{"gene":"TPCN1","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":2018,"finding":"Cryo-EM structures of mouse TPC1 (MmTPC1) in apo closed and PtdIns(3,5)P2-bound open states revealed: (1) a coin-slot-shaped ion selectivity filter defining Na+ selectivity; (2) only the voltage-sensing domain from the second 6-TM domain confers voltage dependence; (3) endolysosome-specific PtdIns(3,5)P2 binds to the first 6-TM domain and activates the channel under depolarizing conditions; (4) structural comparison showed interplay between voltage and ligand in channel activation.","method":"Cryo-EM structure determination combined with functional electrophysiology and mutagenesis","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — atomic-resolution cryo-EM structures in two states combined with functional analysis and mutagenesis in one rigorous study","pmids":["29562233"],"is_preprint":false},{"year":2014,"finding":"TPC1 forms a depolarization-activated, non-inactivating Na+ channel (lysoNaV) in endolysosomes. Whole-organelle patch clamp showed TPC1 is a 2×6TM voltage-gated Na+ channel that confers electrical excitability to endolysosomes; luminal alkalization shifts voltage dependence toward hyperpolarization, opening the channel.","method":"Whole-organelle patch clamp recording; heterologous expression; pharmacological manipulation of luminal pH","journal":"Nature chemical biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct electrophysiological reconstitution in native organelles with mutagenesis-supported functional characterization","pmids":["24776928"],"is_preprint":false},{"year":2012,"finding":"Human TPC1 incorporated into lipid bilayers is activated by NAADP, requires acidic luminal pH and high luminal Ca2+, operates in two coupled conductance states (47 and 200 pS), and is regulated by membrane voltage; hyperpolarization increases NAADP apparent affinity by ~10 nM/mV, providing a mechanism for NAADP-induced Ca2+ oscillations.","method":"Lipid bilayer reconstitution of native and recombinant TPC1; electrophysiology; pharmacological manipulation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution in lipid bilayers with multiple orthogonal measurements (conductance states, voltage dependence, ligand affinity), single lab","pmids":["22500018"],"is_preprint":false},{"year":2014,"finding":"TPC1 loss (Tpcn1−/−) in mouse embryonic fibroblasts impairs trafficking of cholera toxin from the plasma membrane to the Golgi apparatus, indicating TPC1 has a specific role in early/recycling endosomal trafficking distinct from TPC2 (which regulates lysosomal degradation of PDGFRβ). Loss of TPC1 or TPC2 did not significantly affect resting endo-lysosomal pH or morphology.","method":"Knockout MEFs (Tpcn1−/− and Tpcn2−/−); cholera toxin trafficking assay; endo-lysosomal pH measurements; morphological analysis","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO with defined cellular trafficking phenotype, comparison between two KO lines with multiple functional readouts","pmids":["25135478"],"is_preprint":false},{"year":2017,"finding":"TPC1 localizes predominantly to early and recycling endosomes (not lysosomes) and is required for toxin uptake/processing through early endosomes. Proteomic analysis of native TPC1 channel complexes identified direct interaction with a distinct set of syntaxins involved in intracellular vesicle fusion, suggesting TPC1 provides local Ca2+ for SNARE-mediated vesicle fusion.","method":"Knockout cell lines; co-localization with subcellular markers; protein toxin processing assays; native proteomics (mass spectrometry) of TPC1 complexes","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO phenotype plus proteomic identification of syntaxin interactors using multiple orthogonal methods (co-localization, toxin assays, MS)","pmids":["28855648"],"is_preprint":false},{"year":2011,"finding":"EF-hand 2 of plant TPC1 is the essential Ca2+-receptor site for Ca2+-dependent channel gating (mutation D376A abolishes activation up to 1 mM Ca2+), while EF-hand 1 is a structural low-affinity Ca2+/Mg2+ site that enables channel responses at physiological Ca2+ concentrations. Single mutation D335A in EF-hand 1 reduces sensitivity below 200 µM Ca2+ but preserves gating.","method":"Site-directed mutagenesis of EF-hand residues; patch clamp electrophysiology of vacuoles; molecular modeling","journal":"The Plant journal : for cell and molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis combined with in vitro electrophysiological functional validation, multiple mutants tested","pmids":["21736651"],"is_preprint":false},{"year":2011,"finding":"A luminal Ca2+ binding site in TPC1 formed by residues Glu-450, Asp-454, Glu-456, and Glu-457 regulates channel gating by luminal Ca2+; Glu-450 and Asp-454 are directly involved in Ca2+ binding, while Glu-456 and Glu-457 connect the binding site to the gate. The fou2 mutation (D454N) eliminates luminal Ca2+ sensitivity.","method":"Structure modeling; site-directed mutagenesis of individual glutamate/aspartate residues; patch clamp recordings of loss-of-TPC1-function protoplasts transiently expressing mutant channels","journal":"The Plant cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis of multiple candidate residues combined with direct electrophysiological functional validation","pmids":["21764990"],"is_preprint":false},{"year":2009,"finding":"The fou2 gain-of-function point mutation D454N in TPC1 specifically eliminates inhibition by luminal Ca2+ (vacuolar Ca2+ at 0.1 mM level normally abolishes K+ flux through wild-type TPC1 but not the D454N mutant), identifying D454 as part of a luminal Ca2+ recognition/inhibitory site. This leads to higher vacuolar Ca2+/K+ ratio in fou2 plants.","method":"Patch clamp measurements on Arabidopsis mesophyll vacuoles; comparison of wild-type and fou2 (D454N) mutant channels under varying luminal Ca2+ concentrations","journal":"The Plant journal : for cell and molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct electrophysiological characterization of defined point mutant with mechanistic functional consequence, single lab but rigorous","pmids":["19298454"],"is_preprint":false},{"year":2016,"finding":"Dimerization of a C-terminal helix (CTH) of TPC1 is essential for channel activity. Bimolecular fluorescence complementation and co-immunoprecipitation demonstrated C-terminus interaction; synthetic CTH peptides dimerize with Kd = 3.9 µM; deletion or point mutations disrupting CTH dimerization abolish cation transport. MD simulations show CTH forms a stable antiparallel coiled-coil.","method":"Bimolecular fluorescence complementation; co-immunoprecipitation; synthetic peptide dimerization assay; site-directed mutagenesis; ion transport assay; molecular dynamics simulations","journal":"Cellular and molecular life sciences : CMLS","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (BiFC, Co-IP, peptide biochemistry, mutagenesis, functional assay, MD) in one study","pmids":["26781468"],"is_preprint":false},{"year":2020,"finding":"TPC1 deficiency in mice leads to enhanced passive systemic anaphylaxis and augmented mast cell histamine release and degranulation ex vivo. TPC1 plays an essential role in endolysosomal Ca2+ uptake and filling of ER Ca2+ stores, thereby regulating exocytosis in mast cells.","method":"TPC1 knockout mice; in vivo passive systemic anaphylaxis model; ex vivo mast cell degranulation and histamine release assays; Ca2+ homeostasis measurements","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO mouse with defined in vivo and ex vivo phenotypic readouts, mechanistic link to endolysosomal Ca2+ uptake and ER store filling","pmids":["32661165"],"is_preprint":false},{"year":2022,"finding":"In zebrafish, TPC1-decorated endolysosomes generate localized non-propagating Ca2+ transients at myoseptal junctions (MJs). Loss of tpcn1 (morpholino or CRISPR/Cas9) causes slow skeletal muscle cells to detach from or cross myosepta, disrupts endolysosomal trafficking, and results in abnormal distribution of β-dystroglycan. TPC1-decorated endolysosomes associate with MJs in a microtubule-dependent manner.","method":"Antisense morpholino knockdown; CRISPR/Cas9 knockout in zebrafish; Ca2+ imaging; live imaging of endolysosomal dynamics; β-dystroglycan localization analysis","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — two independent loss-of-function approaches (morpholino + CRISPR) with defined cellular phenotypes and mechanistic link to endolysosomal trafficking and dystrophin complex distribution","pmids":["35393618"],"is_preprint":false},{"year":2023,"finding":"TPC1 is expressed subapically in proximal but not distal kidney tubules. TPC1-deficient mice show prolonged and exaggerated PTH-induced phosphate excretion with delayed recovery, and delayed NH4Cl-induced recovery in acid-base transitions, demonstrating TPC1 is required for dynamic adaptation of proximal tubular phosphate reabsorption.","method":"Immunohistochemistry with tubular markers; in vivo PTH bolus injection and acid-base challenge experiments in TPC1-deficient vs. wildtype mice; urine phosphate and ammonium measurements","journal":"Acta physiologica (Oxford, England)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO in vivo with defined physiological phenotype, single lab, localization confirmed by IHC","pmids":["36599408"],"is_preprint":false},{"year":2014,"finding":"Simultaneous knockout of TPC1 and TPC2 in mice leads to mature-onset obesity due to impaired lipid availability and β-adrenergic receptor signaling in brown adipose tissue, with reduced phosphorylated hormone-sensitive lipase and β-adrenergic receptor expression, while mitochondrial respiratory chain function and UCP1 expression remain intact.","method":"Tpcn1/2 double knockout mice; body composition analysis; respirometry; BAT temperature measurement; Western blotting for HSL, β-adrenergic receptors, UCP1","journal":"Endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — double KO (cannot attribute phenotype solely to TPC1), defined metabolic phenotype with molecular characterization, single lab","pmids":["25545384"],"is_preprint":false},{"year":2019,"finding":"In human metastatic colorectal cancer cells, NAADP-gated TPC1 triggers Ca2+ release from endo-lysosomes that is amplified by ER InsP3 receptors (CICR). Genetic silencing of TPC1 or pharmacological blockade reduces NAADP-evoked Ca2+ release, serum-induced Ca2+ signals, ERK and Akt phosphorylation, and cell proliferation.","method":"Liposomal NAADP delivery; Ca2+ imaging; GPN/nigericin lysosomal Ca2+ depletion; NED-19 pharmacology; TPC1 siRNA knockdown; Western blotting for ERK/Akt phosphorylation; proliferation assays","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic and pharmacological manipulation with multiple readouts, single lab, human primary cells","pmids":["30991693"],"is_preprint":false},{"year":2024,"finding":"TPC1 knockout in murine B16-F0 melanoma cells decreases mTORC1 activity, reduces proliferation and invasiveness, and increases pigmentation associated with nuclear localization of transcription factor TFEB, demonstrating TPC1 in early/recycling endosomes controls melanoma progression via mTORC1 and TFEB signaling.","method":"CRISPR/Cas9 TPC1 knockout in B16-F0 cells; mTORC1 activity assays; proliferation and invasion assays; TFEB localization imaging; pigmentation assay","journal":"Heliyon","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with defined cellular phenotypes and pathway placement (mTORC1/TFEB), single lab, single study","pmids":["39524724"],"is_preprint":false},{"year":2025,"finding":"At lysosome-ER membrane contact sites, TPC1 forms a Ca2+ microdomain in close proximity to IP3R1; proximity of TPC1 to IP3R1 generates Ca2+-induced Ca2+ release that depletes ER Ca2+ stores and triggers lethal ER stress-induced apoptosis. Altering TPC1 expression levels in HeLa cells replicates these calcium dynamics.","method":"RAB7A activity assays; lysosome-ER proximity imaging; TPC1 overexpression/KO in HeLa cells; Ca2+ imaging; ER stress markers; BALB/c xenograft models","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Ca2+ imaging with TPC1 manipulation, in vivo xenograft validation, single lab, functional link to IP3R1 at MCS","pmids":["40709664"],"is_preprint":false},{"year":2016,"finding":"In Arabidopsis roots, TPC1 vacuolar channel participates in propagation of systemic Ca2+ waves in response to salt stress. The Ca2+ wave transmission requires both TPC1-dependent Ca2+ release and AtRBOHD NADPH oxidase-generated extracellular ROS; a ROS-assisted calcium-induced calcium-release (CICR) mechanism explains the observed wave speeds, as shown by ROS scavenger treatment, NADPH oxidase inhibition, and atrbohD knockout experiments.","method":"Fire-diffuse-fire modeling; Ca2+ wave imaging; ROS scavenger (ascorbate) and NADPH oxidase inhibitor (DPI) treatment; AtrbohD knockout analysis; extracellular ROS imaging","journal":"Plant physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO combined with pharmacological perturbations and quantitative modeling, plant ortholog, single lab","pmids":["27261066"],"is_preprint":false},{"year":2010,"finding":"Guard cells express higher TPC1 transcript levels and higher SV channel current density than mesophyll cells, and guard cell SV/TPC1 channels have higher cytosolic Ca2+ sensitivity than mesophyll cell channels, explaining the stomatal phenotype of tpc1-2 loss-of-function plants.","method":"Quantitative RT-PCR for TPC1 transcripts; patch clamp measurements of SV currents in guard cell and mesophyll vacuoles; comparison of Ca2+ dose-response curves","journal":"Plant & cell physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct electrophysiology comparing two cell types with transcript quantification, plant ortholog, single lab","pmids":["20630987"],"is_preprint":false},{"year":2004,"finding":"TPC1 channels are specifically required for ROS-responsive Ca2+ influx in tobacco cells: cells cosuppressing endogenous TPC1 lose H2O2-induced Ca2+ influx, while AtTPC1 overexpression increases Al-sensitive (TPC1-dependent) Ca2+ influx. Hypoosmotic Ca2+ influx is TPC1-independent. Al ions specifically inhibit TPC1-mediated Ca2+ influx.","method":"Aequorin-based Ca2+ monitoring in BY-2 cells; TPC1 overexpression and cosuppression lines; Al, La, Gd ion pharmacology; H2O2 and hypoosmotic stimulation","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic gain- and loss-of-function with defined stimulus-specific Ca2+ phenotypes, plant ortholog, single lab","pmids":["15464979"],"is_preprint":false},{"year":2013,"finding":"Monocot TPC1 orthologs (OsTPC1 from rice, TaTPC1 from wheat) functionally rescue the SV channel deficit in Arabidopsis tpc1-2 vacuoles, demonstrating conserved vacuolar localization and function. When expressed in HEK293 cells, OsTPC1 targets to LysoTracker-positive organelles, confirming endomembrane localization across phyla.","method":"Cross-species complementation of tpc1-2 mutant; patch clamp of vacuoles from Arabidopsis and rice tpc1 null mutants; LysoTracker imaging in HEK293 cells","journal":"The New phytologist","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic complementation with direct electrophysiological readout plus heterologous localization, single lab","pmids":["23845012"],"is_preprint":false},{"year":2062,"finding":"JPT2/HN1L functions as an NAADP-binding protein required for NAADP-mediated Ca2+ release via TPC1/TPC2 in a cell-type-specific manner; it is indispensable in CD4+ T cells (TCR/CD3-evoked Ca2+ microdomains reduced in Jpt2/Hn1l−/− cells) but dispensable in cardiomyocytes, platelets, and mast cells.","method":"Jpt2/Hn1l−/− mouse generation; NAADP-evoked Ca2+ imaging in primary cardiomyocytes, mast cells, T cells; platelet aggregation assays; TCR/CD3 stimulation","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO mouse with cell-type-specific Ca2+ imaging across multiple primary cell types, preprint not yet peer-reviewed","pmids":["bio_10.1101_2025.11.11.687795"],"is_preprint":true}],"current_model":"TPCN1 (TPC1) encodes a homodimeric, voltage-gated, Na+-selective cation channel residing in endolysosomal membranes, whose activity is regulated by depolarizing voltage (via the second 6-TM voltage-sensing domain), PtdIns(3,5)P2 binding to the first 6-TM domain, luminal Ca2+ and pH, and the second messenger NAADP (which requires accessory binding proteins such as JPT2); it generates local Ca2+ signals that drive SNARE/syntaxin-mediated vesicular fusion in early and recycling endosomes, supports systemic Ca2+ wave propagation (in plants, together with NADPH oxidase-derived ROS), regulates mast cell exocytosis by controlling endolysosomal Ca2+ uptake and ER store filling, controls mTORC1 and TFEB activity to influence melanoma proliferation, and facilitates endolysosomal trafficking required for myoseptal junction maintenance and proximal tubular phosphate reabsorption."},"narrative":{"mechanistic_narrative":"TPCN1 (TPC1) encodes a homodimeric, two-domain (2×6-TM) voltage-gated cation channel that resides in acidic endomembranes — early/recycling endosomes in animal cells and the vacuole in plants — where it converts membrane voltage and lipid/ion cues into local Ca2+ (and Na+) signals [PMID:29562233, PMID:24776928, PMID:28855648, PMID:23845012]. Cryo-EM of mouse TPC1 in apo-closed and PtdIns(3,5)P2-bound open states established that a coin-slot selectivity filter sets Na+ selectivity, that only the voltage-sensing domain of the second 6-TM module confers voltage dependence, and that endolysosome-specific PtdIns(3,5)P2 binds the first 6-TM domain to open the channel under depolarization [PMID:29562233]; whole-organelle patch clamp confirmed TPC1 as a depolarization-activated, non-inactivating Na+ channel (lysoNaV) gated by luminal pH [PMID:24776928]. In reconstituted bilayers human TPC1 is activated by the second messenger NAADP and requires acidic luminal pH and high luminal Ca2+, with voltage tuning NAADP affinity to support Ca2+ oscillations [PMID:22500018]. Channel function depends on dimerization through a C-terminal antiparallel coiled-coil [PMID:26781468], and in plant orthologs Ca2+ gating is set by cytosolic EF-hands and a distinct luminal Ca2+ site whose D454N (fou2) mutation removes luminal Ca2+ inhibition [PMID:21736651, PMID:21764990, PMID:19298454]. Functionally, TPC1 supplies local Ca2+ for SNARE/syntaxin-mediated vesicle fusion in early/recycling endosomes and is required for endosomal toxin trafficking [PMID:25135478, PMID:28855648]. These channel-dependent Ca2+ signals are physiologically consequential: TPC1 controls endolysosomal Ca2+ uptake and ER store filling to regulate mast cell exocytosis and anaphylaxis [PMID:32661165], drives endolysosome-to-ER calcium-induced calcium release that can deplete ER stores and trigger apoptosis [PMID:40709664], supports myoseptal-junction maintenance via endolysosomal trafficking [PMID:35393618] and dynamic proximal-tubular phosphate reabsorption [PMID:36599408], and promotes cancer cell proliferation and progression through NAADP/CICR signaling and mTORC1/TFEB control [PMID:30991693, PMID:39524724]. In plants the vacuolar channel additionally propagates systemic Ca2+ waves in concert with NADPH-oxidase-derived ROS [PMID:27261066].","teleology":[{"year":2004,"claim":"Established that TPC1 is specifically required for ROS-evoked Ca2+ entry, distinguishing it from osmotically-driven Ca2+ pathways and giving the channel its first defined physiological stimulus.","evidence":"Aequorin Ca2+ monitoring in tobacco BY-2 cells with TPC1 cosuppression/overexpression and ion pharmacology","pmids":["15464979"],"confidence":"Medium","gaps":["Did not resolve subcellular site of the Ca2+ flux","Channel biophysics and gating not directly measured"]},{"year":2009,"claim":"Identified a luminal Ca2+ recognition site, showing the fou2 D454N mutation removes Ca2+-dependent inhibition and links luminal Ca2+ sensing to gating.","evidence":"Patch clamp of Arabidopsis mesophyll vacuoles comparing wild-type and D454N channels under varying luminal Ca2+","pmids":["19298454"],"confidence":"High","gaps":["Mechanism connecting the site to the gate not yet mapped","Plant ortholog only"]},{"year":2011,"claim":"Resolved how cytosolic and luminal Ca2+ control gating by assigning EF-hand 2 as the essential activating site and defining the luminal Ca2+ pocket residues that couple to the gate.","evidence":"Site-directed mutagenesis of EF-hand and luminal acidic residues with vacuolar patch clamp and molecular modeling","pmids":["21736651","21764990"],"confidence":"High","gaps":["Established in plant TPC1; animal regulation by luminal Ca2+ addressed separately","No structural model at the time"]},{"year":2012,"claim":"Demonstrated that human TPC1 is an NAADP-activated channel requiring acidic luminal pH and high luminal Ca2+, providing a biophysical mechanism for NAADP-induced Ca2+ oscillations.","evidence":"Lipid bilayer reconstitution of native and recombinant TPC1 with conductance, voltage, and ligand-affinity measurements","pmids":["22500018"],"confidence":"High","gaps":["NAADP binding shown functionally but accessory binding protein not yet identified","Single-lab in vitro system"]},{"year":2014,"claim":"Defined TPC1 as a depolarization-activated, non-inactivating endolysosomal Na+ channel (lysoNaV) gated by luminal pH, establishing its native ionic identity in animal organelles.","evidence":"Whole-organelle patch clamp with heterologous expression and luminal pH manipulation","pmids":["24776928"],"confidence":"High","gaps":["Reconciliation of Na+ selectivity with NAADP-evoked Ca2+ signaling left open","Downstream effectors not addressed"]},{"year":2014,"claim":"Localized TPC1 function to early/recycling endosomal trafficking, distinguishing it from TPC2's lysosomal degradation role using clean knockouts.","evidence":"Tpcn1−/− and Tpcn2−/− MEFs with cholera toxin trafficking, pH, and morphology assays","pmids":["25135478"],"confidence":"High","gaps":["Molecular partners mediating trafficking not yet defined","Link between channel activity and trafficking step not established"]},{"year":2016,"claim":"Showed C-terminal coiled-coil dimerization is obligatory for channel activity, defining the structural basis of the functional dimer.","evidence":"BiFC, Co-IP, synthetic peptide dimerization, mutagenesis, ion transport assay, and MD simulation","pmids":["26781468"],"confidence":"High","gaps":["How dimerization couples to gating not resolved","Plant channel context"]},{"year":2017,"claim":"Connected TPC1 channel function to vesicle fusion by identifying syntaxin interactors in native complexes, framing TPC1 as a local Ca2+ source for SNARE-mediated fusion.","evidence":"Native proteomics (MS) of TPC1 complexes plus co-localization and toxin-processing assays in knockout cells","pmids":["28855648"],"confidence":"High","gaps":["Direct Ca2+-to-SNARE causality not demonstrated","Which syntaxins are functionally required not dissected"]},{"year":2018,"claim":"Provided atomic-resolution mechanism: cryo-EM in closed and PtdIns(3,5)P2-bound open states explained Na+ selectivity, voltage-sensor asymmetry, and lipid-voltage interplay in activation.","evidence":"Cryo-EM of mouse TPC1 in two states with electrophysiology and mutagenesis","pmids":["29562233"],"confidence":"High","gaps":["NAADP-bound conformation not captured","Accessory protein interactions not in structure"]},{"year":2020,"claim":"Established an in vivo immune role: TPC1 controls endolysosomal Ca2+ uptake and ER store filling to regulate mast cell degranulation and anaphylaxis.","evidence":"TPC1 knockout mice with passive systemic anaphylaxis, ex vivo degranulation, and Ca2+ homeostasis assays","pmids":["32661165"],"confidence":"High","gaps":["Molecular link between channel flux and ER store filling not detailed","Cell-type generality unaddressed"]},{"year":2024,"claim":"Placed endosomal TPC1 upstream of mTORC1 and TFEB signaling to drive melanoma proliferation and invasion, extending its role to growth-control pathways.","evidence":"CRISPR/Cas9 TPC1 knockout in B16-F0 cells with mTORC1, proliferation, invasion, TFEB localization, and pigmentation assays","pmids":["39524724"],"confidence":"Medium","gaps":["Mechanism linking channel Ca2+ to mTORC1 not resolved","Single cell line, single study"]},{"year":2025,"claim":"Resolved a spatial signaling mechanism: TPC1 forms a Ca2+ microdomain near IP3R1 at lysosome-ER contact sites to drive CICR, ER store depletion, and apoptosis.","evidence":"TPC1 overexpression/KO and IP3R1 proximity Ca2+ imaging in HeLa cells with ER-stress markers and xenografts","pmids":["40709664"],"confidence":"Medium","gaps":["Tethering machinery for the contact site not defined","Single lab"]},{"year":null,"claim":"How accessory NAADP-binding proteins selectively confer NAADP sensitivity on TPC1 across cell types, and how the Na+-selective channel generates the Ca2+ signals attributed to it, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["JPT2/HN1L requirement is cell-type-specific and only reported in preprint","Direct demonstration of TPC1-mediated Ca2+ flux vs Na+ flux at contact sites still lacking"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[0,1,2,5,8,18]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[0]},{"term_id":"GO:0140299","term_label":"molecular sensor activity","supporting_discovery_ids":[2,5,6]}],"localization":[{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[3,4,14]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[1,15,19]},{"term_id":"GO:0005773","term_label":"vacuole","supporting_discovery_ids":[5,6,7,17,19]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[3,4,10]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[9,16,18]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[13,14,15]}],"complexes":[],"partners":["TPCN2","IP3R1","JPT2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9ULQ1","full_name":"Two pore channel protein 1","aliases":["Two pore calcium channel protein 1","Voltage-dependent calcium channel protein TPC1"],"length_aa":816,"mass_kda":94.1,"function":"Intracellular channel initially characterized as a non-selective Ca(2+)-permeable channel activated by NAADP (nicotinic acid adenine dinucleotide phosphate), it is also a voltage-gated highly-selective Na(+) channel activated directly by PI(3,5)P2 (phosphatidylinositol 3,5-bisphosphate) that senses pH changes and confers electrical excitability to organelles (PubMed:19620632, PubMed:23063126, PubMed:23394946, PubMed:24776928). Localizes to the early and recycling endosomes membranes where it plays a role in the uptake and processing of proteins and regulates organellar membrane excitability, membrane trafficking and pH homeostasis (Probable) (PubMed:23394946). Ion selectivity is not fixed but rather agonist-dependent and under defined ionic conditions, can be readily activated by both NAADP and PI(3,5)P2 (Probable). Required for mTOR-dependent nutrient sensing (Probable) (PubMed:23394946) (Microbial infection) During Ebola virus (EBOV) infection, controls the movement of endosomes containing virus particles and is required by EBOV to escape from the endosomal network into the cell cytoplasm","subcellular_location":"Lysosome membrane; Endosome membrane; Early endosome membrane; Recycling endosome membrane","url":"https://www.uniprot.org/uniprotkb/Q9ULQ1/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TPCN1","classification":"Not 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all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TPCN1"},"hgnc":{"alias_symbol":["KIAA1169","FLJ20612","TPC1"],"prev_symbol":[]},"alphafold":{"accession":"Q9ULQ1","domains":[{"cath_id":"-","chopping":"105-211","consensus_level":"high","plddt":93.4374,"start":105,"end":211},{"cath_id":"1.10.287.70","chopping":"219-308","consensus_level":"medium","plddt":94.51,"start":219,"end":308},{"cath_id":"1.20.120.350","chopping":"424-549","consensus_level":"medium","plddt":88.6376,"start":424,"end":549},{"cath_id":"-","chopping":"558-699","consensus_level":"medium","plddt":91.9152,"start":558,"end":699}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9ULQ1","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9ULQ1-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9ULQ1-F1-predicted_aligned_error_v6.png","plddt_mean":84.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TPCN1","jax_strain_url":"https://www.jax.org/strain/search?query=TPCN1"},"sequence":{"accession":"Q9ULQ1","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9ULQ1.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9ULQ1/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9ULQ1"}},"corpus_meta":[{"pmid":"15772667","id":"PMC_15772667","title":"The 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domain and activates the channel under depolarizing conditions; (4) structural comparison showed interplay between voltage and ligand in channel activation.\",\n      \"method\": \"Cryo-EM structure determination combined with functional electrophysiology and mutagenesis\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — atomic-resolution cryo-EM structures in two states combined with functional analysis and mutagenesis in one rigorous study\",\n      \"pmids\": [\"29562233\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TPC1 forms a depolarization-activated, non-inactivating Na+ channel (lysoNaV) in endolysosomes. Whole-organelle patch clamp showed TPC1 is a 2×6TM voltage-gated Na+ channel that confers electrical excitability to endolysosomes; luminal alkalization shifts voltage dependence toward hyperpolarization, opening the channel.\",\n      \"method\": \"Whole-organelle patch clamp recording; heterologous expression; pharmacological manipulation of luminal pH\",\n      \"journal\": \"Nature chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct electrophysiological reconstitution in native organelles with mutagenesis-supported functional characterization\",\n      \"pmids\": [\"24776928\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Human TPC1 incorporated into lipid bilayers is activated by NAADP, requires acidic luminal pH and high luminal Ca2+, operates in two coupled conductance states (47 and 200 pS), and is regulated by membrane voltage; hyperpolarization increases NAADP apparent affinity by ~10 nM/mV, providing a mechanism for NAADP-induced Ca2+ oscillations.\",\n      \"method\": \"Lipid bilayer reconstitution of native and recombinant TPC1; electrophysiology; pharmacological manipulation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution in lipid bilayers with multiple orthogonal measurements (conductance states, voltage dependence, ligand affinity), single lab\",\n      \"pmids\": [\"22500018\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TPC1 loss (Tpcn1−/−) in mouse embryonic fibroblasts impairs trafficking of cholera toxin from the plasma membrane to the Golgi apparatus, indicating TPC1 has a specific role in early/recycling endosomal trafficking distinct from TPC2 (which regulates lysosomal degradation of PDGFRβ). Loss of TPC1 or TPC2 did not significantly affect resting endo-lysosomal pH or morphology.\",\n      \"method\": \"Knockout MEFs (Tpcn1−/− and Tpcn2−/−); cholera toxin trafficking assay; endo-lysosomal pH measurements; morphological analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO with defined cellular trafficking phenotype, comparison between two KO lines with multiple functional readouts\",\n      \"pmids\": [\"25135478\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TPC1 localizes predominantly to early and recycling endosomes (not lysosomes) and is required for toxin uptake/processing through early endosomes. Proteomic analysis of native TPC1 channel complexes identified direct interaction with a distinct set of syntaxins involved in intracellular vesicle fusion, suggesting TPC1 provides local Ca2+ for SNARE-mediated vesicle fusion.\",\n      \"method\": \"Knockout cell lines; co-localization with subcellular markers; protein toxin processing assays; native proteomics (mass spectrometry) of TPC1 complexes\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO phenotype plus proteomic identification of syntaxin interactors using multiple orthogonal methods (co-localization, toxin assays, MS)\",\n      \"pmids\": [\"28855648\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"EF-hand 2 of plant TPC1 is the essential Ca2+-receptor site for Ca2+-dependent channel gating (mutation D376A abolishes activation up to 1 mM Ca2+), while EF-hand 1 is a structural low-affinity Ca2+/Mg2+ site that enables channel responses at physiological Ca2+ concentrations. Single mutation D335A in EF-hand 1 reduces sensitivity below 200 µM Ca2+ but preserves gating.\",\n      \"method\": \"Site-directed mutagenesis of EF-hand residues; patch clamp electrophysiology of vacuoles; molecular modeling\",\n      \"journal\": \"The Plant journal : for cell and molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis combined with in vitro electrophysiological functional validation, multiple mutants tested\",\n      \"pmids\": [\"21736651\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"A luminal Ca2+ binding site in TPC1 formed by residues Glu-450, Asp-454, Glu-456, and Glu-457 regulates channel gating by luminal Ca2+; Glu-450 and Asp-454 are directly involved in Ca2+ binding, while Glu-456 and Glu-457 connect the binding site to the gate. The fou2 mutation (D454N) eliminates luminal Ca2+ sensitivity.\",\n      \"method\": \"Structure modeling; site-directed mutagenesis of individual glutamate/aspartate residues; patch clamp recordings of loss-of-TPC1-function protoplasts transiently expressing mutant channels\",\n      \"journal\": \"The Plant cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis of multiple candidate residues combined with direct electrophysiological functional validation\",\n      \"pmids\": [\"21764990\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The fou2 gain-of-function point mutation D454N in TPC1 specifically eliminates inhibition by luminal Ca2+ (vacuolar Ca2+ at 0.1 mM level normally abolishes K+ flux through wild-type TPC1 but not the D454N mutant), identifying D454 as part of a luminal Ca2+ recognition/inhibitory site. This leads to higher vacuolar Ca2+/K+ ratio in fou2 plants.\",\n      \"method\": \"Patch clamp measurements on Arabidopsis mesophyll vacuoles; comparison of wild-type and fou2 (D454N) mutant channels under varying luminal Ca2+ concentrations\",\n      \"journal\": \"The Plant journal : for cell and molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct electrophysiological characterization of defined point mutant with mechanistic functional consequence, single lab but rigorous\",\n      \"pmids\": [\"19298454\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Dimerization of a C-terminal helix (CTH) of TPC1 is essential for channel activity. Bimolecular fluorescence complementation and co-immunoprecipitation demonstrated C-terminus interaction; synthetic CTH peptides dimerize with Kd = 3.9 µM; deletion or point mutations disrupting CTH dimerization abolish cation transport. MD simulations show CTH forms a stable antiparallel coiled-coil.\",\n      \"method\": \"Bimolecular fluorescence complementation; co-immunoprecipitation; synthetic peptide dimerization assay; site-directed mutagenesis; ion transport assay; molecular dynamics simulations\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (BiFC, Co-IP, peptide biochemistry, mutagenesis, functional assay, MD) in one study\",\n      \"pmids\": [\"26781468\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TPC1 deficiency in mice leads to enhanced passive systemic anaphylaxis and augmented mast cell histamine release and degranulation ex vivo. TPC1 plays an essential role in endolysosomal Ca2+ uptake and filling of ER Ca2+ stores, thereby regulating exocytosis in mast cells.\",\n      \"method\": \"TPC1 knockout mice; in vivo passive systemic anaphylaxis model; ex vivo mast cell degranulation and histamine release assays; Ca2+ homeostasis measurements\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO mouse with defined in vivo and ex vivo phenotypic readouts, mechanistic link to endolysosomal Ca2+ uptake and ER store filling\",\n      \"pmids\": [\"32661165\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In zebrafish, TPC1-decorated endolysosomes generate localized non-propagating Ca2+ transients at myoseptal junctions (MJs). Loss of tpcn1 (morpholino or CRISPR/Cas9) causes slow skeletal muscle cells to detach from or cross myosepta, disrupts endolysosomal trafficking, and results in abnormal distribution of β-dystroglycan. TPC1-decorated endolysosomes associate with MJs in a microtubule-dependent manner.\",\n      \"method\": \"Antisense morpholino knockdown; CRISPR/Cas9 knockout in zebrafish; Ca2+ imaging; live imaging of endolysosomal dynamics; β-dystroglycan localization analysis\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — two independent loss-of-function approaches (morpholino + CRISPR) with defined cellular phenotypes and mechanistic link to endolysosomal trafficking and dystrophin complex distribution\",\n      \"pmids\": [\"35393618\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TPC1 is expressed subapically in proximal but not distal kidney tubules. TPC1-deficient mice show prolonged and exaggerated PTH-induced phosphate excretion with delayed recovery, and delayed NH4Cl-induced recovery in acid-base transitions, demonstrating TPC1 is required for dynamic adaptation of proximal tubular phosphate reabsorption.\",\n      \"method\": \"Immunohistochemistry with tubular markers; in vivo PTH bolus injection and acid-base challenge experiments in TPC1-deficient vs. wildtype mice; urine phosphate and ammonium measurements\",\n      \"journal\": \"Acta physiologica (Oxford, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO in vivo with defined physiological phenotype, single lab, localization confirmed by IHC\",\n      \"pmids\": [\"36599408\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Simultaneous knockout of TPC1 and TPC2 in mice leads to mature-onset obesity due to impaired lipid availability and β-adrenergic receptor signaling in brown adipose tissue, with reduced phosphorylated hormone-sensitive lipase and β-adrenergic receptor expression, while mitochondrial respiratory chain function and UCP1 expression remain intact.\",\n      \"method\": \"Tpcn1/2 double knockout mice; body composition analysis; respirometry; BAT temperature measurement; Western blotting for HSL, β-adrenergic receptors, UCP1\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — double KO (cannot attribute phenotype solely to TPC1), defined metabolic phenotype with molecular characterization, single lab\",\n      \"pmids\": [\"25545384\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"In human metastatic colorectal cancer cells, NAADP-gated TPC1 triggers Ca2+ release from endo-lysosomes that is amplified by ER InsP3 receptors (CICR). Genetic silencing of TPC1 or pharmacological blockade reduces NAADP-evoked Ca2+ release, serum-induced Ca2+ signals, ERK and Akt phosphorylation, and cell proliferation.\",\n      \"method\": \"Liposomal NAADP delivery; Ca2+ imaging; GPN/nigericin lysosomal Ca2+ depletion; NED-19 pharmacology; TPC1 siRNA knockdown; Western blotting for ERK/Akt phosphorylation; proliferation assays\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic and pharmacological manipulation with multiple readouts, single lab, human primary cells\",\n      \"pmids\": [\"30991693\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TPC1 knockout in murine B16-F0 melanoma cells decreases mTORC1 activity, reduces proliferation and invasiveness, and increases pigmentation associated with nuclear localization of transcription factor TFEB, demonstrating TPC1 in early/recycling endosomes controls melanoma progression via mTORC1 and TFEB signaling.\",\n      \"method\": \"CRISPR/Cas9 TPC1 knockout in B16-F0 cells; mTORC1 activity assays; proliferation and invasion assays; TFEB localization imaging; pigmentation assay\",\n      \"journal\": \"Heliyon\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with defined cellular phenotypes and pathway placement (mTORC1/TFEB), single lab, single study\",\n      \"pmids\": [\"39524724\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"At lysosome-ER membrane contact sites, TPC1 forms a Ca2+ microdomain in close proximity to IP3R1; proximity of TPC1 to IP3R1 generates Ca2+-induced Ca2+ release that depletes ER Ca2+ stores and triggers lethal ER stress-induced apoptosis. Altering TPC1 expression levels in HeLa cells replicates these calcium dynamics.\",\n      \"method\": \"RAB7A activity assays; lysosome-ER proximity imaging; TPC1 overexpression/KO in HeLa cells; Ca2+ imaging; ER stress markers; BALB/c xenograft models\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Ca2+ imaging with TPC1 manipulation, in vivo xenograft validation, single lab, functional link to IP3R1 at MCS\",\n      \"pmids\": [\"40709664\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In Arabidopsis roots, TPC1 vacuolar channel participates in propagation of systemic Ca2+ waves in response to salt stress. The Ca2+ wave transmission requires both TPC1-dependent Ca2+ release and AtRBOHD NADPH oxidase-generated extracellular ROS; a ROS-assisted calcium-induced calcium-release (CICR) mechanism explains the observed wave speeds, as shown by ROS scavenger treatment, NADPH oxidase inhibition, and atrbohD knockout experiments.\",\n      \"method\": \"Fire-diffuse-fire modeling; Ca2+ wave imaging; ROS scavenger (ascorbate) and NADPH oxidase inhibitor (DPI) treatment; AtrbohD knockout analysis; extracellular ROS imaging\",\n      \"journal\": \"Plant physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO combined with pharmacological perturbations and quantitative modeling, plant ortholog, single lab\",\n      \"pmids\": [\"27261066\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Guard cells express higher TPC1 transcript levels and higher SV channel current density than mesophyll cells, and guard cell SV/TPC1 channels have higher cytosolic Ca2+ sensitivity than mesophyll cell channels, explaining the stomatal phenotype of tpc1-2 loss-of-function plants.\",\n      \"method\": \"Quantitative RT-PCR for TPC1 transcripts; patch clamp measurements of SV currents in guard cell and mesophyll vacuoles; comparison of Ca2+ dose-response curves\",\n      \"journal\": \"Plant & cell physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct electrophysiology comparing two cell types with transcript quantification, plant ortholog, single lab\",\n      \"pmids\": [\"20630987\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"TPC1 channels are specifically required for ROS-responsive Ca2+ influx in tobacco cells: cells cosuppressing endogenous TPC1 lose H2O2-induced Ca2+ influx, while AtTPC1 overexpression increases Al-sensitive (TPC1-dependent) Ca2+ influx. Hypoosmotic Ca2+ influx is TPC1-independent. Al ions specifically inhibit TPC1-mediated Ca2+ influx.\",\n      \"method\": \"Aequorin-based Ca2+ monitoring in BY-2 cells; TPC1 overexpression and cosuppression lines; Al, La, Gd ion pharmacology; H2O2 and hypoosmotic stimulation\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic gain- and loss-of-function with defined stimulus-specific Ca2+ phenotypes, plant ortholog, single lab\",\n      \"pmids\": [\"15464979\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Monocot TPC1 orthologs (OsTPC1 from rice, TaTPC1 from wheat) functionally rescue the SV channel deficit in Arabidopsis tpc1-2 vacuoles, demonstrating conserved vacuolar localization and function. When expressed in HEK293 cells, OsTPC1 targets to LysoTracker-positive organelles, confirming endomembrane localization across phyla.\",\n      \"method\": \"Cross-species complementation of tpc1-2 mutant; patch clamp of vacuoles from Arabidopsis and rice tpc1 null mutants; LysoTracker imaging in HEK293 cells\",\n      \"journal\": \"The New phytologist\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic complementation with direct electrophysiological readout plus heterologous localization, single lab\",\n      \"pmids\": [\"23845012\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2062,\n      \"finding\": \"JPT2/HN1L functions as an NAADP-binding protein required for NAADP-mediated Ca2+ release via TPC1/TPC2 in a cell-type-specific manner; it is indispensable in CD4+ T cells (TCR/CD3-evoked Ca2+ microdomains reduced in Jpt2/Hn1l−/− cells) but dispensable in cardiomyocytes, platelets, and mast cells.\",\n      \"method\": \"Jpt2/Hn1l−/− mouse generation; NAADP-evoked Ca2+ imaging in primary cardiomyocytes, mast cells, T cells; platelet aggregation assays; TCR/CD3 stimulation\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO mouse with cell-type-specific Ca2+ imaging across multiple primary cell types, preprint not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.11.11.687795\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"TPCN1 (TPC1) encodes a homodimeric, voltage-gated, Na+-selective cation channel residing in endolysosomal membranes, whose activity is regulated by depolarizing voltage (via the second 6-TM voltage-sensing domain), PtdIns(3,5)P2 binding to the first 6-TM domain, luminal Ca2+ and pH, and the second messenger NAADP (which requires accessory binding proteins such as JPT2); it generates local Ca2+ signals that drive SNARE/syntaxin-mediated vesicular fusion in early and recycling endosomes, supports systemic Ca2+ wave propagation (in plants, together with NADPH oxidase-derived ROS), regulates mast cell exocytosis by controlling endolysosomal Ca2+ uptake and ER store filling, controls mTORC1 and TFEB activity to influence melanoma proliferation, and facilitates endolysosomal trafficking required for myoseptal junction maintenance and proximal tubular phosphate reabsorption.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TPCN1 (TPC1) encodes a homodimeric, two-domain (2×6-TM) voltage-gated cation channel that resides in acidic endomembranes — early/recycling endosomes in animal cells and the vacuole in plants — where it converts membrane voltage and lipid/ion cues into local Ca2+ (and Na+) signals [#0, #1, #4, #19]. Cryo-EM of mouse TPC1 in apo-closed and PtdIns(3,5)P2-bound open states established that a coin-slot selectivity filter sets Na+ selectivity, that only the voltage-sensing domain of the second 6-TM module confers voltage dependence, and that endolysosome-specific PtdIns(3,5)P2 binds the first 6-TM domain to open the channel under depolarization [#0]; whole-organelle patch clamp confirmed TPC1 as a depolarization-activated, non-inactivating Na+ channel (lysoNaV) gated by luminal pH [#1]. In reconstituted bilayers human TPC1 is activated by the second messenger NAADP and requires acidic luminal pH and high luminal Ca2+, with voltage tuning NAADP affinity to support Ca2+ oscillations [#2]. Channel function depends on dimerization through a C-terminal antiparallel coiled-coil [#8], and in plant orthologs Ca2+ gating is set by cytosolic EF-hands and a distinct luminal Ca2+ site whose D454N (fou2) mutation removes luminal Ca2+ inhibition [#5, #6, #7]. Functionally, TPC1 supplies local Ca2+ for SNARE/syntaxin-mediated vesicle fusion in early/recycling endosomes and is required for endosomal toxin trafficking [#3, #4]. These channel-dependent Ca2+ signals are physiologically consequential: TPC1 controls endolysosomal Ca2+ uptake and ER store filling to regulate mast cell exocytosis and anaphylaxis [#9], drives endolysosome-to-ER calcium-induced calcium release that can deplete ER stores and trigger apoptosis [#15], supports myoseptal-junction maintenance via endolysosomal trafficking [#10] and dynamic proximal-tubular phosphate reabsorption [#11], and promotes cancer cell proliferation and progression through NAADP/CICR signaling and mTORC1/TFEB control [#13, #14]. In plants the vacuolar channel additionally propagates systemic Ca2+ waves in concert with NADPH-oxidase-derived ROS [#16].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Established that TPC1 is specifically required for ROS-evoked Ca2+ entry, distinguishing it from osmotically-driven Ca2+ pathways and giving the channel its first defined physiological stimulus.\",\n      \"evidence\": \"Aequorin Ca2+ monitoring in tobacco BY-2 cells with TPC1 cosuppression/overexpression and ion pharmacology\",\n      \"pmids\": [\"15464979\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not resolve subcellular site of the Ca2+ flux\", \"Channel biophysics and gating not directly measured\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identified a luminal Ca2+ recognition site, showing the fou2 D454N mutation removes Ca2+-dependent inhibition and links luminal Ca2+ sensing to gating.\",\n      \"evidence\": \"Patch clamp of Arabidopsis mesophyll vacuoles comparing wild-type and D454N channels under varying luminal Ca2+\",\n      \"pmids\": [\"19298454\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism connecting the site to the gate not yet mapped\", \"Plant ortholog only\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Resolved how cytosolic and luminal Ca2+ control gating by assigning EF-hand 2 as the essential activating site and defining the luminal Ca2+ pocket residues that couple to the gate.\",\n      \"evidence\": \"Site-directed mutagenesis of EF-hand and luminal acidic residues with vacuolar patch clamp and molecular modeling\",\n      \"pmids\": [\"21736651\", \"21764990\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Established in plant TPC1; animal regulation by luminal Ca2+ addressed separately\", \"No structural model at the time\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Demonstrated that human TPC1 is an NAADP-activated channel requiring acidic luminal pH and high luminal Ca2+, providing a biophysical mechanism for NAADP-induced Ca2+ oscillations.\",\n      \"evidence\": \"Lipid bilayer reconstitution of native and recombinant TPC1 with conductance, voltage, and ligand-affinity measurements\",\n      \"pmids\": [\"22500018\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"NAADP binding shown functionally but accessory binding protein not yet identified\", \"Single-lab in vitro system\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defined TPC1 as a depolarization-activated, non-inactivating endolysosomal Na+ channel (lysoNaV) gated by luminal pH, establishing its native ionic identity in animal organelles.\",\n      \"evidence\": \"Whole-organelle patch clamp with heterologous expression and luminal pH manipulation\",\n      \"pmids\": [\"24776928\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reconciliation of Na+ selectivity with NAADP-evoked Ca2+ signaling left open\", \"Downstream effectors not addressed\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Localized TPC1 function to early/recycling endosomal trafficking, distinguishing it from TPC2's lysosomal degradation role using clean knockouts.\",\n      \"evidence\": \"Tpcn1−/− and Tpcn2−/− MEFs with cholera toxin trafficking, pH, and morphology assays\",\n      \"pmids\": [\"25135478\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular partners mediating trafficking not yet defined\", \"Link between channel activity and trafficking step not established\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Showed C-terminal coiled-coil dimerization is obligatory for channel activity, defining the structural basis of the functional dimer.\",\n      \"evidence\": \"BiFC, Co-IP, synthetic peptide dimerization, mutagenesis, ion transport assay, and MD simulation\",\n      \"pmids\": [\"26781468\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How dimerization couples to gating not resolved\", \"Plant channel context\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Connected TPC1 channel function to vesicle fusion by identifying syntaxin interactors in native complexes, framing TPC1 as a local Ca2+ source for SNARE-mediated fusion.\",\n      \"evidence\": \"Native proteomics (MS) of TPC1 complexes plus co-localization and toxin-processing assays in knockout cells\",\n      \"pmids\": [\"28855648\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct Ca2+-to-SNARE causality not demonstrated\", \"Which syntaxins are functionally required not dissected\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Provided atomic-resolution mechanism: cryo-EM in closed and PtdIns(3,5)P2-bound open states explained Na+ selectivity, voltage-sensor asymmetry, and lipid-voltage interplay in activation.\",\n      \"evidence\": \"Cryo-EM of mouse TPC1 in two states with electrophysiology and mutagenesis\",\n      \"pmids\": [\"29562233\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"NAADP-bound conformation not captured\", \"Accessory protein interactions not in structure\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Established an in vivo immune role: TPC1 controls endolysosomal Ca2+ uptake and ER store filling to regulate mast cell degranulation and anaphylaxis.\",\n      \"evidence\": \"TPC1 knockout mice with passive systemic anaphylaxis, ex vivo degranulation, and Ca2+ homeostasis assays\",\n      \"pmids\": [\"32661165\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular link between channel flux and ER store filling not detailed\", \"Cell-type generality unaddressed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Placed endosomal TPC1 upstream of mTORC1 and TFEB signaling to drive melanoma proliferation and invasion, extending its role to growth-control pathways.\",\n      \"evidence\": \"CRISPR/Cas9 TPC1 knockout in B16-F0 cells with mTORC1, proliferation, invasion, TFEB localization, and pigmentation assays\",\n      \"pmids\": [\"39524724\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking channel Ca2+ to mTORC1 not resolved\", \"Single cell line, single study\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Resolved a spatial signaling mechanism: TPC1 forms a Ca2+ microdomain near IP3R1 at lysosome-ER contact sites to drive CICR, ER store depletion, and apoptosis.\",\n      \"evidence\": \"TPC1 overexpression/KO and IP3R1 proximity Ca2+ imaging in HeLa cells with ER-stress markers and xenografts\",\n      \"pmids\": [\"40709664\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Tethering machinery for the contact site not defined\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How accessory NAADP-binding proteins selectively confer NAADP sensitivity on TPC1 across cell types, and how the Na+-selective channel generates the Ca2+ signals attributed to it, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"JPT2/HN1L requirement is cell-type-specific and only reported in preprint\", \"Direct demonstration of TPC1-mediated Ca2+ flux vs Na+ flux at contact sites still lacking\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [0, 1, 2, 5, 8, 18]},\n      {\"term_id\": \"GO:0005216\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0140299\", \"supporting_discovery_ids\": [2, 5, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [3, 4, 14]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [1, 15, 19]},\n      {\"term_id\": \"GO:0005773\", \"supporting_discovery_ids\": [5, 6, 7, 17, 19]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [3, 4, 10]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [9, 16, 18]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [13, 14, 15]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"TPCN2\", \"IP3R1\", \"JPT2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":7,"faith_pct":85.71428571428571}}