expression locale, or deficient in Rab binding, failed to replicate these outcomes. Nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca2+ release was also impaired using either a Rab binding-defective TPC2 mutant or a Rab inhibitor. These data suggest a fundamental role for the ancient TPC complex in trafficking that holds relevance for lysosomal proliferative scenarios observed in disease.
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convergent regulation of the lysosomal two pore Channel 2 by mg2 naadp pi 3 5 p2 and multiple protein kinases
The EMBO Journal, 2014Co-Authors: Archana Jha, Sandip Patel, Malini Ahuja, Eugen Brailoiu, Shmuel MuallemAbstract:Lysosomal Ca2+ homeostasis is implicated in disease and controls many lysosomal functions. A key in understanding lysosomal Ca2+ signaling was the discovery of the Two-Pore Channels (TPCs) and their potential activation by NAADP. Recent work concluded that the TPCs function as a PI(3,5)P2 activated Channels regulated by mTORC1, but not by NAADP. Here, we identified Mg2+ and the MAPKs, JNK and P38 as novel regulators of TPC2. Cytoplasmic Mg2+ specifically inhibited TPC2 outward current, whereas lysosomal Mg2+ partially inhibited both outward and inward currents in a lysosomal lumen pH-dependent manner. Under controlled Mg2+, TPC2 is readily activated by NAADP with Channel properties identical to those in response to PI(3,5)P2. Moreover, TPC2 is robustly regulated by P38 and JNK. Notably, NAADP-mediated Ca2+ release in intact cells is regulated by Mg2+, PI(3,5)P2, and P38/JNK kinases, thus paralleling regulation of TPC2 currents. Our data affirm a key role for TPC2 in NAADP-mediated Ca2+ signaling and link this pathway to Mg2+ homeostasis and MAP kinases, pointing to roles for lysosomal Ca2+ in cell growth, inflammation and cancer.
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membrane potential regulates nicotinic acid adenine dinucleotide phosphate naadp dependence of the ph and ca2 sensitive organellar two pore Channel tpc1
Journal of Biological Chemistry, 2012Co-Authors: Volodymyr Rybalchenko, Sandip Patel, Dev Churamani, Malini Ahuja, Jessica Coblentz, Kirill Kiselyov, Shmuel MuallemAbstract:Abstract Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent second messenger that mobilizes Ca2+ from the acidic endolysosomes by activation of the Two-Pore Channels TPC1 and TPC2. The Channel properties of human TPC1 have not been studied before, and its cellular function is not known. In the present study, we characterized TPC1 incorporated into lipid bilayers. The native and recombinant TPC1 Channels are activated by NAADP. TPC1 activity requires acidic luminal pH and high luminal Ca2+. With Ba2+ as the permeable ion, luminal Ca2+ activates TPC1 with an apparent Km of 180 μm. TPC1 operates in two tightly coupled conductance states of 47 ± 8 and 200 ± 9 picosiemens. Importantly, opening of the large conductance markedly increases the small conductance mean open time. Changes in membrane potential from 0 to −60 mV increased linearly both the small and the large conductances and NPo, indicating that TPC1 is regulated by voltage. Intriguingly, the apparent affinity for activation of TPC1 by its ligand NAADP is not constant. Rather, hyperpolarization increases the apparent affinity of TPC1 for NAADP by 10 nm/mV. The concerted regulation of TPC1 activity by luminal Ca2+ and by membrane potential thus provides a potential mechanism to explain NAADP-induced Ca2+ oscillations. These findings reveal unique properties of TPC1 to explain its role in Ca2+ oscillations and cell function.
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an naadp gated two pore Channel targeted to the plasma membrane uncouples triggering from amplifying ca2 signals
Journal of Biological Chemistry, 2010Co-Authors: Eugen Brailoiu, Dev Churamani, Robert Hooper, Taufiq Rahman, Cristina G Brailoiu, David L Prole, Colin W Taylor, Sandip PatelAbstract:Nicotinic acid adenine dinucleotide phosphate (NAADP) is a ubiquitous messenger proposed to stimulate Ca2+ release from acidic organelles via Two-Pore Channels (TPCs). It has been difficult to resolve this trigger event from its amplification via endoplasmic reticulum Ca2+ stores, fuelling speculation that archetypal intracellular Ca2+ Channels are the primary targets of NAADP. Here, we redirect TPC2 from lysosomes to the plasma membrane and show that NAADP evokes Ca2+ influx independent of ryanodine receptors and that it activates a Ca2+-permeable Channel whose conductance is reduced by mutation of a residue within a putative pore. We therefore uncouple TPC2 from amplification pathways and prove that it is a pore-forming subunit of an NAADP-gated Ca2+ Channel.
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an ancestral deuterostome family of two pore Channels mediates nicotinic acid adenine dinucleotide phosphate dependent calcium release from acidic organelles
Journal of Biological Chemistry, 2010Co-Authors: Eugen Brailoiu, Robert Hooper, Xinjiang Cai, Cristina G Brailoiu, Nae J Dun, Jonathan S Marchant, Michael V Keebler, Sandip PatelAbstract:Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent and widespread calcium-mobilizing messenger, the properties of which have been most extensively described in sea urchin eggs. The molecular basis for calcium release by NAADP, however, is not clear and subject to controversy. Recent studies have provided evidence that members of the Two-Pore Channel (TPC) family in mammals are the long sought after target Channels for NAADP. Here, we show that the TPC3 gene, which has yet to be functionally characterized, is present throughout the deuterostome lineage but is a pseudogene in humans and other primates. We report the molecular cloning of the complete ancestral TPC gene family from the sea urchin and demonstrate that all three isoforms localize to acidic organelles to mediate NAADP-dependent calcium release. Our data highlight the functional divergence of this novel gene family during deuterostome evolution and provide further evidence that NAADP mediates calcium release from acidic stores through activation of TPCs.
John Parrington - One of the best experts on this subject based on the ideXlab platform.
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loss of two pore Channel 2 tpc2 expression increases the metastatic traits of melanoma cells by a mechanism involving the hippo signalling pathway and store operated calcium entry
Cancers, 2020Co-Authors: Antonella Damore, Fioretta Palombi, Ali Hanbashi, Silvia Di Agostino, Andrea Sacconi, Aniruddha Voruganti, Marilena Taggi, Rita Canipari, Giovanni Blandino, John ParringtonAbstract:Melanoma is one of the most aggressive and treatment-resistant human cancers. The Two-Pore Channel 2 (TPC2) is located on late endosomes, lysosomes and melanosomes. Here, we characterized how TPC2 knockout (KO) affected human melanoma cells derived from a metastatic site. TPC2 KO increased these cells’ ability to invade the extracelullar matrix and was associated with the increased expression of mesenchymal markers ZEB-1, Vimentin and N-Cadherin, and the enhanced secretion of MMP9. TPC2 KO also activated genes regulated by YAP/TAZ, which are key regulators of tumourigenesis and metastasis. Expression levels of ORAI1, a component of store-operated Ca2+ entry (SOCE), and PKC-βII, part of the HIPPO pathway that negatively regulates YAP/TAZ activity, were reduced by TPC2 KO and RNA interference knockdown. We propose a cellular mechanism mediated by ORAI1/Ca2+/PKC-βII to explain these findings. Highlighting their potential clinical significance, patients with metastatic tumours showed a reduction in TPC2 expression. Our research indicates a novel role of TPC2 in melanoma. While TPC2 loss may not activate YAP/TAZ target genes in primary melanoma, in metastatic melanoma it could activate such genes and increase cancer aggressiveness. These findings aid the understanding of tumourigenesis mechanisms and could provide new diagnostic and treatment strategies for skin cancer and other metastatic cancers.
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tpcn2 knockout mice have improved insulin sensitivity and are protected against high fat diet induced weight gain
Physiological Genomics, 2018Co-Authors: Katie Holl, John Parrington, Margarida Ruas, Sarah Debehnke, Chay Teng Yeo, Polly A Hansen, Abraham K Gebre, Sandra Leonekabler, John S Parks, Leah Solberg C WoodsAbstract:Type 2 diabetes is a complex disorder affected by multiple genes and the environment. Our laboratory has shown that in response to a glucose challenge, Two-Pore Channel 2 (Tpcn2) knockout mice exhi...
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ca2 release via two pore Channel type 2 tpc2 is required for slow muscle cell myofibrillogenesis and myotomal patterning in intact zebrafish embryos
Developmental Biology, 2017Co-Authors: Jeffrey J Kelu, John Parrington, Antony Galione, Sarah E Webb, Andrew L MillerAbstract:We recently demonstrated a critical role for Two-Pore Channel type 2 (TPC2)-mediated Ca2+ release during the differentiation of slow (skeletal) muscle cells (SMC) in intact zebrafish embryos, via the introduction of a translational-blocking morpholino antisense oligonucleotide (MO). Here, we extend our study and demonstrate that knockdown of TPC2 with a non-overlapping splice-blocking MO, knockout of TPC2 (via the generation of a tpcn2dhkz1a mutant line of zebrafish using CRISPR/Cas9 gene-editing), or the pharmacological inhibition of TPC2 action with bafilomycin A1 or trans-ned-19, also lead to a significant attenuation of SMC differentiation, characterized by a disruption of SMC myofibrillogenesis and gross morphological changes in the trunk musculature. When the morphants were injected with tpcn2-mRNA or were treated with IP3/BM or caffeine (agonists of the inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR), respectively), many aspects of myofibrillogenesis and myotomal patterning (and in the case of the pharmacological treatments, the Ca2+ signals generated in the SMCs), were rescued. STED super-resolution microscopy revealed a close physical relationship between clusters of RyR in the terminal cisternae of the sarcoplasmic reticulum (SR), and TPC2 in lysosomes, with a mean estimated separation of ~52–87 nm. Our data therefore add to the increasing body of evidence, which indicate that localized Ca2+ release via TPC2 might trigger the generation of more global Ca2+ release from the SR via Ca2+-induced Ca2+ release.
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VEGF-induced neoangiogenesis is mediated by NAADP and Two-Pore Channel-2-dependent Ca2+ signaling
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Annarita Favia, John Parrington, Marianna Desideri, Guido Gambara, Alessio D'alessio, Margarida Ruas, Bianca Esposito, Donatella Del Bufalo, Elio Ziparo, Fioretta PalombiAbstract:Vascular endothelial growth factor (VEGF) and its receptors VEGFR1/VEGFR2 play major roles in controlling angiogenesis, including vascularization of solid tumors. Here we describe a specific Ca2+ signaling pathway linked to the VEGFR2 receptor subtype, controlling the critical angiogenic responses of endothelial cells (ECs) to VEGF. Key steps of this pathway are the involvement of the potent Ca2+ mobilizing messenger, nicotinic acid adenine-dinucleotide phosphate (NAADP), and the specific engagement of the Two-Pore Channel TPC2 subtype on acidic intracellular Ca2+ stores, resulting in Ca2+ release and angiogenic responses. Targeting this intracellular pathway pharmacologically using the NAADP antagonist Ned-19 or genetically using Tpcn2−/− mice was found to inhibit angiogenic responses to VEGF in vitro and in vivo. In human umbilical vein endothelial cells (HUVECs) Ned-19 abolished VEGF-induced Ca2+ release, impairing phosphorylation of ERK1/2, Akt, eNOS, JNK, cell proliferation, cell migration, and capillary-like tube formation. Interestingly, Tpcn2 shRNA treatment abolished VEGF-induced Ca2+ release and capillary-like tube formation. Importantly, in vivo VEGF-induced vessel formation in matrigel plugs in mice was abolished by Ned-19 and, most notably, failed to occur in Tpcn2−/− mice, but was unaffected in Tpcn1−/− animals. These results demonstrate that a VEGFR2/NAADP/TPC2/Ca2+ signaling pathway is critical for VEGF-induced angiogenesis in vitro and in vivo. Given that VEGF can elicit both pro- and antiangiogenic responses depending upon the balance of signal transduction pathways activated, targeting specific VEGFR2 downstream signaling pathways could modify this balance, potentially leading to more finely tailored therapeutic strategies.
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tpc2 proteins mediate nicotinic acid adenine dinucleotide phosphate naadp and agonist evoked contractions of smooth muscle
Journal of Biological Chemistry, 2010Co-Authors: John Parrington, Margarida Ruas, Nezahat Tugba Durlukandilci, Kaiting Chuang, Alison F Brading, Antony GalioneAbstract:Agonists such as those acting at muscarinic receptors are thought to induce contraction of smooth muscle primarily through inositol 1,4,5-trisphosphate production and release of Ca2+ from sarcoplasmic reticulum. However, the additional Ca2+-mobilizing messengers cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) may also be involved in this process, the former acting on the sarcoplasmic reticulum, the latter acting on lysosome-related organelles. In this study, we provide the first systematic analysis of the capacity of inositol 1,4,5-trisphosphate, cADPR, and NAADP to cause contraction in smooth muscle. Using permeabilized guinea pig detrusor and taenia caecum, we show that all three Ca2+-mobilizing messengers cause contractions in both types of smooth muscle. We demonstrate that cADPR and NAADP play differential roles in mediating contraction in response to muscarinic receptor activation, with a sizeable role for NAADP and acidic calcium stores in detrusor muscle but not in taenia caecum, underscoring the heterogeneity of smooth muscle signal transduction systems. Two-Pore Channel proteins (TPCs) have recently been shown to be key components of the NAADP receptor. We show that contractile responses to NAADP were completely abolished, and agonist-evoked contractions were reduced and now became independent of acidic calcium stores in Tpcn2−/− mouse detrusor smooth muscle. Our findings provide the first evidence that TPC proteins mediate a key NAADP-regulated tissue response brought about by agonist activation of a cell surface receptor.
Antony Galione - One of the best experts on this subject based on the ideXlab platform.
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A Two-Pore Channel protein required for regulating mTORC1 activity on starvation
BMC Biology, 2020Co-Authors: Fu-sheng Chang, Antony Galione, Yuntao Wang, Phillip Dmitriev, Julian Gross, Catherine PearsAbstract:Background Two-Pore Channels (TPCs) release Ca^2+ from acidic intracellular stores and are implicated in a number of diseases, but their role in development is unclear. The social amoeba Dictyostelium discoideum proliferates as single cells that aggregate to form a multicellular organism on starvation. Starvation is sensed by the mTORC1 complex which, like TPC proteins, is found on acidic vesicles. Here, we address the role of TPCs in development and under starvation. Results We report that disruption of the gene encoding the single Dictyostelium TPC protein, TPC2, leads to a delay in early development and prolonged growth in culture with delayed expression of early developmental genes, although a rapid starvation-induced increase in autophagy is still apparent. Ca^2+ signals induced by extracellular cAMP are delayed in developing tpc2 ^ − cells, and aggregation shows increased sensitivity to weak bases, consistent with reduced acidity of the vesicles. In mammalian cells, the mTORC1 protein kinase has been proposed to suppress TPC Channel opening. Here, we show a reciprocal effect as tpc2 ^ − cells show an increased level of phosphorylation of an mTORC1 substrate, 4E-BP1. mTORC1 inhibition reverses the prolonged growth and increases the efficiency of aggregation of tpc2 ^ − cells. Conclusion TPC2 is required for efficient growth development transition in Dictyostelium and acts through modulation of mTORC1 activity revealing a novel mode of regulation.
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TPC: the NAADP discovery Channel?
2020Co-Authors: Anthony J Morgan, Margarida Ruas, Lianne C Davis, Antony GalioneAbstract:Abstract The Ca 2 + -mobilizing second messenger, NAADP (nicotinic acid adenine dinucleotide phosphate), has been with us for nearly 20 years and yet we still cannot fully agree on the identity of its target Ca 2 + -release Channel. In spite of some recent robust challenges to the idea that Two-Pore Channels (TPCs) represent the elusive "NAADP receptor", evidence continues to accumulate that TPCs are important for NAADP-mediated responses. This article will briefly outline the background and review more recent work pertaining to the TPC story. Acidic Ca 2 + stores Ca 2 + mobilization from intracellular stores is an ancient and conserved signal transduction pathway from plants to animals, and while the study of the endoplasmic reticulum (ER) Ca 2 + store has dominated this arena for decades, another organelle Ca 2 + store family -the acidic Ca 2 + stores -is stepping out from the shadow of its bigger brother and assuming an importance in its own right. The so-called acidic Ca 2 + stores encompass a spectrum of organelles with an acidic lumen such as endosomes, lysosomes, secretory vesicles and lysosome-related organelles (that include vacuoles, secretory lysosomes and yolk platelets) [1]. That these organelles are key for diverse cellular functions is without doubt, that they also store and release Ca 2 + may be less familiar but arguably just as important. How Ca 2 + is sequestered into these acidic vesicles is unclear for higher organisms but, by analogy with the better understood plants and yeast systems [2], it is anticipated to involve some form of Ca NAADP, a unique messenger The archetypal ER Ca 2 + store is mobilized by IP 3 or cADPR and, in a comparable manner, acidic Ca 2 + stores are mobilized by their own unique second messenger, NAADP (nicotinic acid adenine dinucleotide phosphate) [1]. Key words: NAADP, TPC, Ca 2 + , lysosome, endosome. Abbreviations: ER, endoplasmic reticulum; NAADP, nicotinic acid adenine dinucleotide phosphate; pHL, luminal pH; PI(3,5)P2, phosphatidylinositol 3,5-bisphosphate; RyRs, ryanodine receptors; TPCs, Two-Pore Channels; TRPML1, mucolipin-1. 1 To whom correspondence should be addressed (email Anthony.morgan@pharm. ox.ac.uk). Coupled to a burgeoning list of extracellular stimuli, NAADP plays a unique role and is physiologically important in contexts as broad as fertilization, immunology, angiogenesis, cardiac function and autophagy [9]. However, the identity of the Ca 2 + -release Channel targeted by NAADP has long been sought and debated, with several candidate Channels proposed over the years including ryanodine receptors (RyRs), mucolipin-1 (TRPML1), TRPM2 and the Two-Pore Channel family (TPCs) [1]. TPCs have received the lion's share of recent attention owing to a substantial body of evidence linking it to NAADP, as we shall briefly discuss. . This was a tantalizing development because the vacuole is the acidic Ca 2 + store in plants, and pre-empted a cluster of papers in 2009 that proposed that the animal orthologues of TPC were endo-lysosomal Ca 2 + -release Channels activated by NAADP i.e. TPCs were the long sought after "NAADP receptors" [13][14][15]. TPCs: a new Channel family Building on this foundation, different laboratories subsequently appeared to confirm and extend these observations using Ca 2 + imaging and/or electrophysiology and revealed that TPCs recapitulated the properties expected of the "NAADP receptor": they were permeable to Ca 2 + (or Ca 2 + surrogates), associated with NAADP-binding sites [7] and were activated by NAADP (in mammalian cells, with the characteristic bell-shaped concentration-response curve) [16]. In view of this compelling body of evidence, it came as something of a surprise in late 2012/early 2013 when this premise was challenged by a couple of high profile papers that asserted that TPCs had nothing to do with either NAADP or Ca 2 + : TPCs were described as lipid-regulated Na + Channel
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ca2 release via two pore Channel type 2 tpc2 is required for slow muscle cell myofibrillogenesis and myotomal patterning in intact zebrafish embryos
Developmental Biology, 2017Co-Authors: Jeffrey J Kelu, John Parrington, Antony Galione, Sarah E Webb, Andrew L MillerAbstract:We recently demonstrated a critical role for Two-Pore Channel type 2 (TPC2)-mediated Ca2+ release during the differentiation of slow (skeletal) muscle cells (SMC) in intact zebrafish embryos, via the introduction of a translational-blocking morpholino antisense oligonucleotide (MO). Here, we extend our study and demonstrate that knockdown of TPC2 with a non-overlapping splice-blocking MO, knockout of TPC2 (via the generation of a tpcn2dhkz1a mutant line of zebrafish using CRISPR/Cas9 gene-editing), or the pharmacological inhibition of TPC2 action with bafilomycin A1 or trans-ned-19, also lead to a significant attenuation of SMC differentiation, characterized by a disruption of SMC myofibrillogenesis and gross morphological changes in the trunk musculature. When the morphants were injected with tpcn2-mRNA or were treated with IP3/BM or caffeine (agonists of the inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR), respectively), many aspects of myofibrillogenesis and myotomal patterning (and in the case of the pharmacological treatments, the Ca2+ signals generated in the SMCs), were rescued. STED super-resolution microscopy revealed a close physical relationship between clusters of RyR in the terminal cisternae of the sarcoplasmic reticulum (SR), and TPC2 in lysosomes, with a mean estimated separation of ~52–87 nm. Our data therefore add to the increasing body of evidence, which indicate that localized Ca2+ release via TPC2 might trigger the generation of more global Ca2+ release from the SR via Ca2+-induced Ca2+ release.
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intracellular sphingosine releases calcium from lysosomes
eLife, 2015Co-Authors: Doris Hoglinger, Antony Galione, Per Haberkant, Auxiliadora Aguileraromero, Howard Riezman, Forbes D Porter, Frances M Platt, Carsten SchultzAbstract:To elucidate new functions of sphingosine (Sph), we demonstrate that the spontaneous elevation of intracellular Sph levels via caged Sph leads to a significant and transient calcium release from acidic stores that is independent of sphingosine 1-phosphate, extracellular and ER calcium levels. This photo-induced Sph-driven calcium release requires the Two-Pore Channel 1 (TPC1) residing on endosomes and lysosomes. Further, uncaging of Sph leads to the translocation of the autophagy-relevant transcription factor EB (TFEB) to the nucleus specifically after lysosomal calcium release. We confirm that Sph accumulates in late endosomes and lysosomes of cells derived from Niemann-Pick disease type C (NPC) patients and demonstrate a greatly reduced calcium release upon Sph uncaging. We conclude that sphingosine is a positive regulator of calcium release from acidic stores and that understanding the interplay between Sph homeostasis, calcium signaling and autophagy will be crucial in developing new therapies for lipid storage disorders such as NPC.
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tpc2 proteins mediate nicotinic acid adenine dinucleotide phosphate naadp and agonist evoked contractions of smooth muscle
Journal of Biological Chemistry, 2010Co-Authors: John Parrington, Margarida Ruas, Nezahat Tugba Durlukandilci, Kaiting Chuang, Alison F Brading, Antony GalioneAbstract:Agonists such as those acting at muscarinic receptors are thought to induce contraction of smooth muscle primarily through inositol 1,4,5-trisphosphate production and release of Ca2+ from sarcoplasmic reticulum. However, the additional Ca2+-mobilizing messengers cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) may also be involved in this process, the former acting on the sarcoplasmic reticulum, the latter acting on lysosome-related organelles. In this study, we provide the first systematic analysis of the capacity of inositol 1,4,5-trisphosphate, cADPR, and NAADP to cause contraction in smooth muscle. Using permeabilized guinea pig detrusor and taenia caecum, we show that all three Ca2+-mobilizing messengers cause contractions in both types of smooth muscle. We demonstrate that cADPR and NAADP play differential roles in mediating contraction in response to muscarinic receptor activation, with a sizeable role for NAADP and acidic calcium stores in detrusor muscle but not in taenia caecum, underscoring the heterogeneity of smooth muscle signal transduction systems. Two-Pore Channel proteins (TPCs) have recently been shown to be key components of the NAADP receptor. We show that contractile responses to NAADP were completely abolished, and agonist-evoked contractions were reduced and now became independent of acidic calcium stores in Tpcn2−/− mouse detrusor smooth muscle. Our findings provide the first evidence that TPC proteins mediate a key NAADP-regulated tissue response brought about by agonist activation of a cell surface receptor.
Eugen Brailoiu - One of the best experts on this subject based on the ideXlab platform.
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the two pore Channel tpc interactome unmasks isoform specific roles for tpcs in endolysosomal morphology and cell pigmentation
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Yaping Linmoshier, Dev Churamani, Robert Hooper, Eugen Brailoiu, Michael J Boulware, Michael V Keebler, Xiaolong Liu, Mary E Abood, Timothy F Walseth, Sandip PatelAbstract:The Two-Pore Channels (TPC1 and TPC2) belong to an ancient family of intracellular ion Channels expressed in the endolysosomal system. Little is known about how regulatory inputs converge to modulate TPC activity, and proposed activation mechanisms are controversial. Here, we compiled a proteomic characterization of the human TPC interactome, which revealed that TPCs complex with many proteins involved in Ca2+ homeostasis, trafficking, and membrane organization. Among these interactors, TPCs were resolved to scaffold Rab GTPases and regulate endomembrane dynamics in an isoform-specific manner. TPC2, but not TPC1, caused a proliferation of endolysosomal structures, dysregulating intracellular trafficking, and cellular pigmentation. These outcomes required both TPC2 and Rab activity, as well as their interactivity, because TPC2 mutants that were inactive, or rerouted away from their endogenous expression locale, or deficient in Rab binding, failed to replicate these outcomes. Nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca2+ release was also impaired using either a Rab binding-defective TPC2 mutant or a Rab inhibitor. These data suggest a fundamental role for the ancient TPC complex in trafficking that holds relevance for lysosomal proliferative scenarios observed in disease.
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convergent regulation of the lysosomal two pore Channel 2 by mg2 naadp pi 3 5 p2 and multiple protein kinases
The EMBO Journal, 2014Co-Authors: Archana Jha, Sandip Patel, Malini Ahuja, Eugen Brailoiu, Shmuel MuallemAbstract:Lysosomal Ca2+ homeostasis is implicated in disease and controls many lysosomal functions. A key in understanding lysosomal Ca2+ signaling was the discovery of the Two-Pore Channels (TPCs) and their potential activation by NAADP. Recent work concluded that the TPCs function as a PI(3,5)P2 activated Channels regulated by mTORC1, but not by NAADP. Here, we identified Mg2+ and the MAPKs, JNK and P38 as novel regulators of TPC2. Cytoplasmic Mg2+ specifically inhibited TPC2 outward current, whereas lysosomal Mg2+ partially inhibited both outward and inward currents in a lysosomal lumen pH-dependent manner. Under controlled Mg2+, TPC2 is readily activated by NAADP with Channel properties identical to those in response to PI(3,5)P2. Moreover, TPC2 is robustly regulated by P38 and JNK. Notably, NAADP-mediated Ca2+ release in intact cells is regulated by Mg2+, PI(3,5)P2, and P38/JNK kinases, thus paralleling regulation of TPC2 currents. Our data affirm a key role for TPC2 in NAADP-mediated Ca2+ signaling and link this pathway to Mg2+ homeostasis and MAP kinases, pointing to roles for lysosomal Ca2+ in cell growth, inflammation and cancer.
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an naadp gated two pore Channel targeted to the plasma membrane uncouples triggering from amplifying ca2 signals
Journal of Biological Chemistry, 2010Co-Authors: Eugen Brailoiu, Dev Churamani, Robert Hooper, Taufiq Rahman, Cristina G Brailoiu, David L Prole, Colin W Taylor, Sandip PatelAbstract:Nicotinic acid adenine dinucleotide phosphate (NAADP) is a ubiquitous messenger proposed to stimulate Ca2+ release from acidic organelles via Two-Pore Channels (TPCs). It has been difficult to resolve this trigger event from its amplification via endoplasmic reticulum Ca2+ stores, fuelling speculation that archetypal intracellular Ca2+ Channels are the primary targets of NAADP. Here, we redirect TPC2 from lysosomes to the plasma membrane and show that NAADP evokes Ca2+ influx independent of ryanodine receptors and that it activates a Ca2+-permeable Channel whose conductance is reduced by mutation of a residue within a putative pore. We therefore uncouple TPC2 from amplification pathways and prove that it is a pore-forming subunit of an NAADP-gated Ca2+ Channel.
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an ancestral deuterostome family of two pore Channels mediates nicotinic acid adenine dinucleotide phosphate dependent calcium release from acidic organelles
Journal of Biological Chemistry, 2010Co-Authors: Eugen Brailoiu, Robert Hooper, Xinjiang Cai, Cristina G Brailoiu, Nae J Dun, Jonathan S Marchant, Michael V Keebler, Sandip PatelAbstract:Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent and widespread calcium-mobilizing messenger, the properties of which have been most extensively described in sea urchin eggs. The molecular basis for calcium release by NAADP, however, is not clear and subject to controversy. Recent studies have provided evidence that members of the Two-Pore Channel (TPC) family in mammals are the long sought after target Channels for NAADP. Here, we show that the TPC3 gene, which has yet to be functionally characterized, is present throughout the deuterostome lineage but is a pseudogene in humans and other primates. We report the molecular cloning of the complete ancestral TPC gene family from the sea urchin and demonstrate that all three isoforms localize to acidic organelles to mediate NAADP-dependent calcium release. Our data highlight the functional divergence of this novel gene family during deuterostome evolution and provide further evidence that NAADP mediates calcium release from acidic stores through activation of TPCs.
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essential requirement for two pore Channel 1 in naadp mediated calcium signaling
Journal of Cell Biology, 2009Co-Authors: Eugen Brailoiu, Dev Churamani, Robert Hooper, Xinjiang Cai, Michael G Schrlau, Cristina G Brailoiu, Xin Gao, Michael J Boulware, Nae J Dun, Jonathan S MarchantAbstract:Nicotinic acid adenine dinucleotide phosphate (NAADP) is a widespread and potent calcium-mobilizing messenger that is highly unusual in activating calcium Channels located on acidic stores. However, the molecular identity of the target protein is unclear. In this study, we show that the previously uncharacterized human Two-Pore Channels (TPC1 and TPC2) are endolysosomal proteins, that NAADP-mediated calcium signals are enhanced by overexpression of TPC1 and attenuated after knockdown of TPC1, and that mutation of a single highly conserved residue within a putative pore region abrogated calcium release by NAADP. Thus, TPC1 is critical for NAADP action and is likely the long sought after target Channel for NAADP.
Andrew L Miller - One of the best experts on this subject based on the ideXlab platform.
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ca2 release via two pore Channel type 2 tpc2 is required for slow muscle cell myofibrillogenesis and myotomal patterning in intact zebrafish embryos
Developmental Biology, 2017Co-Authors: Jeffrey J Kelu, John Parrington, Antony Galione, Sarah E Webb, Andrew L MillerAbstract:We recently demonstrated a critical role for Two-Pore Channel type 2 (TPC2)-mediated Ca2+ release during the differentiation of slow (skeletal) muscle cells (SMC) in intact zebrafish embryos, via the introduction of a translational-blocking morpholino antisense oligonucleotide (MO). Here, we extend our study and demonstrate that knockdown of TPC2 with a non-overlapping splice-blocking MO, knockout of TPC2 (via the generation of a tpcn2dhkz1a mutant line of zebrafish using CRISPR/Cas9 gene-editing), or the pharmacological inhibition of TPC2 action with bafilomycin A1 or trans-ned-19, also lead to a significant attenuation of SMC differentiation, characterized by a disruption of SMC myofibrillogenesis and gross morphological changes in the trunk musculature. When the morphants were injected with tpcn2-mRNA or were treated with IP3/BM or caffeine (agonists of the inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR), respectively), many aspects of myofibrillogenesis and myotomal patterning (and in the case of the pharmacological treatments, the Ca2+ signals generated in the SMCs), were rescued. STED super-resolution microscopy revealed a close physical relationship between clusters of RyR in the terminal cisternae of the sarcoplasmic reticulum (SR), and TPC2 in lysosomes, with a mean estimated separation of ~52–87 nm. Our data therefore add to the increasing body of evidence, which indicate that localized Ca2+ release via TPC2 might trigger the generation of more global Ca2+ release from the SR via Ca2+-induced Ca2+ release.
