{"gene":"TRPM2","run_date":"2026-04-28T21:43:00","timeline":{"discoveries":[{"year":2001,"finding":"LTRPC2/TRPM2 contains a C-terminal NUDT9 homology domain that functions as an ADP-ribose pyrophosphatase; free ADP-ribose (ADPR) directly gates TRPM2 as a Ca2+-permeable cation channel, establishing ADPR as a novel intracellular second messenger for Ca2+ entry.","method":"Whole-cell and single-channel patch-clamp in HEK-293 cells expressing LTRPC2; biochemical identification of NUDT9 domain; native current recording in U937 monocytes","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 — reconstituted channel activity with defined ligand, replicated in native cells, foundational paper with 782 citations","pmids":["11385575"],"is_preprint":false},{"year":2001,"finding":"Intracellular ADPR and NAD directly activate TRPM2 as a Ca2+-permeable nonselective cation channel in immunocytes; intracellular ATP suppresses this activation.","method":"Whole-cell patch-clamp and Ca2+ imaging in LTRPC2-expressing cells and native immunocytes","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 — electrophysiological reconstitution in heterologous and native cells, 389 citations, independent replication of ADPR gating","pmids":["11509734"],"is_preprint":false},{"year":2002,"finding":"TRPM2 is activated by H2O2 and reactive oxygen/nitrogen species; sensitivity to redox-state modifiers is attributable to agonistic binding of NAD+ to the MutT (Nudix) motif; arachidonic acid and Ca2+ are positive regulators; TRPM2 activation confers susceptibility to cell death.","method":"Whole-cell patch-clamp, Ca2+ imaging, antisense oligonucleotide knockdown in HEK cells and native cells; H2O2 and TNFα treatment","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal methods (electrophysiology, imaging, knockdown), replicated across cell types, 682 citations","pmids":["11804595"],"is_preprint":false},{"year":2002,"finding":"A splice variant of TRPM2 lacking amino acids 1292–1325 (C-terminal deletion) found in neutrophils is activated by H2O2 but not by ADPR, demonstrating that H2O2-mediated activation is independent of the ADPR-binding Nudix domain.","method":"Whole-cell patch-clamp in HEK-293 cells expressing wild-type or splice variant TRPM2; Ca2+ imaging; identification of splice variant by molecular cloning","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — functional dissection of activation modes with defined deletion variant, mechanistically rigorous","pmids":["11960981"],"is_preprint":false},{"year":2003,"finding":"A short isoform of TRPM2 (TRPM2-S), arising from alternative splicing that removes the four C-terminal transmembrane domains and pore region, localizes to the plasma membrane and directly interacts with full-length TRPM2-L (shown by co-immunoprecipitation); co-expression of TRPM2-S suppresses H2O2-induced Ca2+ influx through TRPM2-L and inhibits cell death, functioning as a dominant negative.","method":"Co-immunoprecipitation, confocal microscopy for localization, Ca2+ influx assay, cell death/apoptosis assay in HEK 293T cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP plus functional rescue, multiple readouts","pmids":["12594222"],"is_preprint":false},{"year":2005,"finding":"TRPM2 activation by oxidative stress or TNFα mediates cell death through increased intracellular Ca2+, followed by activation of caspases-8, -9, -3, -7 and PARP cleavage; chelation of intracellular Ca2+ with BAPTA blocked caspase/PARP cleavage and cell death, demonstrating Ca2+ is the critical intermediary.","method":"Retroviral overexpression/knockdown of TRPM2 isoforms in U937 cells; Ca2+ chelation; caspase/PARP western blot; viability and apoptosis assays","journal":"American journal of physiology. Cell physiology","confidence":"High","confidence_rationale":"Tier 2 — genetic manipulation plus pharmacological rescue, multiple mechanistic readouts","pmids":["16306129"],"is_preprint":false},{"year":2006,"finding":"Cyclic ADP-ribose (cADPR) and NAADP strongly activate natively expressed TRPM2 channels in Jurkat T cells; their activity is partially suppressed by the ADPR antagonist AMP and fully suppressed by 8-Br-cADPR, suggesting cADPR and NAADP share a common binding site on TRPM2 that acts synergistically with ADPR.","method":"Patch-clamp electrophysiology in Jurkat T cells; pharmacological antagonism with AMP and 8-Br-cADPR","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 — native cell electrophysiology with pharmacological dissection, single lab","pmids":["16585058"],"is_preprint":false},{"year":2007,"finding":"TRPM2 mediates H2O2-induced Ca2+ entry and increased endothelial permeability through an ADP-ribose–dependent mechanism; H2O2 stimulates ADPR formation, which opens TRPM2; siRNA depletion or dominant-negative TRPM2-S expression inhibited the cationic current, Ca2+ entry, and transendothelial electrical resistance decrease.","method":"Patch-clamp recording, fura-2 Ca2+ imaging, transendothelial electrical resistance measurement, siRNA knockdown, overexpression in endothelial cells","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods with genetic and pharmacological controls in native endothelial cells","pmids":["18048770"],"is_preprint":false},{"year":2009,"finding":"Intracellular Ca2+ activates TRPM2 via a calmodulin (CaM)-binding domain in the N-terminus, independent of ADPR-binding sites; Ca2+-mediated gating is the only known activator of ADPR-insensitive splice isoforms; both intracellular Ca2+ release and receptor-activation-induced Ca2+ release can activate endogenous TRPM2 in intact cells.","method":"Whole-cell patch-clamp, Ca2+ imaging, mutagenesis of ADPR-binding sites, CaM-binding domain identification, intact cell activation assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis combined with electrophysiology and imaging in multiple systems","pmids":["19372375"],"is_preprint":false},{"year":2011,"finding":"TRPM2 in dendritic cells localizes to endolysosomal vesicles (not the plasma membrane) and functions as a Ca2+ release channel from lysosomes, contributing to DC chemotaxis; TRPM2-deficient DCs show impaired maturation and directional migration in response to chemokines.","method":"siRNA knockdown and TRPM2-knockout mice; endolysosomal localization by confocal immunofluorescence; Ca2+ signaling assays; in vitro and in vivo chemotaxis assays","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 2 — genetic KO plus localization plus functional readouts in native DCs; replicated with siRNA and KO","pmids":["21753080"],"is_preprint":false},{"year":2012,"finding":"EFHC1 (mutated in juvenile myoclonic epilepsy) physically interacts with both N- and C-terminal cytoplasmic regions of TRPM2 (shown by co-immunoprecipitation) and potentiates H2O2- and ADPR-induced Ca2+ responses and cationic currents through TRPM2; JME mutations in EFHC1 reverse this potentiation.","method":"Co-immunoprecipitation, whole-cell patch-clamp, Ca2+ imaging in HEK293 cells; in situ hybridization for co-expression","journal":"Cell calcium","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP plus electrophysiological functional validation with disease mutations, single lab","pmids":["22226147"],"is_preprint":false},{"year":2013,"finding":"TRPM2 deficiency in mice modulates NMDAR subunit composition: TRPM2-null neurons upregulate GluN2A-containing NMDARs and downregulate GluN2B-containing NMDARs, augmenting prosurvival Akt/ERK signaling and inhibiting GSK3β, resulting in reduced ischemic infarct volume.","method":"TRPM2-knockout mice; middle cerebral artery occlusion model; field potential electrophysiology in brain slices; western blot for signaling proteins; pharmacological NMDAR subunit inhibitors","journal":"The Journal of neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 — genetic KO with mechanistic pathway placement, but mechanism is indirect (TRPM2 regulates NMDAR expression)","pmids":["24174660"],"is_preprint":false},{"year":2013,"finding":"TRPM2 channels mediate ischemic kidney injury through RAC1 activation: TRPM2 promotes RAC1 activation after ischemia; active RAC1 physically interacts with TRPM2 and increases TRPM2 plasma membrane expression, creating a positive feedback loop that amplifies NADPH oxidase activity and oxidative stress.","method":"TRPM2-knockout mice; chimeric mouse models; Co-IP of active RAC1 with TRPM2; pharmacological RAC1 inhibition in vivo; NADPH oxidase activity assays","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, genetic KO, pharmacological inhibition, chimeric mice for cell-type specificity; strong mechanistic evidence","pmids":["25295536"],"is_preprint":false},{"year":2015,"finding":"TRPM2-mediated Ca2+ influx is required to maintain cardiac myocyte mitochondrial function and protect against hypoxia-reoxygenation injury; loss-of-function TRPM2 mutants (E960D, P1018L) fail to reduce mitochondrial ROS in knockout myocytes, demonstrating Ca2+ entry (not reverse NCX) is the operative mechanism.","method":"Adenoviral rescue with WT and loss-of-function TRPM2 mutants in global TRPM2-KO hearts; mitochondrial superoxide measurements; oxygen consumption rate; cardiac-specific TRPM2-KO in ischemia-reperfusion","journal":"American journal of physiology. Heart and circulatory physiology","confidence":"High","confidence_rationale":"Tier 1–2 — mutagenesis rescue in KO background with multiple functional readouts; rigorous controls","pmids":["25576627"],"is_preprint":false},{"year":2015,"finding":"TRPM2 activation by H2O2 induces intracellular Ca2+ and Zn2+ elevation in pancreatic β-cells; Zn2+ originates from TRPM2-expressing lysosomes, and Ca2+-potentiated Zn2+ release (not Ca2+ alone) is primarily responsible for apoptotic β-cell death; TRPM2-KO mice are protected from streptozotocin-induced β-cell death.","method":"Zn2+ chelation experiments, lysosomal TRPM2 localization, Ca2+ and Zn2+ imaging, TRPM2-KO mouse model, INS1 and HEK293 cells, isolated islets","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal approaches (chelation, imaging, KO), mechanistically identifies Zn2+ as primary death mediator","pmids":["25562606"],"is_preprint":false},{"year":2015,"finding":"TRPM2 activation by oxidative stress regulates GSK-3 phosphorylation in brain via calcineurin: TRPM2 activation leads to Ca2+-dependent calcineurin activation, which dephosphorylates GSK-3; the bipolar disorder-associated D543E loss-of-function TRPM2 mutant fails to dephosphorylate GSK-3, and TRPM2-KO mice show increased GSK-3 phosphorylation and BD-like behavior.","method":"TRPM2-KO mice; overexpression of WT and D543E mutant; calcineurin inhibitor experiments; western blot for p-GSK-3; behavioral assays; EEG recordings","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 — genetic KO, disease mutant, pharmacological pathway placement via calcineurin inhibitors; multiple orthogonal methods","pmids":["26311765"],"is_preprint":false},{"year":2016,"finding":"Oxidative stress activates a TRPM2-Ca2+-CAMK2-BECN1 signaling cascade to inhibit autophagy: TRPM2-mediated Ca2+ influx activates CAMK2 (by phosphorylation and oxidation), which phosphorylates BECN1/Beclin-1 at Ser295, reducing BECN1–PIK3C3 association while increasing BECN1–BCL2 binding, thereby blocking early autophagy induction.","method":"Pharmacological TRPM2 inhibition and KO; CAMK2 phosphorylation/oxidation assays; BECN1 co-immunoprecipitation with PIK3C3 and BCL2; site-directed mutagenesis of Ser295; autophagy flux assays; validated in acetaminophen liver injury model","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 1–2 — mechanistic pathway with mutagenesis, Co-IP, multiple inhibitors, validated in two cell-death contexts","pmids":["27245989"],"is_preprint":false},{"year":2017,"finding":"2'-Deoxy-ADPR is an endogenous TRPM2 superagonist producing 10.4-fold higher whole-cell currents than ADPR at saturation, due to decreased inactivation rate and higher open probability; 2'-deoxy-ADPR is synthesized endogenously by NMNAT-2 and CD38 acting sequentially on NMN and 2'-deoxy-ATP, and was detected in Jurkat T cells by HPLC/mass spectrometry.","method":"Whole-cell patch-clamp; single-channel analysis; HPLC and mass spectrometry detection of endogenous 2'-deoxy-ADPR; in vitro enzymatic synthesis assay with NMNAT-2 and CD38","journal":"Nature chemical biology","confidence":"High","confidence_rationale":"Tier 1 — in vitro enzymatic reconstitution, mass spectrometry identification of endogenous molecule, electrophysiological characterization","pmids":["28671679"],"is_preprint":false},{"year":2017,"finding":"TRPM2 channel activation by Zn2+ in microglia requires a PKC/NADPH oxidase-mediated ROS generation and PARP-1 stimulation pathway; subsequently, TRPM2-mediated Ca2+ entry triggers a PYK2/MEK/ERK positive feedback mechanism that amplifies further TRPM2 activation, driving Ca2+ overload and cell death.","method":"TRPM2-KO microglia; pharmacological inhibitors of PKC (chelerythrine), NOX (DPI, GKT137831, Phox-I2), PARP (PJ34, DPQ), PYK2 (PF431396), MEK/ERK (U0126); Ca2+ imaging; cell death assays","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 — KO plus pharmacological pathway dissection, multiple inhibitors; single lab","pmids":["28322340"],"is_preprint":false},{"year":2017,"finding":"Neutrophil-derived reactive oxygen species activate TRPM2 in endothelial cells through ADPR generation (requiring PARP); TRPM2-mediated Ca2+ entry triggers VE-cadherin phosphorylation, adherens junction disassembly, and transendothelial neutrophil migration; conditional EC-specific TRPM2 deletion prevents PMN transmigration in LPS-challenged mice.","method":"Conditional EC-specific TRPM2 knockout mice; gp91phox-/- PMNs; ADPR-insensitive TRPM2 mutant (C1008A) overexpression; PARP silencing; Ca2+ entry and VE-cadherin phosphorylation assays; in vivo PMN transmigration","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 2 — conditional KO, functional mutant, multiple genetic/pharmacological controls, in vivo validation","pmids":["28790198"],"is_preprint":false},{"year":2017,"finding":"Ligand-induced TRPM2 activation critically depends on hydrogen bonding of Arg1433 and Tyr1349 in the NUDT9H domain with the terminal ribose of ADPR; all ADPR derivatives modified at the terminal ribose abolished agonist activity; these residues were identified by structure-guided mutagenesis combined with synthetic ADPR analogues.","method":"Site-directed mutagenesis of NUDT9H domain; patch-clamp electrophysiology in TRPM2-expressing HEK293 cells; synthetic ADPR analogue library; structure-activity relationship analysis","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 — complementary chemical biology (SAR with synthetic ligands) and mutagenesis approach, mechanistically defines key binding residues","pmids":["28515263"],"is_preprint":false},{"year":2017,"finding":"Palmitate-induced β-cell death involves TRPM2-mediated rise in mitochondrial Zn2+, which drives Drp-1 recruitment to mitochondria, mitochondrial fission and membrane potential loss; Zn2+ chelation alone prevents fission, and TRPM2 inhibition/KO prevents palmitate-induced fission in rodent and human islets.","method":"TRPM2 inhibition, RNAi silencing, and KO; mitochondrial Zn2+ and Ca2+ imaging; Drp-1 recruitment assay; membrane potential measurement; INS1 cells, mouse and human islets","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 — multiple genetic and pharmacological approaches, mechanistically identifies Zn2+ as mediator of mitochondrial fission, validated in human islets","pmids":["28753206"],"is_preprint":false},{"year":2018,"finding":"Cryo-EM structures of zebrafish TRPM2 in apo/closed and ADPR+Ca2+-bound/open states reveal that ADPR binds to a bi-lobed MHR1/2 domain (not exclusively NUDT9-H), and that NUDT9-H domains hang beneath MHR1/2; Ca2+ binding in the transmembrane region, together with ADPR at MHR1/2, is required for channel opening.","method":"Single-particle cryo-electron microscopy at near-atomic resolution; functional validation by electrophysiology","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 — high-resolution structure with functional validation, defines activation mechanism at atomic level","pmids":["30250252"],"is_preprint":false},{"year":2018,"finding":"Cryo-EM structures of human TRPM2 alone, with ADPR, and with ADPR+Ca2+ reveal the gating mechanism: NUDT9H forms intra- and intersubunit interactions with MHR1/2/3 in the apo state; ADPR binding causes MHR1/2 rotation and disrupts intersubunit interactions; Ca2+ binding further engages transmembrane helices and the TRP helix to open the lower gate.","method":"Single-particle cryo-electron microscopy of human TRPM2 in three states; structural comparison; functional electrophysiology","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 — high-resolution cryo-EM of human protein in multiple states, mechanistically defines concerted ADPR+Ca2+ gating","pmids":["30467180"],"is_preprint":false},{"year":2019,"finding":"TRPM2 Nudix motifs are canonical (enzymatically active ADPRases) in invertebrates but vestigial (inactive) in vertebrates; invertebrate TRPM2 (from Nematostella and Salpingoeca) possesses active ADPR phosphohydrolase activity; loss of ADPRase activity and loss of pore stability (inactivation) co-occurred in early vertebrate TRPM2 evolution.","method":"Comparative sequence analysis; in vitro ADPRase activity assays with isolated domains; whole-cell patch-clamp of TRPM2 orthologues (nvTRPM2, srTRPM2, drTRPM2, hsTRPM2); pore sequence mutagenesis","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 — in vitro enzymatic assay plus electrophysiology across multiple orthologues with mutagenesis, defines evolutionary loss of catalysis","pmids":["30938679"],"is_preprint":false},{"year":2020,"finding":"In dendritic cells, TRPM2 is required for NLRP3 inflammasome activation in response to ROS; TRPM2-mediated Ca2+ permeation links oxidative stress sensing to NLRP3 activation.","method":"TRPM2-KO macrophages/DCs; NLRP3 inflammasome activation assays; Ca2+ imaging; cytokine secretion (IL-1β); ROS measurements (review synthesizing experimental findings)","journal":"Current opinion in immunology","confidence":"Medium","confidence_rationale":"Tier 3 — review summarizing experimental data from multiple labs, no new primary data; moderate confidence based on cited primary experiments","pmids":["32058297"],"is_preprint":false},{"year":2021,"finding":"TRPM2 promotes atherogenesis through a TRPM2-CD36 axis: oxLDL activates CD36, which induces TRPM2 channel activity, and conversely TRPM2 promotes CD36 signaling; macrophage-specific or global Trpm2 deletion protects ApoE-/- mice from atherosclerosis by reducing oxLDL uptake, foam cell formation, and macrophage infiltration.","method":"Global and macrophage-specific Trpm2-KO in ApoE-/- atherosclerosis model; oxLDL and thrombospondin-1 stimulation assays; TRPM2 current recordings; CD36 signaling assays in KO and WT macrophages","journal":"Nature cardiovascular research","confidence":"High","confidence_rationale":"Tier 2 — cell-type-specific KO in disease model, bidirectional functional coupling demonstrated, multiple mechanistic readouts","pmids":["35421327"],"is_preprint":false},{"year":2022,"finding":"TRPM2 physically associates with extrasynaptic NMDARs; this TRPM2-NMDAR complex promotes surface expression of extrasynaptic NMDARs and enhances NMDAR-mediated excitotoxicity during ischemia; a membrane-permeable peptide targeting the NMDAR-interacting motif on TRPM2 disrupts the complex and protects neurons in vitro and in vivo.","method":"Co-immunoprecipitation of TRPM2-NMDAR complex; surface biotinylation assays for NMDAR expression; electrophysiology; cell death assays; in vivo MCAO stroke model with therapeutic peptide","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 2 — Co-IP, surface expression assays, in vitro and in vivo rescue with disrupting peptide; rigorous mechanistic validation","pmids":["35421327"],"is_preprint":false},{"year":2023,"finding":"Bilirubin acts as a direct extracellular agonist of TRPM2, gating the channel independently of canonical intracellular agonists (ADPR/Ca2+) by binding to a specific cavity; a knock-in loss-of-binding mutation (D1066A) antagonizes ischemic neurotoxicity; ischemic insults induce endogenous bilirubin release, creating a vicious cycle of TRPM2-mediated Ca2+-dependent brain injury.","method":"TRPM2-knockout mice; TRPM2 antagonist A23; D1066A knock-in mice; electrophysiology with bilirubin application; competitive binding assays; MCAO stroke model","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 1–2 — novel direct agonist identified by electrophysiology, validated by KO, antagonist, and point mutant knock-in mice in vivo","pmids":["36921602"],"is_preprint":false},{"year":2021,"finding":"TRPM2 protein is ubiquitinated by the E3 ligase TRIM21 at K1218; lncTRPM2-AS (a non-coding antisense RNA from the same locus) blocks TRIM21-mediated TRPM2 ubiquitination and degradation, maintaining TRPM2 protein levels and intracellular calcium signaling in macrophages.","method":"RNA-immunoprecipitation, mass spectrometry, Co-IP of TRIM21 with TRPM2, ubiquitination assays, K1218 mutation analysis, intracellular calcium measurement","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 — identifies E3 ligase and specific ubiquitination site on TRPM2 protein; mechanistically relevant to TRPM2 protein stability","pmids":["33903714"],"is_preprint":false},{"year":2023,"finding":"TRPM2-mediated Ca2+ influx promotes hepatic ischemia-reperfusion ferroptosis via Ca2+-induced mitochondrial Ca2+ accumulation (through the mitochondrial uniporter MCU) and upregulation of ALOX12, leading to mitochondrial lipid peroxidation; TRPM2 inhibition or Ca2+ depletion relieved ferroptosis in vitro and in vivo.","method":"TRPM2-KO mice; RNA sequencing; ALOX12 expression analysis; MCU-dependent mitochondrial Ca2+ measurement; ferroptosis assays; TRPM2 inhibitor treatment in vivo","journal":"Research (Washington, D.C.)","confidence":"Medium","confidence_rationale":"Tier 2 — KO plus mechanistic pathway (TRPM2→MCU→ALOX12→ferroptosis) with multiple readouts, single lab","pmids":["37275121"],"is_preprint":false}],"current_model":"TRPM2 is a homotetrameric, Ca2+-permeable non-selective cation channel activated by intracellular ADPR (binding the C-terminal NUDT9-H/MHR1-2 domain) and Ca2+ acting synergistically, by oxidative stress (H2O2/ROS) through PARP-dependent ADPR generation, by direct gating from bilirubin, and by intracellular Ca2+ alone via a CaM-binding N-terminal domain; cryo-EM structures define a two-step gating mechanism in which ADPR binding rotates MHR1/2 and disrupts NUDT9-H intersubunit contacts, while Ca2+ binding to transmembrane sites opens the lower gate, and downstream Ca2+ (and Zn2+) signals mediate diverse physiological and pathological outcomes including cell death via caspase/PARP activation, autophagy inhibition via CAMK2-BECN1 phosphorylation, NLRP3 inflammasome activation, GSK-3 regulation via calcineurin, ferroptosis via MCU-ALOX12, lysosomal Ca2+/Zn2+ release, and functional coupling with NMDARs and CD36 to amplify excitotoxicity and atherogenesis."},"narrative":{"teleology":[{"year":2001,"claim":"The foundational question of TRPM2's ligand identity and ion selectivity was answered: ADPR directly gates TRPM2 as a Ca2+-permeable nonselective cation channel via a C-terminal NUDT9 homology domain, establishing ADPR as a novel intracellular second messenger for Ca2+ entry.","evidence":"Whole-cell and single-channel patch-clamp in HEK-293 cells and native U937 monocytes/immunocytes, biochemical identification of NUDT9-H domain","pmids":["11385575","11509734"],"confidence":"High","gaps":["Whether NUDT9-H retains enzymatic ADPRase activity in vertebrate TRPM2 was unknown","Structural basis of ADPR recognition unresolved","Physiological source of ADPR in vivo not established"]},{"year":2002,"claim":"TRPM2 was shown to be a sensor of oxidative stress: H2O2 and ROS activate the channel through a mechanism partially independent of the ADPR-binding Nudix domain, as demonstrated by a splice variant lacking this region, linking TRPM2 to redox-sensitive cell death.","evidence":"Patch-clamp and Ca2+ imaging with H2O2/TNFα stimulation; antisense knockdown; functional characterization of NUDT9-H-deleted splice variant in HEK-293 cells","pmids":["11804595","11960981"],"confidence":"High","gaps":["Exact ROS-sensing mechanism (direct oxidation vs. intermediate metabolite generation) unresolved","Whether PARP-dependent ADPR generation is required for all ROS-mediated activation unclear"]},{"year":2003,"claim":"Discovery of the dominant-negative splice isoform TRPM2-S demonstrated that channel oligomerization is functionally regulatable: TRPM2-S directly interacts with full-length TRPM2-L and suppresses H2O2-induced Ca2+ influx and cell death.","evidence":"Co-immunoprecipitation, confocal localization, Ca2+ influx and cell death assays in HEK-293T cells","pmids":["12594222"],"confidence":"High","gaps":["Stoichiometry of TRPM2-S/L heteromers unknown","Physiological regulation of TRPM2-S expression not defined"]},{"year":2005,"claim":"The downstream cell-death mechanism was mapped: TRPM2-dependent Ca2+ entry activates caspases-8, -9, -3, -7 and PARP cleavage, and intracellular Ca2+ chelation fully blocks this cascade, establishing Ca2+ as the critical intermediary between channel opening and apoptosis.","evidence":"Retroviral overexpression/knockdown of TRPM2 isoforms in U937 cells; BAPTA-AM chelation; caspase/PARP western blot","pmids":["16306129"],"confidence":"High","gaps":["Whether Zn2+ contributes alongside Ca2+ was not yet explored","Cell-type specificity of the apoptotic pathway not addressed"]},{"year":2007,"claim":"TRPM2 was placed in endothelial barrier function: H2O2-stimulated ADPR formation opens TRPM2 in endothelial cells, causing Ca2+ entry and increased permeability; siRNA or dominant-negative TRPM2-S suppresses this response.","evidence":"Patch-clamp, fura-2 imaging, transendothelial electrical resistance in primary endothelial cells with siRNA and dominant-negative approaches","pmids":["18048770"],"confidence":"High","gaps":["Downstream targets linking TRPM2 Ca2+ to junctional disassembly (e.g., VE-cadherin phosphorylation) not yet identified"]},{"year":2009,"claim":"An ADPR-independent gating mechanism was uncovered: intracellular Ca2+ alone activates TRPM2 via an N-terminal calmodulin-binding domain, explaining how ADPR-insensitive splice variants retain channel function and establishing Ca2+ as both a co-agonist and a standalone gating signal.","evidence":"Patch-clamp with ADPR-binding-site mutations; CaM-binding domain identification; intact cell activation assays","pmids":["19372375"],"confidence":"High","gaps":["CaM-TRPM2 interface not structurally resolved","Relative contribution of CaM-dependent vs. transmembrane Ca2+-binding-site gating unknown"]},{"year":2011,"claim":"TRPM2 was shown to function as an intracellular (endolysosomal) Ca2+ release channel in dendritic cells, expanding its role beyond plasma membrane Ca2+ entry and linking it to immune cell chemotaxis and maturation.","evidence":"TRPM2-KO mice and siRNA knockdown; endolysosomal confocal localization; Ca2+ signaling and in vitro/in vivo chemotaxis assays in DCs","pmids":["21753080"],"confidence":"High","gaps":["How TRPM2 is targeted to lysosomes vs. plasma membrane not defined","Whether lysosomal TRPM2 gating uses the same ADPR/Ca2+ mechanism unclear"]},{"year":2015,"claim":"Three key downstream signaling axes were mapped: (1) lysosomal Zn2+ release (not Ca2+ alone) mediates β-cell apoptosis; (2) Ca2+-dependent calcineurin activation dephosphorylates GSK-3β (relevant to bipolar disorder); (3) TRPM2-mediated Ca2+ maintains cardiac mitochondrial function during ischemia-reperfusion, as confirmed by loss-of-function mutant rescue in KO cardiomyocytes.","evidence":"TRPM2-KO mice, Zn2+ chelation/imaging in islets, calcineurin inhibitor experiments plus D543E mutant analysis, and adenoviral rescue with E960D/P1018L mutants in KO hearts","pmids":["25562606","26311765","25576627"],"confidence":"High","gaps":["Mechanism of TRPM2-dependent lysosomal Zn2+ permeation not structurally resolved","Whether GSK-3β regulation is cell-type-specific beyond neurons unclear","Identity of cardiac TRPM2 agonist in ischemia-reperfusion not determined"]},{"year":2016,"claim":"TRPM2 was positioned as an autophagy regulator: TRPM2-mediated Ca2+ activates CAMK2, which phosphorylates BECN1 at Ser295 to disrupt BECN1-PIK3C3 association and promote BECN1-BCL2 binding, thereby inhibiting autophagy induction during oxidative stress.","evidence":"TRPM2 KO/inhibition; CAMK2 phosphorylation assays; BECN1 Co-IP with PIK3C3/BCL2; S295 mutagenesis; autophagy flux assays; acetaminophen liver injury model","pmids":["27245989"],"confidence":"High","gaps":["Whether autophagy inhibition is a general consequence of TRPM2 activation across tissues not tested","CAMK2 isoform specificity not established"]},{"year":2017,"claim":"Multiple advances refined TRPM2 pharmacology, structure-activity relationships, and tissue-specific signaling: 2′-deoxy-ADPR was identified as an endogenous superagonist; Arg1433/Tyr1349 in NUDT9-H were identified as critical ADPR-binding residues; lysosomal Zn2+ was linked to palmitate-induced mitochondrial fission via Drp-1; neutrophil ROS-TRPM2-VE-cadherin phosphorylation was established as the mechanism for endothelial barrier opening and transendothelial migration; and a PKC/NOX/PARP-1→TRPM2→PYK2/MEK/ERK positive feedback loop was mapped in microglia.","evidence":"Electrophysiology with synthetic ADPR analogues; mass spectrometry for endogenous 2′-deoxy-ADPR; conditional EC-TRPM2-KO mice with VE-cadherin phosphorylation assays; TRPM2-KO microglia with pharmacological inhibitor panels; Zn2+/Ca2+ imaging with Drp-1 recruitment in human islets","pmids":["28671679","28515263","28753206","28790198","28322340"],"confidence":"High","gaps":["Binding site for 2′-deoxy-ADPR not structurally resolved","Relative physiological concentrations of ADPR vs. 2′-deoxy-ADPR in most tissues unknown","PYK2-TRPM2 physical interaction not demonstrated"]},{"year":2018,"claim":"Cryo-EM structures of zebrafish and human TRPM2 in multiple ligand-bound states revealed a two-step gating mechanism: ADPR binds the MHR1/2 cleft (not solely NUDT9-H), rotating MHR1/2 and disrupting intersubunit NUDT9-H contacts, while Ca2+ binding at a transmembrane site engages the TRP helix to open the lower gate.","evidence":"Single-particle cryo-EM at near-atomic resolution of zebrafish and human TRPM2 in apo, ADPR-bound, and ADPR+Ca2+-bound states; functional validation by electrophysiology","pmids":["30250252","30467180"],"confidence":"High","gaps":["Open-state structure at true atomic resolution still lacking","CaM-binding domain and N-terminal gating contributions not resolved in structures","Mechanism of inactivation not captured"]},{"year":2019,"claim":"The evolutionary trajectory of TRPM2 was clarified: invertebrate TRPM2 retains active ADPRase catalysis in the Nudix domain, but this activity was lost in early vertebrate evolution concurrently with loss of pore stability, explaining why vertebrate NUDT9-H is vestigial for hydrolysis but critical for ligand-dependent gating.","evidence":"Comparative in vitro ADPRase assays with isolated domains from Nematostella, Salpingoeca, zebrafish, and human; patch-clamp of orthologues; pore mutagenesis","pmids":["30938679"],"confidence":"High","gaps":["Selective pressure driving loss of catalysis not established","Whether vestigial NUDT9-H has any residual product release function unknown"]},{"year":2021,"claim":"Two new functional partnerships were defined: TRPM2 is ubiquitinated by the E3 ligase TRIM21 at K1218 (regulated by the antisense lncRNA TRPM2-AS), and TRPM2 functionally couples with CD36 to amplify oxLDL uptake and foam cell formation, promoting atherogenesis in macrophages.","evidence":"TRIM21-TRPM2 Co-IP, K1218 mutation, ubiquitination assays, lncRNA-immunoprecipitation; global and macrophage-specific TRPM2-KO in ApoE−/− atherosclerosis model with CD36 signaling assays","pmids":["33903714","35421327"],"confidence":"High","gaps":["Whether TRIM21-mediated degradation is regulated by oxidative stress not tested","Structural basis of TRPM2-CD36 functional coupling unknown"]},{"year":2022,"claim":"TRPM2 was shown to physically associate with extrasynaptic NMDARs, promoting their surface expression and enhancing excitotoxic neuronal death during ischemia; a peptide disrupting this complex is neuroprotective in vivo.","evidence":"Co-immunoprecipitation of TRPM2-NMDAR complex; surface biotinylation; electrophysiology; therapeutic peptide in MCAO stroke model","pmids":["35421327"],"confidence":"High","gaps":["Interaction interface between TRPM2 and specific NMDAR subunits not structurally defined","Whether TRPM2-NMDAR coupling occurs in non-neuronal cells unknown"]},{"year":2023,"claim":"Bilirubin was identified as a direct extracellular agonist of TRPM2, gating the channel independently of ADPR/Ca2+ through a specific cavity (D1066), establishing a new non-canonical activation mode that amplifies ischemic brain injury in a feed-forward loop; additionally, TRPM2-mediated Ca2+ was linked to ferroptosis via an MCU-ALOX12 axis in hepatic ischemia-reperfusion.","evidence":"D1066A knock-in mice; TRPM2-KO; electrophysiology with bilirubin; competitive binding assays; MCAO model; RNA-seq with ALOX12 upregulation; MCU-dependent mitochondrial Ca2+ measurement; ferroptosis assays in vivo","pmids":["36921602","37275121"],"confidence":"High","gaps":["Bilirubin binding site not structurally resolved at atomic level","Whether bilirubin-TRPM2 signaling operates in non-neuronal tissues not tested","Relative contribution of ferroptosis vs. apoptosis in TRPM2-dependent cell death not quantified"]},{"year":null,"claim":"Key unresolved questions include: the structural basis of CaM-dependent gating, the mechanism by which TRPM2 is differentially sorted to plasma membrane vs. lysosomes, how bilirubin and ADPR gating interact when both are present, and whether distinct downstream death programs (apoptosis, ferroptosis, necrosis) are independently tunable by different TRPM2 activation modes.","evidence":"","pmids":[],"confidence":"Low","gaps":["CaM-TRPM2 structural interface unresolved","Lysosomal vs. plasma membrane sorting mechanism unknown","Integrated model of multi-agonist gating lacking","Cell-type-specific wiring of downstream death programs not systematically mapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[0,1,2,22,23]},{"term_id":"GO:0140299","term_label":"molecular sensor activity","supporting_discovery_ids":[2,8,28]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,1,4,7,12,19]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[9,14]}],"pathway":[{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[5,14,16,30]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[8,15,16,26]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[9,19,25]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[16]},{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[0,1,22,23]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[2,13,30]}],"complexes":["TRPM2 homotetramer"],"partners":["EFHC1","RAC1","TRIM21","CD36","GRIN2B","CAM"],"other_free_text":[]},"mechanistic_narrative":"TRPM2 is a Ca2+-permeable, non-selective cation channel that couples intracellular ADP-ribose (ADPR) sensing to Ca2+ (and Zn2+) entry, thereby transducing oxidative stress, metabolic, and inflammatory signals into diverse cellular outcomes including cell death, autophagy inhibition, inflammasome activation, and endothelial barrier regulation. The channel is gated by ADPR binding to the MHR1/2 cleft and by Ca2+ binding to transmembrane sites; cryo-EM structures show that ADPR rotates MHR1/2 and disrupts intersubunit NUDT9-H contacts while Ca2+ opens the lower gate, and additional agonists including 2′-deoxy-ADPR (a superagonist), cADPR, and bilirubin (an extracellular agonist acting independently of ADPR) expand the activation repertoire [PMID:30250252, PMID:30467180, PMID:28671679, PMID:36921602]. TRPM2-mediated Ca2+ influx drives downstream signaling through CAMK2-dependent BECN1 phosphorylation to inhibit autophagy, calcineurin-dependent GSK-3β dephosphorylation relevant to bipolar disorder-like phenotypes, caspase/PARP-mediated apoptosis, NLRP3 inflammasome priming, and MCU/ALOX12-dependent ferroptosis [PMID:27245989, PMID:26311765, PMID:16306129, PMID:37275121]. Beyond plasma-membrane Ca2+ entry, TRPM2 functions as a lysosomal Ca2+/Zn2+ release channel—lysosomal Zn2+ mobilization drives mitochondrial fission and β-cell death, while lysosomal TRPM2 supports dendritic cell chemotaxis—and physically associates with extrasynaptic NMDARs to promote their surface expression and excitotoxicity, and with CD36 to amplify oxLDL uptake and atherogenesis [PMID:25562606, PMID:28753206, PMID:21753080, PMID:35421327]."},"prefetch_data":{"uniprot":{"accession":"O94759","full_name":"Transient receptor potential cation channel subfamily M member 2","aliases":["Estrogen-responsive element-associated gene 1 protein","Long transient receptor potential channel 2","LTrpC-2","LTrpC2","Transient receptor potential channel 7","TrpC7","Transient receptor potential melastatin 2"],"length_aa":1503,"mass_kda":171.2,"function":"Nonselective, voltage-independent cation channel that mediates Na(+) and Ca(2+) influx, leading to increased cytoplasmic Ca(2+) levels (PubMed:11385575, PubMed:11509734, PubMed:11804595, PubMed:12594222, PubMed:15561722, PubMed:16601673, PubMed:19171771, PubMed:20660597, PubMed:25620041, PubMed:27068538, PubMed:27383051, PubMed:28775320, PubMed:29745897, PubMed:30467180, PubMed:31513012, PubMed:34788616). Functions as a ligand-gated ion channel, gated by intracellular adenosine diphosphate ribose (ADP-ribose), Ca(2+), warm temperature, and oxidative stress (PubMed:19171771, PubMed:25620041, PubMed:28775320, PubMed:30467180). The precise physiological activators are under debate; the true, physiological activators may be ADP-ribose and ADP-ribose-2'-phosphate (PubMed:20650899, PubMed:25918360). Binding of ADP-ribose to the cytoplasmic Nudix domain causes a conformation change; the channel is primed but still requires Ca(2+) binding to trigger channel opening (PubMed:19171771, PubMed:25620041, PubMed:28775320, PubMed:29745897, PubMed:30467180). Extracellular Ca(2+) passes through the channel and increases channel activity (PubMed:19171771). Contributes to Ca(2+) release from intracellular stores in response to ADP-ribose (PubMed:19454650). Plays a role in numerous processes that involve signaling via intracellular Ca(2+) levels (Probable). Besides, mediates the release of lysosomal Zn(2+) stores in response to reactive oxygen species, leading to increased cytosolic Zn(2+) levels (PubMed:25562606, PubMed:27068538). Plays a role in mediating behavorial and physiological responses to moderate heat and thereby contributes to body temperature homeostasis. Plays a role in insulin secretion, a process that requires increased cytoplasmic Ca(2+) levels (By similarity). Required for normal IFNG and cytokine secretion and normal innate immune immunity in response to bacterial infection. Required for normal phagocytosis and cytokine release by macrophages exposed to zymosan (in vitro) (PubMed:22493272). Plays a role in dendritic cell differentiation and maturation, and in dendritic cell chemotaxis via its role in regulating cytoplasmic Ca(2+) levels (By similarity). Plays a role in the regulation of the reorganization of the actin cytoskeleton and filopodia formation in response to reactive oxygen species via its role in increasing cytoplasmic Ca(2+) and Zn(2+) levels (PubMed:27068538). Confers susceptibility to cell death following oxidative stress (PubMed:12594222, PubMed:25562606) Lacks cation channel activity. Does not mediate cation transport in response to oxidative stress or ADP-ribose Lacks cation channel activity and negatively regulates the channel activity of isoform 1. Negatively regulates susceptibility to cell death in reposponse to oxidative stress","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/O94759/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TRPM2","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/TRPM2","total_profiled":1310},"omim":[{"mim_id":"616252","title":"TRPM8 CHANNEL-ASSOCIATED FACTOR 2; TCAF2","url":"https://www.omim.org/entry/616252"},{"mim_id":"616251","title":"TRPM8 CHANNEL-ASSOCIATED FACTOR 1; TCAF1","url":"https://www.omim.org/entry/616251"},{"mim_id":"612489","title":"RING FINGER PROTEIN 24; RNF24","url":"https://www.omim.org/entry/612489"},{"mim_id":"609633","title":"MAJOR AFFECTIVE DISORDER 3; MAFD3","url":"https://www.omim.org/entry/609633"},{"mim_id":"606678","title":"TRANSIENT RECEPTOR POTENTIAL CATION CHANNEL, SUBFAMILY M, MEMBER 8; TRPM8","url":"https://www.omim.org/entry/606678"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"bone marrow","ntpm":19.0}],"url":"https://www.proteinatlas.org/search/TRPM2"},"hgnc":{"alias_symbol":["KNP3","LTRPC2","NUDT9L1","NUDT9H","EREG1"],"prev_symbol":["TRPC7"]},"alphafold":{"accession":"O94759","domains":[{"cath_id":"-","chopping":"66-124","consensus_level":"medium","plddt":75.8722,"start":66,"end":124},{"cath_id":"-","chopping":"129-165_204-329","consensus_level":"medium","plddt":85.3925,"start":129,"end":329},{"cath_id":"-","chopping":"510-541_557-579_618-742","consensus_level":"high","plddt":88.5962,"start":510,"end":742},{"cath_id":"-","chopping":"748-928_1062-1099","consensus_level":"medium","plddt":82.9433,"start":748,"end":1099},{"cath_id":"-","chopping":"937-1041","consensus_level":"high","plddt":78.6916,"start":937,"end":1041},{"cath_id":"3.90.79.10","chopping":"1242-1500","consensus_level":"medium","plddt":81.4557,"start":1242,"end":1500},{"cath_id":"1.20.5","chopping":"1144-1184","consensus_level":"medium","plddt":70.8966,"start":1144,"end":1184}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O94759","model_url":"https://alphafold.ebi.ac.uk/files/AF-O94759-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O94759-F1-predicted_aligned_error_v6.png","plddt_mean":78.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TRPM2","jax_strain_url":"https://www.jax.org/strain/search?query=TRPM2"},"sequence":{"accession":"O94759","fasta_url":"https://rest.uniprot.org/uniprotkb/O94759.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O94759/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O94759"}},"corpus_meta":[{"pmid":"11385575","id":"PMC_11385575","title":"ADP-ribose gating of the calcium-permeable LTRPC2 channel revealed by Nudix motif homology.","date":"2001","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/11385575","citation_count":782,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"11804595","id":"PMC_11804595","title":"LTRPC2 Ca2+-permeable channel activated by changes in redox status confers susceptibility to cell death.","date":"2002","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/11804595","citation_count":682,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"11509734","id":"PMC_11509734","title":"Immunocyte Ca2+ influx system mediated by LTRPC2.","date":"2001","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/11509734","citation_count":389,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"11960981","id":"PMC_11960981","title":"Activation of the cation channel long transient receptor potential channel 2 (LTRPC2) by hydrogen peroxide. A splice variant reveals a mode of activation independent of ADP-ribose.","date":"2002","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11960981","citation_count":299,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"12594222","id":"PMC_12594222","title":"A novel TRPM2 isoform inhibits calcium influx and susceptibility to cell death.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12594222","citation_count":202,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"18048770","id":"PMC_18048770","title":"Role of TRPM2 channel in mediating H2O2-induced Ca2+ entry and endothelial hyperpermeability.","date":"2007","source":"Circulation research","url":"https://pubmed.ncbi.nlm.nih.gov/18048770","citation_count":191,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"30250252","id":"PMC_30250252","title":"Architecture of the TRPM2 channel and its activation mechanism by ADP-ribose and calcium.","date":"2018","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/30250252","citation_count":152,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"16585058","id":"PMC_16585058","title":"Nicotinic acid adenine dinucleotide phosphate and cyclic ADP-ribose regulate TRPM2 channels in T lymphocytes.","date":"2006","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/16585058","citation_count":148,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"30467180","id":"PMC_30467180","title":"Structures and gating mechanism of human TRPM2.","date":"2018","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/30467180","citation_count":146,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"21616534","id":"PMC_21616534","title":"Roles of TRPM2 in oxidative stress.","date":"2011","source":"Cell calcium","url":"https://pubmed.ncbi.nlm.nih.gov/21616534","citation_count":137,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"12765697","id":"PMC_12765697","title":"TRPM2 Ca2+ permeable cation channels: from gene to biological function.","date":"2003","source":"Cell calcium","url":"https://pubmed.ncbi.nlm.nih.gov/12765697","citation_count":136,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"19372375","id":"PMC_19372375","title":"Intracellular calcium activates TRPM2 and its alternative spliced isoforms.","date":"2009","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/19372375","citation_count":127,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"21753080","id":"PMC_21753080","title":"Dendritic cell maturation and chemotaxis is regulated by TRPM2-mediated lysosomal Ca2+ release.","date":"2011","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/21753080","citation_count":124,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"21140288","id":"PMC_21140288","title":"TRPM2 cation channels, oxidative stress and neurological diseases: where are we now?","date":"2010","source":"Neurochemical research","url":"https://pubmed.ncbi.nlm.nih.gov/21140288","citation_count":124,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"22975787","id":"PMC_22975787","title":"The TRPM2 ion channel, an oxidative stress and metabolic sensor regulating innate immunity and inflammation.","date":"2013","source":"Immunologic research","url":"https://pubmed.ncbi.nlm.nih.gov/22975787","citation_count":117,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"30925291","id":"PMC_30925291","title":"TRPM2 in Cancer.","date":"2019","source":"Cell calcium","url":"https://pubmed.ncbi.nlm.nih.gov/30925291","citation_count":107,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"16306129","id":"PMC_16306129","title":"TRPM2 is an ion channel that modulates hematopoietic cell death through activation of caspases and PARP cleavage.","date":"2005","source":"American journal of physiology. Cell physiology","url":"https://pubmed.ncbi.nlm.nih.gov/16306129","citation_count":106,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"25295536","id":"PMC_25295536","title":"TRPM2 mediates ischemic kidney injury and oxidant stress through RAC1.","date":"2014","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/25295536","citation_count":104,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"33130440","id":"PMC_33130440","title":"TRPM2 channel-mediated cell death: An important mechanism linking oxidative stress-inducing pathological factors to associated pathological conditions.","date":"2020","source":"Redox biology","url":"https://pubmed.ncbi.nlm.nih.gov/33130440","citation_count":102,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"15952035","id":"PMC_15952035","title":"TRPM2: a calcium influx pathway regulated by oxidative stress and the novel second messenger ADP-ribose.","date":"2005","source":"Pflugers Archiv : European journal of physiology","url":"https://pubmed.ncbi.nlm.nih.gov/15952035","citation_count":94,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"27245989","id":"PMC_27245989","title":"Mechanistic study of TRPM2-Ca(2+)-CAMK2-BECN1 signaling in oxidative stress-induced autophagy inhibition.","date":"2016","source":"Autophagy","url":"https://pubmed.ncbi.nlm.nih.gov/27245989","citation_count":89,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"24174660","id":"PMC_24174660","title":"Modulation of NMDAR subunit expression by TRPM2 channels regulates neuronal vulnerability to ischemic cell death.","date":"2013","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/24174660","citation_count":89,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"27957685","id":"PMC_27957685","title":"TRPM2 Promotes Neurotoxin MPP+/MPTP-Induced Cell Death.","date":"2016","source":"Molecular neurobiology","url":"https://pubmed.ncbi.nlm.nih.gov/27957685","citation_count":86,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"35421327","id":"PMC_35421327","title":"Functional coupling of TRPM2 and extrasynaptic NMDARs exacerbates excitotoxicity in ischemic brain injury.","date":"2022","source":"Neuron","url":"https://pubmed.ncbi.nlm.nih.gov/35421327","citation_count":86,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"32058297","id":"PMC_32058297","title":"TRPM2, linking oxidative stress and Ca2+ permeation to NLRP3 inflammasome activation.","date":"2020","source":"Current opinion in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/32058297","citation_count":82,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"20670202","id":"PMC_20670202","title":"TRPM2 channel properties, functions and therapeutic potentials.","date":"2010","source":"Expert opinion on therapeutic 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lesions.","date":"2003","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/14630798","citation_count":616,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"19056867","id":"PMC_19056867","title":"Large-scale proteomics and phosphoproteomics of urinary exosomes.","date":"2008","source":"Journal of the American Society of Nephrology : JASN","url":"https://pubmed.ncbi.nlm.nih.gov/19056867","citation_count":607,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"33845483","id":"PMC_33845483","title":"Multilevel proteomics reveals host perturbations by SARS-CoV-2 and SARS-CoV.","date":"2021","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/33845483","citation_count":532,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"11906815","id":"PMC_11906815","title":"Clusterin.","date":"2002","source":"The international journal of biochemistry & cell 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intracellular free ADP-ribose (ADPR), which binds to a C-terminal NUDT9 homology domain with ADP-ribose pyrophosphatase activity.\",\n      \"method\": \"Whole-cell and single-channel patch-clamp electrophysiology in HEK-293 cells expressing LTRPC2; NUDT9 domain homology identification; in vitro pyrophosphatase activity assay\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstitution in heterologous cells, enzymatic domain characterization, multiple orthogonal methods, foundational paper with 782 citations\",\n      \"pmids\": [\"11385575\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Intracellular ADPR and NAD directly activate TRPM2 as a Ca2+-permeable nonselective cation channel, enabling Ca2+ influx into immunocytes; this activation is suppressed by intracellular ATP.\",\n      \"method\": \"Whole-cell patch-clamp recording and Ca2+ imaging in LTRPC2-expressing cells and native immunocytes\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — direct electrophysiological and Ca2+ imaging, replicated across labs\",\n      \"pmids\": [\"11509734\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"TRPM2 is activated by H2O2 through a mechanism independent of ADPR binding; a splice variant lacking C-terminal amino acids 1292–1325 retains H2O2 sensitivity but loses ADPR responsiveness, indicating two distinct activation pathways.\",\n      \"method\": \"Whole-cell patch-clamp and intracellular Ca2+ measurements in HEK293 cells expressing wild-type or splice variant TRPM2; H2O2 and ADPR stimulation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — direct electrophysiology with splice variant dissection, multiple orthogonal methods\",\n      \"pmids\": [\"11960981\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"TRPM2 activation by H2O2 and redox-state modifiers (including beta-NAD+) confers susceptibility to cell death; arachidonic acid and Ca2+ are positive regulators; antisense knockdown reduces H2O2- and TNFα-induced Ca2+ influx and cell death.\",\n      \"method\": \"Heterologous expression in HEK cells; antisense oligonucleotide knockdown; Ca2+ imaging; cell viability assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including KD with defined phenotypic readout, 682 citations\",\n      \"pmids\": [\"11804595\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"A short isoform TRPM2-S (lacking the four C-terminal transmembrane domains and pore region) physically interacts with full-length TRPM2-L, localizes to the plasma membrane similarly, and acts as a dominant negative to suppress H2O2-induced Ca2+ influx and reduce apoptosis.\",\n      \"method\": \"Immunoprecipitation; confocal microscopy; Ca2+ influx assays; cell viability assays in HEK293T cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, localization, functional Ca2+ and apoptosis assays, multiple methods\",\n      \"pmids\": [\"12594222\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"TRPM2 activation by H2O2 or TNFα induces Ca2+ overload that triggers caspase-8, -9, -3, and -7 activation and PARP cleavage; chelation of intracellular Ca2+ with BAPTA blocks this caspase/PARP cascade and cell death.\",\n      \"method\": \"Retroviral overexpression and RNAi knockdown of TRPM2 isoforms in U937 cells; caspase activity assays; PARP cleavage immunoblotting; Ca2+ chelation experiments\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods with defined molecular mechanism, BAPTA rescue experiment\",\n      \"pmids\": [\"16306129\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"cADPR and NAADP strongly activate native TRPM2 channels in Jurkat T cells, partially via mobilization of endogenous ADPR; a remaining component represents direct gating that is blocked by 8-Br-cADPR, suggesting cADPR and NAADP share a common binding site and co-activate TRPM2 synergistically with ADPR.\",\n      \"method\": \"Patch-clamp electrophysiology on native T cells; pharmacological dissection with AMP antagonist and 8-Br-cADPR\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — native cell electrophysiology with pharmacological dissection, multiple agonist/antagonist tools\",\n      \"pmids\": [\"16585058\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"TRPM2 mediates H2O2-induced Ca2+ entry and endothelial hyperpermeability; H2O2 stimulates ADP-ribose formation, which opens TRPM2; siRNA depletion, dominant-negative splice variant overexpression, or inhibition of ADP-ribose formation all attenuate the cationic current, Ca2+ entry, and decrease in transendothelial electrical resistance.\",\n      \"method\": \"Patch-clamp recording; fura-2 Ca2+ imaging; transendothelial electrical resistance measurement; siRNA knockdown; dominant-negative overexpression in endothelial cell monolayers\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal approaches in native endothelial cells with defined functional readout\",\n      \"pmids\": [\"18048770\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Intracellular Ca2+ activates TRPM2 independently of ADPR through a calmodulin (CaM)-binding domain in the TRPM2 N-terminus; both Ca2+ and the CaM-binding motif are required for ADPR-mediated gating; this Ca2+-dependent mechanism is also the primary activator of ADPR-insensitive TRPM2 splice isoforms.\",\n      \"method\": \"Patch-clamp electrophysiology; site-directed mutagenesis of ADPR-binding site; CaM-binding domain mutants; Ca2+ uncaging in intact cells\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis combined with electrophysiology, mechanistic dissection of two independent activation pathways\",\n      \"pmids\": [\"19372375\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"In dendritic cells, TRPM2 localizes to endolysosomal vesicles (not plasma membrane) and functions as a Ca2+ release channel contributing to chemokine-stimulated Ca2+ signaling; TRPM2-deficient DCs show impaired maturation and chemotaxis, while in neutrophils TRPM2 localizes to the plasma membrane.\",\n      \"method\": \"siRNA and knockout mice; subcellular localization imaging; Ca2+ signaling measurements; chemotaxis assays; bacterial trafficking experiments in vivo\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO mice plus localization plus functional phenotype, multiple orthogonal approaches\",\n      \"pmids\": [\"21753080\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"EFHC1, a protein mutated in juvenile myoclonic epilepsy, physically interacts with both the N- and C-terminal cytoplasmic regions of TRPM2 and potentiates H2O2- and ADPR-induced Ca2+ responses and cationic currents; JME mutations in EFHC1 reverse this potentiation.\",\n      \"method\": \"Co-immunoprecipitation; patch-clamp electrophysiology; Ca2+ imaging; coexpression in HEK293 cells; in situ hybridization\",\n      \"journal\": \"Cell calcium\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus functional electrophysiology plus mutagenesis validation\",\n      \"pmids\": [\"22226147\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"TRPM2 activation promotes NMDAR-modulated neuronal vulnerability to ischemia; TRPM2-null mice have smaller infarcts after middle cerebral artery occlusion; loss of TRPM2 upregulates GluN2A-containing NMDARs and augments prosurvival Akt/ERK signaling while inhibiting GSK3β.\",\n      \"method\": \"TRPM2-null mice; middle cerebral artery occlusion model; field potential recordings in brain slices; GluN2A/GluN2B subunit-selective antagonists; Akt/ERK/GSK3β immunoblotting\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse with defined infarct phenotype plus molecular pathway analysis\",\n      \"pmids\": [\"24174660\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ADPR activates TRPM2 by binding to the cytosolic C-terminal NUDT9-H domain; chemical biology mapping identified that the terminal ribose of ADPR and residues Arg1433 and Tyr1349 are critical for channel activation; 8-Ph-ADPR acts as an antagonist and inhibits Ca2+ signaling and chemotaxis in human neutrophils.\",\n      \"method\": \"Patch-clamp in HEK293 cells overexpressing TRPM2; systematic ADPR analogue synthesis and testing; site-directed mutagenesis of NUDT9-H domain residues\",\n      \"journal\": \"Journal of medicinal chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro assay combined with mutagenesis of defined binding-site residues\",\n      \"pmids\": [\"24304219\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Androgen signaling is required for TRPM2-mediated neuronal death in male ischemic brain; PARP-1 activity drives male-specific TRPM2 activation; PARP-1 knockout reduces ischemic damage and abolishes TRPM2 inhibitor protection, placing PARP-1 upstream of TRPM2 in this cell-death pathway.\",\n      \"method\": \"TRPM2 inhibitor (clotrimazole) in vivo; castration and androgen replacement; PARP-1 knockout mice; PARP activity assays in peri-infarct brain\",\n      \"journal\": \"Journal of cerebral blood flow and metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — epistasis with KO mice and pharmacological rescue, multiple orthogonal in vivo approaches\",\n      \"pmids\": [\"23801245\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TRPM2 mediates ischemic kidney injury through RAC1; TRPM2 promotes RAC1 activation after ischemia, active RAC1 physically interacts with TRPM2 and increases TRPM2 plasma membrane expression, forming a positive feedback loop that amplifies NADPH oxidase-dependent oxidative stress.\",\n      \"method\": \"TRPM2-deficient mice; chimeric bone-marrow transplant; RAC1 activity assays; Co-immunoprecipitation of RAC1 and TRPM2; NADPH oxidase activity; RAC1 inhibition in vivo\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO mice with chimeric controls plus Co-IP establishing physical interaction and feedback loop\",\n      \"pmids\": [\"25295536\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TRPM2 activation regulates GSK-3 phosphorylation via calcineurin, a Ca2+-dependent phosphatase; the BD-associated D543E loss-of-function mutation fails to induce GSK-3 dephosphorylation; Trpm2-deficient mice show increased phospho-GSK-3 and bipolar disorder-related behavior unresponsive to lithium.\",\n      \"method\": \"Trpm2-knockout and D543E knock-in mice; behavioral testing; EEG; calcineurin pharmacological inhibition; GSK-3 phosphorylation immunoblotting\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO mice with defined behavioral phenotype plus molecular pathway placement via calcineurin inhibitor and phosphorylation assays\",\n      \"pmids\": [\"26311765\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Ca2+ entry via TRPM2 is required to maintain cardiac myocyte mitochondrial function and bioenergetics; loss-of-function mutations (E960D, P1018L) abolish this protection; Ca2+ influx (not Na+/Ca2+ exchange) is the critical mediator, and cardiac-specific TRPM2 KO worsens ischemia-reperfusion injury.\",\n      \"method\": \"Adenoviral expression of WT and loss-of-function TRPM2 mutants in global KO hearts; oxygen consumption rate measurements; mitochondrial ROS imaging; cardiac-specific KO in I/R model\",\n      \"journal\": \"American journal of physiology. Heart and circulatory physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — mutagenesis reconstitution in KO background plus in vivo cardiac phenotype\",\n      \"pmids\": [\"25576627\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TRPM2-mediated lysosomal Zn2+ release (in addition to Ca2+) is the primary cytotoxic signal in ROS-induced pancreatic β-cell death; TRPM2 channels on lysosomes release Zn2+ in a Ca2+-potentiated manner; chelation of Zn2+ alone prevents apoptosis, and TRPM2-KO mice are protected from streptozotocin-induced β-cell loss.\",\n      \"method\": \"INS1 cells, HEK293-TRPM2 cells, pancreatic islets; Zn2+-specific fluorescent chelation; lysosomal TRPM2 localization; TRPM2-KO mice with streptozotocin model\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO mice, lysosomal localization, Zn2+ chelation rescue, multiple cell types\",\n      \"pmids\": [\"25562606\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Oxidative stress activates TRPM2-mediated Ca2+ influx, which activates CaMKII (by both phosphorylation and oxidation); activated CaMKII phosphorylates BECN1/Beclin1 at Ser295, reducing BECN1-PIK3C3 association and increasing BECN1-BCL2 binding, thereby inhibiting autophagy and promoting cell death.\",\n      \"method\": \"TRPM2 KD/inhibition; CaMKII phosphorylation and oxidation assays; BECN1 phosphorylation mutants; Co-IP of BECN1 with VPS34 and BCL2; primary hepatocyte APAP model\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — defined signaling cascade with phospho-site mutagenesis, Co-IP, and in vitro pathway mapping\",\n      \"pmids\": [\"27245989\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"2'-Deoxy-ADPR is an endogenous TRPM2 superagonist (10.4-fold higher whole-cell currents than ADPR at saturation) that acts by decreasing channel inactivation rate and increasing open probability; NMNAT-2 and CD38 sequentially synthesize 2'-deoxy-ADPR from NMN and 2'-deoxy-ATP in vitro.\",\n      \"method\": \"Patch-clamp electrophysiology; HPLC and mass spectrometry detection in Jurkat T cells; in vitro enzymatic synthesis assays with NMNAT-2 and CD38\",\n      \"journal\": \"Nature chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro enzymatic reconstitution, endogenous molecule detection by MS, single-channel gating analysis\",\n      \"pmids\": [\"28671679\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Ligand-induced TRPM2 activation critically requires the terminal ribose of ADPR; hydrogen bonding of Arg1433 and Tyr1349 in the NUDT9-H domain with the terminal ribose is essential for channel gating.\",\n      \"method\": \"Patch-clamp; synthetic ADPR analogues with terminal ribose modifications; site-directed mutagenesis of NUDT9-H domain residues\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — complementary chemical biology (ligand modification) and mutagenesis, defining molecular contacts for gating\",\n      \"pmids\": [\"28515263\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Zn2+ signaling through TRPM2 promotes palmitate-induced mitochondrial fission in pancreatic β-cells; palmitate activates NOX2 to generate ROS, which activates TRPM2, causing a rise in mitochondrial Zn2+ that recruits Drp-1 and induces fission; Ca2+ entry alone is insufficient without Zn2+.\",\n      \"method\": \"TRPM2 RNAi and KO; NOX2 inhibition; mitochondrial Zn2+/Ca2+ imaging; Drp-1 recruitment assay; islet studies\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO/RNAi plus mechanistic dissection of Zn2+ vs Ca2+ with chelators, multiple cell systems\",\n      \"pmids\": [\"28753206\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"In endothelial cells, PMN-generated ROS activate TRPM2 via ADPR, causing Ca2+ entry, VE-cadherin phosphorylation, adherens junction disassembly, and transendothelial neutrophil migration; the ADPR-insensitive C1008A TRPM2 mutant and PARP silencing prevent PMN transmigration.\",\n      \"method\": \"Conditional endothelial Trpm2 deletion; C1008A gain-of-function mutant; gp91phox-/- PMNs; PARP silencing; Ca2+ imaging; VE-cadherin phosphorylation assays\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO, dominant mutant, and PARP epistasis, multiple orthogonal approaches\",\n      \"pmids\": [\"28790198\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Cryo-EM structures of zebrafish TRPM2 in apo (closed) and ADPR/Ca2+-bound (open) states reveal that ADPR binds in the bi-lobed MHR1/2 domain and that NUDT9-H domains hang underneath MHR1/2; Ca2+ binding cooperates with ADPR to gate the channel open.\",\n      \"method\": \"Cryo-electron microscopy structure determination of zebrafish TRPM2 in multiple states\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structures in both apo and ligand-bound states, defining binding site architecture\",\n      \"pmids\": [\"30250252\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Cryo-EM structures of human TRPM2 reveal that in the apo state, NUDT9-H forms both intra- and intersubunit interactions with MHR1/2/3; ADPR binding disrupts intersubunit NUDT9-H contacts and rotates MHR1/2; subsequent Ca2+ binding engages transmembrane helices and the TRP helix to open the lower gate, defining the concerted two-ligand gating mechanism.\",\n      \"method\": \"Cryo-EM of human TRPM2 alone, with ADPR, and with ADPR+Ca2+\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple cryo-EM states capturing stepwise conformational changes, rigorous structural biochemistry\",\n      \"pmids\": [\"30467180\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Evolutionarily, vertebrate TRPM2 simultaneously lost its NUDT9-H ADPRase catalytic activity and acquired pore inactivation; invertebrate TRPM2 (Nematostella, Salpingoeca) retains active ADPRase; disrupting enzymatic activity in nvTRPM2 does not affect ADPR-dependent gating but enables H2O2 sensitivity, indicating catalytic and gating functions are uncoupled.\",\n      \"method\": \"ADPRase activity assays of isolated NUDT9-H domains; patch-clamp of invertebrate and vertebrate TRPM2 orthologs; mutagenesis of Nudix box; deletion of NUDT9-H in nvTRPM2\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro enzymatic reconstitution combined with electrophysiology and mutagenesis across multiple orthologs\",\n      \"pmids\": [\"30938679\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TRPM2 deficiency in neural progenitor cells (NPCs) reduces proliferation and increases premature neuronal differentiation during hyperthermia; mechanistically, TRPM2 inhibits β-catenin phosphorylation and increases β-catenin expression to maintain SP5 levels and NPC self-renewal.\",\n      \"method\": \"TRPM2 knockdown and knockout in mouse embryonic brain; NPC proliferation assays; SP5 and β-catenin pathway analysis; rescue with constitutive TRPM2 or SP5 expression\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO with defined cellular phenotype, pathway rescue with specific genes\",\n      \"pmids\": [\"31911949\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TRPM2 ubiquitination at K1218 by the E3 ubiquitin ligase TRIM21 targets TRPM2 for degradation; lncTRPM2-AS blocks TRIM21-TRPM2 interaction, preventing ubiquitination and stabilizing TRPM2 protein; loss of lncTRPM2-AS reduces TRPM2 expression, decreases intracellular Ca2+, and promotes autophagy-induced macrophage apoptosis.\",\n      \"method\": \"RIP and mass spectrometry identifying TRIM21 interaction; TRPM2 K1218 mutation analysis; Co-IP of TRIM21-TRPM2; ubiquitination assays; Ca2+ and ROS measurements\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mass spectrometry-identified interaction, site-specific ubiquitination mutagenesis, multiple functional readouts\",\n      \"pmids\": [\"34903714\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TRPM2 physically and functionally couples to extrasynaptic NMDARs via a specific NMDAR-interacting motif on TRPM2; this coupling promotes surface expression of extrasynaptic NMDARs and enhances NMDAR activity during ischemia; a membrane-permeable disrupting peptide targeting this interface reduces neuronal death in vitro and infarct volume in vivo.\",\n      \"method\": \"Co-immunoprecipitation; surface NMDAR expression assays; disrupting peptide design; in vitro ischemia model; mouse MCAO stroke model\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP establishing physical complex, peptide disruption with defined molecular and in vivo functional consequences\",\n      \"pmids\": [\"35421327\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TRPM2 and CD36 activate each other in a positive feedback loop in macrophages; CD36 ligands (oxLDL and TSP1) activate TRPM2, and TRPM2 activity in turn sustains CD36 signaling; global and macrophage-specific Trpm2 deletion reduces oxLDL uptake, foam cell formation, and atherogenesis in Apoe-/- mice.\",\n      \"method\": \"Global and macrophage-specific Trpm2 KO in Apoe-/- atherosclerosis model; patch-clamp with CD36 ligands; CD36 signaling cascade analysis; foam cell formation assays\",\n      \"journal\": \"Nature cardiovascular research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — macrophage-specific KO plus electrophysiology establishing bidirectional TRPM2-CD36 relationship\",\n      \"pmids\": [\"35445217\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Bilirubin directly gates the TRPM2 channel as an agonist, independent of canonical intracellular agonists ADPR and Ca2+; bilirubin binds to a cavity also recognized by antagonist A23; a knock-in point mutation D1066A abolishing bilirubin binding protects mice against ischemic neurotoxicity.\",\n      \"method\": \"Patch-clamp electrophysiology with bilirubin and metabolites; competitive binding with antagonist A23; TRPM2-D1066A knock-in mice; mouse ischemic stroke model\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — novel agonist identification by electrophysiology, binding-site mutagenesis in vivo, functional validation in knock-in model\",\n      \"pmids\": [\"36921602\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TRPM2-mediated Ca2+ influx causes mitochondrial Ca2+ accumulation via the mitochondrial Ca2+ uniporter, which increases ALOX12 expression leading to mitochondrial lipid peroxidation and ferroptotic hepatocyte death during ischemia-reperfusion injury.\",\n      \"method\": \"TRPM2 KO mice; TRPM2 inhibitor; mitochondrial Ca2+ uniporter blockade; ALOX12 expression analysis; ferroptosis pathway assays; RSL3 treatment\",\n      \"journal\": \"Research (Washington, D.C.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO plus mechanistic pathway from TRPM2→Ca2+→MCU→ALOX12→ferroptosis with pharmacological validation\",\n      \"pmids\": [\"37275121\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TRPM2-mediated Ca2+ influx activates the AKT-mTOR signaling pathway to suppress autophagy; TRPM2 deficiency in cisplatin-induced AKI exacerbates mitochondrial damage by blocking Ca2+ influx-dependent autophagy; mTOR inhibition rescues TRPM2-deficient mice from cisplatin nephrotoxicity.\",\n      \"method\": \"TRPM2-KO mice; RNA sequencing; mTOR inhibitor rescue; Ca2+ flux measurements; autophagy flux assays\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse with mTOR inhibitor epistasis rescue defining pathway position\",\n      \"pmids\": [\"37649595\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In Nematostella vectensis TRPM2, the NUDT9-H domain retains ADPRase enzymatic activity and exerts catalytic function independent of gating; ADP-ribose activates nvTRPM2 through a novel mechanism not requiring the NUDT9-H domain for gating, since deletion of NUDT9-H does not abolish ADPR-dependent channel opening.\",\n      \"method\": \"Patch-clamp; NUDT9-H deletion and domain-swap experiments; Ca2+ imaging; Western blot; ADPRase co-expression experiments\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — domain deletion and swap mutagenesis with functional electrophysiology, enzymatic activity assays\",\n      \"pmids\": [\"27333281\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Zn2+ activates TRPM2 in microglia through a PKC/NOX-mediated ROS and PARP-1 pathway; TRPM2-mediated Ca2+ entry then triggers PYK2/MEK/ERK signaling as a positive feedback mechanism amplifying TRPM2 activation and leading to Ca2+ overload and cell death.\",\n      \"method\": \"TRPM2-KO microglia; PARP inhibitors; PKC inhibitor; NOX inhibitors; PYK2 and MEK/ERK inhibitors; Ca2+ imaging; patch-clamp\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO with systematic pharmacological pathway dissection establishing signaling cascade\",\n      \"pmids\": [\"28322340\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In cerebral endothelial cells, TRPM2 and CD36 promote reciprocal activation; OGD and TSP1 activate TRPM2 in a CD36-dependent manner; endothelial-specific Trpm2 deletion reduces BBB degradation, Ca2+ overload, oxidative stress, and infarct size in ischemic stroke mice.\",\n      \"method\": \"Cdh5-Cre endothelial-specific Trpm2 KO; HEK293T transfection with TRPM2 and CD36; OGD model; TSP1 stimulation; BBB permeability assays; MCAO model\",\n      \"journal\": \"Cardiovascular research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — endothelial cell-specific KO with mechanistic cell biology establishing TRPM2-CD36 reciprocal activation\",\n      \"pmids\": [\"37595268\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TRPM2 is a tetrameric, Ca2+-permeable non-selective cation channel with a C-terminal NUDT9-H domain that binds ADP-ribose (ADPR) as the primary activating ligand; channel opening requires synergistic binding of intracellular ADPR (or the superagonist 2'-deoxy-ADPR) and Ca2+ (acting through a calmodulin-binding N-terminal domain), as revealed by cryo-EM structures defining the conformational transitions from apo-closed to ADPR/Ca2+-bound-open states; H2O2 and other ROS activate the channel via PARP-1-dependent generation of ADPR, while additional direct agonists include bilirubin and NAADP/cADPR; in different cell types TRPM2 resides at the plasma membrane or on endolysosomes, mediating Ca2+ entry or Ca2+/Zn2+ release respectively; downstream, TRPM2-mediated Ca2+ influx drives caspase/PARP cell death cascades, activates CaMKII→BECN1 to suppress autophagy, dephosphorylates GSK-3 via calcineurin, couples to NMDARs and CD36 through direct protein interactions, and engages ALOX12-dependent ferroptosis, while its protein stability is regulated by TRIM21-mediated ubiquitination at K1218.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2001,\n      \"finding\": \"LTRPC2/TRPM2 contains a C-terminal NUDT9 homology domain that functions as an ADP-ribose pyrophosphatase; free ADP-ribose (ADPR) directly gates TRPM2 as a Ca2+-permeable cation channel, establishing ADPR as a novel intracellular second messenger for Ca2+ entry.\",\n      \"method\": \"Whole-cell and single-channel patch-clamp in HEK-293 cells expressing LTRPC2; biochemical identification of NUDT9 domain; native current recording in U937 monocytes\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted channel activity with defined ligand, replicated in native cells, foundational paper with 782 citations\",\n      \"pmids\": [\"11385575\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Intracellular ADPR and NAD directly activate TRPM2 as a Ca2+-permeable nonselective cation channel in immunocytes; intracellular ATP suppresses this activation.\",\n      \"method\": \"Whole-cell patch-clamp and Ca2+ imaging in LTRPC2-expressing cells and native immunocytes\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — electrophysiological reconstitution in heterologous and native cells, 389 citations, independent replication of ADPR gating\",\n      \"pmids\": [\"11509734\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"TRPM2 is activated by H2O2 and reactive oxygen/nitrogen species; sensitivity to redox-state modifiers is attributable to agonistic binding of NAD+ to the MutT (Nudix) motif; arachidonic acid and Ca2+ are positive regulators; TRPM2 activation confers susceptibility to cell death.\",\n      \"method\": \"Whole-cell patch-clamp, Ca2+ imaging, antisense oligonucleotide knockdown in HEK cells and native cells; H2O2 and TNFα treatment\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal methods (electrophysiology, imaging, knockdown), replicated across cell types, 682 citations\",\n      \"pmids\": [\"11804595\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"A splice variant of TRPM2 lacking amino acids 1292–1325 (C-terminal deletion) found in neutrophils is activated by H2O2 but not by ADPR, demonstrating that H2O2-mediated activation is independent of the ADPR-binding Nudix domain.\",\n      \"method\": \"Whole-cell patch-clamp in HEK-293 cells expressing wild-type or splice variant TRPM2; Ca2+ imaging; identification of splice variant by molecular cloning\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — functional dissection of activation modes with defined deletion variant, mechanistically rigorous\",\n      \"pmids\": [\"11960981\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"A short isoform of TRPM2 (TRPM2-S), arising from alternative splicing that removes the four C-terminal transmembrane domains and pore region, localizes to the plasma membrane and directly interacts with full-length TRPM2-L (shown by co-immunoprecipitation); co-expression of TRPM2-S suppresses H2O2-induced Ca2+ influx through TRPM2-L and inhibits cell death, functioning as a dominant negative.\",\n      \"method\": \"Co-immunoprecipitation, confocal microscopy for localization, Ca2+ influx assay, cell death/apoptosis assay in HEK 293T cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP plus functional rescue, multiple readouts\",\n      \"pmids\": [\"12594222\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"TRPM2 activation by oxidative stress or TNFα mediates cell death through increased intracellular Ca2+, followed by activation of caspases-8, -9, -3, -7 and PARP cleavage; chelation of intracellular Ca2+ with BAPTA blocked caspase/PARP cleavage and cell death, demonstrating Ca2+ is the critical intermediary.\",\n      \"method\": \"Retroviral overexpression/knockdown of TRPM2 isoforms in U937 cells; Ca2+ chelation; caspase/PARP western blot; viability and apoptosis assays\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic manipulation plus pharmacological rescue, multiple mechanistic readouts\",\n      \"pmids\": [\"16306129\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Cyclic ADP-ribose (cADPR) and NAADP strongly activate natively expressed TRPM2 channels in Jurkat T cells; their activity is partially suppressed by the ADPR antagonist AMP and fully suppressed by 8-Br-cADPR, suggesting cADPR and NAADP share a common binding site on TRPM2 that acts synergistically with ADPR.\",\n      \"method\": \"Patch-clamp electrophysiology in Jurkat T cells; pharmacological antagonism with AMP and 8-Br-cADPR\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — native cell electrophysiology with pharmacological dissection, single lab\",\n      \"pmids\": [\"16585058\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"TRPM2 mediates H2O2-induced Ca2+ entry and increased endothelial permeability through an ADP-ribose–dependent mechanism; H2O2 stimulates ADPR formation, which opens TRPM2; siRNA depletion or dominant-negative TRPM2-S expression inhibited the cationic current, Ca2+ entry, and transendothelial electrical resistance decrease.\",\n      \"method\": \"Patch-clamp recording, fura-2 Ca2+ imaging, transendothelial electrical resistance measurement, siRNA knockdown, overexpression in endothelial cells\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods with genetic and pharmacological controls in native endothelial cells\",\n      \"pmids\": [\"18048770\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Intracellular Ca2+ activates TRPM2 via a calmodulin (CaM)-binding domain in the N-terminus, independent of ADPR-binding sites; Ca2+-mediated gating is the only known activator of ADPR-insensitive splice isoforms; both intracellular Ca2+ release and receptor-activation-induced Ca2+ release can activate endogenous TRPM2 in intact cells.\",\n      \"method\": \"Whole-cell patch-clamp, Ca2+ imaging, mutagenesis of ADPR-binding sites, CaM-binding domain identification, intact cell activation assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis combined with electrophysiology and imaging in multiple systems\",\n      \"pmids\": [\"19372375\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"TRPM2 in dendritic cells localizes to endolysosomal vesicles (not the plasma membrane) and functions as a Ca2+ release channel from lysosomes, contributing to DC chemotaxis; TRPM2-deficient DCs show impaired maturation and directional migration in response to chemokines.\",\n      \"method\": \"siRNA knockdown and TRPM2-knockout mice; endolysosomal localization by confocal immunofluorescence; Ca2+ signaling assays; in vitro and in vivo chemotaxis assays\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO plus localization plus functional readouts in native DCs; replicated with siRNA and KO\",\n      \"pmids\": [\"21753080\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"EFHC1 (mutated in juvenile myoclonic epilepsy) physically interacts with both N- and C-terminal cytoplasmic regions of TRPM2 (shown by co-immunoprecipitation) and potentiates H2O2- and ADPR-induced Ca2+ responses and cationic currents through TRPM2; JME mutations in EFHC1 reverse this potentiation.\",\n      \"method\": \"Co-immunoprecipitation, whole-cell patch-clamp, Ca2+ imaging in HEK293 cells; in situ hybridization for co-expression\",\n      \"journal\": \"Cell calcium\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus electrophysiological functional validation with disease mutations, single lab\",\n      \"pmids\": [\"22226147\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"TRPM2 deficiency in mice modulates NMDAR subunit composition: TRPM2-null neurons upregulate GluN2A-containing NMDARs and downregulate GluN2B-containing NMDARs, augmenting prosurvival Akt/ERK signaling and inhibiting GSK3β, resulting in reduced ischemic infarct volume.\",\n      \"method\": \"TRPM2-knockout mice; middle cerebral artery occlusion model; field potential electrophysiology in brain slices; western blot for signaling proteins; pharmacological NMDAR subunit inhibitors\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with mechanistic pathway placement, but mechanism is indirect (TRPM2 regulates NMDAR expression)\",\n      \"pmids\": [\"24174660\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"TRPM2 channels mediate ischemic kidney injury through RAC1 activation: TRPM2 promotes RAC1 activation after ischemia; active RAC1 physically interacts with TRPM2 and increases TRPM2 plasma membrane expression, creating a positive feedback loop that amplifies NADPH oxidase activity and oxidative stress.\",\n      \"method\": \"TRPM2-knockout mice; chimeric mouse models; Co-IP of active RAC1 with TRPM2; pharmacological RAC1 inhibition in vivo; NADPH oxidase activity assays\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, genetic KO, pharmacological inhibition, chimeric mice for cell-type specificity; strong mechanistic evidence\",\n      \"pmids\": [\"25295536\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TRPM2-mediated Ca2+ influx is required to maintain cardiac myocyte mitochondrial function and protect against hypoxia-reoxygenation injury; loss-of-function TRPM2 mutants (E960D, P1018L) fail to reduce mitochondrial ROS in knockout myocytes, demonstrating Ca2+ entry (not reverse NCX) is the operative mechanism.\",\n      \"method\": \"Adenoviral rescue with WT and loss-of-function TRPM2 mutants in global TRPM2-KO hearts; mitochondrial superoxide measurements; oxygen consumption rate; cardiac-specific TRPM2-KO in ischemia-reperfusion\",\n      \"journal\": \"American journal of physiology. Heart and circulatory physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — mutagenesis rescue in KO background with multiple functional readouts; rigorous controls\",\n      \"pmids\": [\"25576627\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TRPM2 activation by H2O2 induces intracellular Ca2+ and Zn2+ elevation in pancreatic β-cells; Zn2+ originates from TRPM2-expressing lysosomes, and Ca2+-potentiated Zn2+ release (not Ca2+ alone) is primarily responsible for apoptotic β-cell death; TRPM2-KO mice are protected from streptozotocin-induced β-cell death.\",\n      \"method\": \"Zn2+ chelation experiments, lysosomal TRPM2 localization, Ca2+ and Zn2+ imaging, TRPM2-KO mouse model, INS1 and HEK293 cells, isolated islets\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal approaches (chelation, imaging, KO), mechanistically identifies Zn2+ as primary death mediator\",\n      \"pmids\": [\"25562606\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TRPM2 activation by oxidative stress regulates GSK-3 phosphorylation in brain via calcineurin: TRPM2 activation leads to Ca2+-dependent calcineurin activation, which dephosphorylates GSK-3; the bipolar disorder-associated D543E loss-of-function TRPM2 mutant fails to dephosphorylate GSK-3, and TRPM2-KO mice show increased GSK-3 phosphorylation and BD-like behavior.\",\n      \"method\": \"TRPM2-KO mice; overexpression of WT and D543E mutant; calcineurin inhibitor experiments; western blot for p-GSK-3; behavioral assays; EEG recordings\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO, disease mutant, pharmacological pathway placement via calcineurin inhibitors; multiple orthogonal methods\",\n      \"pmids\": [\"26311765\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Oxidative stress activates a TRPM2-Ca2+-CAMK2-BECN1 signaling cascade to inhibit autophagy: TRPM2-mediated Ca2+ influx activates CAMK2 (by phosphorylation and oxidation), which phosphorylates BECN1/Beclin-1 at Ser295, reducing BECN1–PIK3C3 association while increasing BECN1–BCL2 binding, thereby blocking early autophagy induction.\",\n      \"method\": \"Pharmacological TRPM2 inhibition and KO; CAMK2 phosphorylation/oxidation assays; BECN1 co-immunoprecipitation with PIK3C3 and BCL2; site-directed mutagenesis of Ser295; autophagy flux assays; validated in acetaminophen liver injury model\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — mechanistic pathway with mutagenesis, Co-IP, multiple inhibitors, validated in two cell-death contexts\",\n      \"pmids\": [\"27245989\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"2'-Deoxy-ADPR is an endogenous TRPM2 superagonist producing 10.4-fold higher whole-cell currents than ADPR at saturation, due to decreased inactivation rate and higher open probability; 2'-deoxy-ADPR is synthesized endogenously by NMNAT-2 and CD38 acting sequentially on NMN and 2'-deoxy-ATP, and was detected in Jurkat T cells by HPLC/mass spectrometry.\",\n      \"method\": \"Whole-cell patch-clamp; single-channel analysis; HPLC and mass spectrometry detection of endogenous 2'-deoxy-ADPR; in vitro enzymatic synthesis assay with NMNAT-2 and CD38\",\n      \"journal\": \"Nature chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro enzymatic reconstitution, mass spectrometry identification of endogenous molecule, electrophysiological characterization\",\n      \"pmids\": [\"28671679\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TRPM2 channel activation by Zn2+ in microglia requires a PKC/NADPH oxidase-mediated ROS generation and PARP-1 stimulation pathway; subsequently, TRPM2-mediated Ca2+ entry triggers a PYK2/MEK/ERK positive feedback mechanism that amplifies further TRPM2 activation, driving Ca2+ overload and cell death.\",\n      \"method\": \"TRPM2-KO microglia; pharmacological inhibitors of PKC (chelerythrine), NOX (DPI, GKT137831, Phox-I2), PARP (PJ34, DPQ), PYK2 (PF431396), MEK/ERK (U0126); Ca2+ imaging; cell death assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO plus pharmacological pathway dissection, multiple inhibitors; single lab\",\n      \"pmids\": [\"28322340\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Neutrophil-derived reactive oxygen species activate TRPM2 in endothelial cells through ADPR generation (requiring PARP); TRPM2-mediated Ca2+ entry triggers VE-cadherin phosphorylation, adherens junction disassembly, and transendothelial neutrophil migration; conditional EC-specific TRPM2 deletion prevents PMN transmigration in LPS-challenged mice.\",\n      \"method\": \"Conditional EC-specific TRPM2 knockout mice; gp91phox-/- PMNs; ADPR-insensitive TRPM2 mutant (C1008A) overexpression; PARP silencing; Ca2+ entry and VE-cadherin phosphorylation assays; in vivo PMN transmigration\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO, functional mutant, multiple genetic/pharmacological controls, in vivo validation\",\n      \"pmids\": [\"28790198\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Ligand-induced TRPM2 activation critically depends on hydrogen bonding of Arg1433 and Tyr1349 in the NUDT9H domain with the terminal ribose of ADPR; all ADPR derivatives modified at the terminal ribose abolished agonist activity; these residues were identified by structure-guided mutagenesis combined with synthetic ADPR analogues.\",\n      \"method\": \"Site-directed mutagenesis of NUDT9H domain; patch-clamp electrophysiology in TRPM2-expressing HEK293 cells; synthetic ADPR analogue library; structure-activity relationship analysis\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — complementary chemical biology (SAR with synthetic ligands) and mutagenesis approach, mechanistically defines key binding residues\",\n      \"pmids\": [\"28515263\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Palmitate-induced β-cell death involves TRPM2-mediated rise in mitochondrial Zn2+, which drives Drp-1 recruitment to mitochondria, mitochondrial fission and membrane potential loss; Zn2+ chelation alone prevents fission, and TRPM2 inhibition/KO prevents palmitate-induced fission in rodent and human islets.\",\n      \"method\": \"TRPM2 inhibition, RNAi silencing, and KO; mitochondrial Zn2+ and Ca2+ imaging; Drp-1 recruitment assay; membrane potential measurement; INS1 cells, mouse and human islets\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple genetic and pharmacological approaches, mechanistically identifies Zn2+ as mediator of mitochondrial fission, validated in human islets\",\n      \"pmids\": [\"28753206\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Cryo-EM structures of zebrafish TRPM2 in apo/closed and ADPR+Ca2+-bound/open states reveal that ADPR binds to a bi-lobed MHR1/2 domain (not exclusively NUDT9-H), and that NUDT9-H domains hang beneath MHR1/2; Ca2+ binding in the transmembrane region, together with ADPR at MHR1/2, is required for channel opening.\",\n      \"method\": \"Single-particle cryo-electron microscopy at near-atomic resolution; functional validation by electrophysiology\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — high-resolution structure with functional validation, defines activation mechanism at atomic level\",\n      \"pmids\": [\"30250252\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Cryo-EM structures of human TRPM2 alone, with ADPR, and with ADPR+Ca2+ reveal the gating mechanism: NUDT9H forms intra- and intersubunit interactions with MHR1/2/3 in the apo state; ADPR binding causes MHR1/2 rotation and disrupts intersubunit interactions; Ca2+ binding further engages transmembrane helices and the TRP helix to open the lower gate.\",\n      \"method\": \"Single-particle cryo-electron microscopy of human TRPM2 in three states; structural comparison; functional electrophysiology\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — high-resolution cryo-EM of human protein in multiple states, mechanistically defines concerted ADPR+Ca2+ gating\",\n      \"pmids\": [\"30467180\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TRPM2 Nudix motifs are canonical (enzymatically active ADPRases) in invertebrates but vestigial (inactive) in vertebrates; invertebrate TRPM2 (from Nematostella and Salpingoeca) possesses active ADPR phosphohydrolase activity; loss of ADPRase activity and loss of pore stability (inactivation) co-occurred in early vertebrate TRPM2 evolution.\",\n      \"method\": \"Comparative sequence analysis; in vitro ADPRase activity assays with isolated domains; whole-cell patch-clamp of TRPM2 orthologues (nvTRPM2, srTRPM2, drTRPM2, hsTRPM2); pore sequence mutagenesis\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro enzymatic assay plus electrophysiology across multiple orthologues with mutagenesis, defines evolutionary loss of catalysis\",\n      \"pmids\": [\"30938679\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In dendritic cells, TRPM2 is required for NLRP3 inflammasome activation in response to ROS; TRPM2-mediated Ca2+ permeation links oxidative stress sensing to NLRP3 activation.\",\n      \"method\": \"TRPM2-KO macrophages/DCs; NLRP3 inflammasome activation assays; Ca2+ imaging; cytokine secretion (IL-1β); ROS measurements (review synthesizing experimental findings)\",\n      \"journal\": \"Current opinion in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — review summarizing experimental data from multiple labs, no new primary data; moderate confidence based on cited primary experiments\",\n      \"pmids\": [\"32058297\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TRPM2 promotes atherogenesis through a TRPM2-CD36 axis: oxLDL activates CD36, which induces TRPM2 channel activity, and conversely TRPM2 promotes CD36 signaling; macrophage-specific or global Trpm2 deletion protects ApoE-/- mice from atherosclerosis by reducing oxLDL uptake, foam cell formation, and macrophage infiltration.\",\n      \"method\": \"Global and macrophage-specific Trpm2-KO in ApoE-/- atherosclerosis model; oxLDL and thrombospondin-1 stimulation assays; TRPM2 current recordings; CD36 signaling assays in KO and WT macrophages\",\n      \"journal\": \"Nature cardiovascular research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — cell-type-specific KO in disease model, bidirectional functional coupling demonstrated, multiple mechanistic readouts\",\n      \"pmids\": [\"35421327\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TRPM2 physically associates with extrasynaptic NMDARs; this TRPM2-NMDAR complex promotes surface expression of extrasynaptic NMDARs and enhances NMDAR-mediated excitotoxicity during ischemia; a membrane-permeable peptide targeting the NMDAR-interacting motif on TRPM2 disrupts the complex and protects neurons in vitro and in vivo.\",\n      \"method\": \"Co-immunoprecipitation of TRPM2-NMDAR complex; surface biotinylation assays for NMDAR expression; electrophysiology; cell death assays; in vivo MCAO stroke model with therapeutic peptide\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP, surface expression assays, in vitro and in vivo rescue with disrupting peptide; rigorous mechanistic validation\",\n      \"pmids\": [\"35421327\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Bilirubin acts as a direct extracellular agonist of TRPM2, gating the channel independently of canonical intracellular agonists (ADPR/Ca2+) by binding to a specific cavity; a knock-in loss-of-binding mutation (D1066A) antagonizes ischemic neurotoxicity; ischemic insults induce endogenous bilirubin release, creating a vicious cycle of TRPM2-mediated Ca2+-dependent brain injury.\",\n      \"method\": \"TRPM2-knockout mice; TRPM2 antagonist A23; D1066A knock-in mice; electrophysiology with bilirubin application; competitive binding assays; MCAO stroke model\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — novel direct agonist identified by electrophysiology, validated by KO, antagonist, and point mutant knock-in mice in vivo\",\n      \"pmids\": [\"36921602\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TRPM2 protein is ubiquitinated by the E3 ligase TRIM21 at K1218; lncTRPM2-AS (a non-coding antisense RNA from the same locus) blocks TRIM21-mediated TRPM2 ubiquitination and degradation, maintaining TRPM2 protein levels and intracellular calcium signaling in macrophages.\",\n      \"method\": \"RNA-immunoprecipitation, mass spectrometry, Co-IP of TRIM21 with TRPM2, ubiquitination assays, K1218 mutation analysis, intracellular calcium measurement\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — identifies E3 ligase and specific ubiquitination site on TRPM2 protein; mechanistically relevant to TRPM2 protein stability\",\n      \"pmids\": [\"33903714\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TRPM2-mediated Ca2+ influx promotes hepatic ischemia-reperfusion ferroptosis via Ca2+-induced mitochondrial Ca2+ accumulation (through the mitochondrial uniporter MCU) and upregulation of ALOX12, leading to mitochondrial lipid peroxidation; TRPM2 inhibition or Ca2+ depletion relieved ferroptosis in vitro and in vivo.\",\n      \"method\": \"TRPM2-KO mice; RNA sequencing; ALOX12 expression analysis; MCU-dependent mitochondrial Ca2+ measurement; ferroptosis assays; TRPM2 inhibitor treatment in vivo\",\n      \"journal\": \"Research (Washington, D.C.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO plus mechanistic pathway (TRPM2→MCU→ALOX12→ferroptosis) with multiple readouts, single lab\",\n      \"pmids\": [\"37275121\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TRPM2 is a homotetrameric, Ca2+-permeable non-selective cation channel activated by intracellular ADPR (binding the C-terminal NUDT9-H/MHR1-2 domain) and Ca2+ acting synergistically, by oxidative stress (H2O2/ROS) through PARP-dependent ADPR generation, by direct gating from bilirubin, and by intracellular Ca2+ alone via a CaM-binding N-terminal domain; cryo-EM structures define a two-step gating mechanism in which ADPR binding rotates MHR1/2 and disrupts NUDT9-H intersubunit contacts, while Ca2+ binding to transmembrane sites opens the lower gate, and downstream Ca2+ (and Zn2+) signals mediate diverse physiological and pathological outcomes including cell death via caspase/PARP activation, autophagy inhibition via CAMK2-BECN1 phosphorylation, NLRP3 inflammasome activation, GSK-3 regulation via calcineurin, ferroptosis via MCU-ALOX12, lysosomal Ca2+/Zn2+ release, and functional coupling with NMDARs and CD36 to amplify excitotoxicity and atherogenesis.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"TRPM2 is a Ca2+-permeable nonselective cation channel that transduces oxidative stress and metabolic signals into intracellular Ca2+ and Zn2+ fluxes, coupling cellular redox state to diverse downstream outcomes including cell death, autophagy regulation, immune cell chemotaxis, and endothelial barrier function. The channel is gated by synergistic binding of intracellular ADP-ribose (ADPR) to the C-terminal NUDT9-H domain and Ca2+ acting through an N-terminal calmodulin-binding site, with additional direct agonists including the superagonist 2'-deoxy-ADPR and bilirubin; H2O2 activates TRPM2 indirectly via PARP-1-dependent ADPR generation [PMID:11385575, PMID:19372375, PMID:28671679, PMID:36921602, PMID:23801245]. Cryo-EM structures of both zebrafish and human TRPM2 define a two-step gating mechanism in which ADPR binding to the MHR1/2 cleft disrupts intersubunit NUDT9-H contacts and subsequent Ca2+ binding repositions transmembrane helices to open the pore [PMID:30250252, PMID:30467180]. Depending on cell type, TRPM2 resides at the plasma membrane or on endolysosomes where it releases Ca2+ and Zn2+; downstream, TRPM2-mediated cation fluxes engage caspase/PARP apoptotic cascades, CaMKII-BECN1-dependent autophagy suppression, calcineurin-mediated GSK-3β dephosphorylation, ALOX12-dependent ferroptosis, and reciprocal signaling with NMDARs and CD36 [PMID:21753080, PMID:25562606, PMID:27245989, PMID:26311765, PMID:37275121, PMID:35421327, PMID:35445217].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"The fundamental identity of TRPM2 as an ADPR-gated Ca2+-permeable channel was established, resolving how a TRP-family protein could be directly activated by a nucleotide metabolite via its unique C-terminal NUDT9 homology domain.\",\n      \"evidence\": \"Patch-clamp electrophysiology in HEK-293 cells with ADPR perfusion; NUDT9 domain homology analysis and pyrophosphatase assay; parallel validation in immunocytes\",\n      \"pmids\": [\"11385575\", \"11509734\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether NUDT9-H enzymatic activity is required for gating was unresolved\", \"Structural basis of ADPR binding unknown\", \"Physiological roles in native tissues not yet addressed\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"H2O2 was identified as an activator of TRPM2 through a pathway partly independent of ADPR, establishing TRPM2 as a cellular oxidative stress sensor and linking its activation to Ca2+ overload-dependent cell death.\",\n      \"evidence\": \"Patch-clamp and Ca2+ imaging with H2O2 stimulation; splice variant lacking ADPR-binding residues retains H2O2 sensitivity; antisense knockdown reduces H2O2- and TNFα-induced death\",\n      \"pmids\": [\"11960981\", \"11804595\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The molecular intermediary between H2O2 and channel opening was undefined\", \"Whether ROS-dependent activation occurs via PARP-generated ADPR was unknown\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"The downstream cell-death mechanism was defined: TRPM2-mediated Ca2+ influx triggers a caspase-8/-9/-3/-7 cascade and PARP cleavage, with BAPTA rescue proving Ca2+ dependence of the apoptotic pathway.\",\n      \"evidence\": \"Retroviral overexpression and RNAi of TRPM2 in U937 monocytes; caspase activity assays; PARP cleavage immunoblotting; Ca2+ chelation rescue\",\n      \"pmids\": [\"16306129\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Ca2+ alone or additional ions (e.g., Zn2+) contribute to death was not tested\", \"Pathway specificity in different cell types not established\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Ca2+ was shown to activate TRPM2 independently of ADPR through an N-terminal calmodulin-binding domain, establishing the two-input (ADPR + Ca2+) coincidence-detection model for channel gating.\",\n      \"evidence\": \"Patch-clamp with site-directed mutagenesis of ADPR-binding and CaM-binding domains; Ca2+ uncaging in intact cells\",\n      \"pmids\": [\"19372375\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural visualization of the Ca2+/CaM-binding site was lacking\", \"Whether CaM acts as a bridge or Ca2+ binds directly to the channel was unresolved\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"TRPM2 was found on endolysosomes in dendritic cells (contrasting plasma membrane localization in neutrophils), revealing it functions as an intracellular Ca2+ release channel that drives DC maturation and chemotaxis.\",\n      \"evidence\": \"TRPM2-KO mice and siRNA; subcellular fractionation and imaging; Ca2+ signaling and chemotaxis assays in DCs vs. neutrophils\",\n      \"pmids\": [\"21753080\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Targeting signals determining plasma membrane vs. endolysosomal localization unknown\", \"Whether endolysosomal TRPM2 also conducts Zn2+ was not tested\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"PARP-1 was placed upstream of TRPM2 in ischemic neuronal death, and the ADPR-binding pocket was mapped to critical residues (Arg1433, Tyr1349) contacting the terminal ribose of ADPR, linking ligand chemistry to channel gating.\",\n      \"evidence\": \"PARP-1 KO mice with TRPM2 inhibitor epistasis in stroke model; systematic ADPR analogue screening with site-directed mutagenesis of NUDT9-H domain\",\n      \"pmids\": [\"23801245\", \"24304219\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full 3D structure of the binding pocket not yet available\", \"Whether PARP-1 is the sole source of gating ADPR in all cell types was untested\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"TRPM2-conducted Zn2+ release from lysosomes was identified as the primary cytotoxic signal (beyond Ca2+) in ROS-induced β-cell death, and a calcineurin→GSK-3β signaling axis downstream of TRPM2-mediated Ca2+ was linked to bipolar disorder-related behavior in mice.\",\n      \"evidence\": \"TRPM2-KO mice with Zn2+ chelation rescue in streptozotocin model; Trpm2-KO and D543E knock-in mice with calcineurin inhibitor and GSK-3β phosphorylation assays; behavioral testing\",\n      \"pmids\": [\"25562606\", \"26311765\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How lysosomal Zn2+ reaches mitochondria to trigger fission was not fully resolved\", \"Whether TRPM2 D543E variant is causal for human bipolar disorder requires genetic evidence\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"TRPM2-mediated Ca2+ influx was shown to activate CaMKII, which phosphorylates BECN1 at Ser295 to suppress autophagy, establishing a direct link between oxidative Ca2+ entry and autophagy inhibition.\",\n      \"evidence\": \"TRPM2 KD/inhibition; CaMKII phosphorylation/oxidation assays; BECN1 Ser295 phospho-mutants; Co-IP of BECN1-VPS34 and BECN1-BCL2\",\n      \"pmids\": [\"27245989\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this pathway operates in all TRPM2-expressing tissues was unknown\", \"Relative contribution of CaMKII phosphorylation vs. oxidation not fully dissected\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"2'-Deoxy-ADPR was discovered as an endogenous superagonist with ~10-fold greater efficacy than ADPR, produced by NMNAT-2 and CD38, expanding the physiological ligand repertoire and revealing that metabolic context tunes channel activity.\",\n      \"evidence\": \"Patch-clamp single-channel analysis; HPLC/MS detection in Jurkat T cells; in vitro enzymatic reconstitution with NMNAT-2 and CD38\",\n      \"pmids\": [\"28671679\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo concentrations of 2'-deoxy-ADPR in different tissues not quantified\", \"Whether 2'-deoxy-ADPR uses the same or overlapping binding site as ADPR was structurally unresolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Cryo-EM structures of both zebrafish and human TRPM2 defined the two-step gating mechanism: ADPR binds the MHR1/2 cleft disrupting intersubunit NUDT9-H contacts, then Ca2+ binding induces transmembrane helix rearrangement to open the pore.\",\n      \"evidence\": \"Cryo-EM structures in apo, ADPR-bound, and ADPR+Ca2+-bound states for zebrafish and human TRPM2\",\n      \"pmids\": [\"30250252\", \"30467180\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How bilirubin or 2'-deoxy-ADPR engage the same or distinct binding pockets was not resolved\", \"Dynamic gating intermediates between the captured states remain uncharacterized\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Evolutionary analysis revealed that vertebrate TRPM2 lost NUDT9-H catalytic ADPRase activity while gaining pore inactivation; invertebrate TRPM2 retains enzymatic function uncoupled from gating, demonstrating that the NUDT9-H domain was repurposed from enzyme to ligand sensor.\",\n      \"evidence\": \"ADPRase assays on isolated NUDT9-H domains from multiple species; patch-clamp of invertebrate orthologs; Nudix box mutagenesis\",\n      \"pmids\": [\"30938679\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Selective pressures driving the loss of enzymatic activity remain speculative\", \"Whether intermediate evolutionary states exist in non-model vertebrates was not explored\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"TRPM2 was found to physically couple to extrasynaptic NMDARs to promote their surface expression and activity during ischemia, and to engage in a reciprocal activation loop with CD36 in macrophages driving foam cell formation and atherogenesis.\",\n      \"evidence\": \"Co-IP of TRPM2-NMDAR complex; disrupting peptide reducing infarcts in MCAO model; macrophage-specific Trpm2 KO in Apoe−/− atherosclerosis model; patch-clamp with CD36 ligands\",\n      \"pmids\": [\"35421327\", \"35445217\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the TRPM2-NMDAR interface not determined\", \"Whether TRPM2-CD36 coupling operates in non-macrophage contexts is unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Bilirubin was identified as a direct TRPM2 agonist independent of ADPR and Ca2+, binding a pocket shared with the antagonist A23; meanwhile, TRPM2→MCU→ALOX12 ferroptotic signaling and AKT-mTOR-mediated autophagy suppression were defined as distinct downstream pathways.\",\n      \"evidence\": \"Patch-clamp with bilirubin; D1066A knock-in mice protected from ischemic neurotoxicity; TRPM2-KO with MCU blockade and ALOX12 pathway analysis in hepatic I/R; mTOR inhibitor rescue in cisplatin AKI model\",\n      \"pmids\": [\"36921602\", \"37275121\", \"37649595\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether bilirubin gating is physiologically relevant at normal serum concentrations is unclear\", \"Structural basis of bilirubin binding not resolved at atomic resolution\", \"Cross-talk between ferroptosis and autophagy branches downstream of TRPM2 not addressed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include: the structural mechanism by which bilirubin and 2'-deoxy-ADPR gate the channel; the molecular determinants of plasma membrane vs. endolysosomal targeting; and whether TRPM2 variants are causative for human neuropsychiatric disease.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No high-resolution structures with bilirubin or 2'-deoxy-ADPR bound\", \"Trafficking signals for differential subcellular localization not mapped\", \"No human genetic studies directly linking TRPM2 mutations to Mendelian disease\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [0, 1, 2, 6, 8, 23, 24]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 25, 33]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 1, 4, 7, 9, 14]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [9, 17]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [3, 5, 17, 31]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [11, 15, 18, 26, 32, 34]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [18, 27, 32]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [9, 22, 29]},\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [0, 1, 6, 8, 17]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"EFHC1\",\n      \"RAC1\",\n      \"TRIM21\",\n      \"CD36\",\n      \"GRIN2A\",\n      \"CALM1\",\n      \"BECN1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"TRPM2 is a Ca2+-permeable, non-selective cation channel that couples intracellular ADP-ribose (ADPR) sensing to Ca2+ (and Zn2+) entry, thereby transducing oxidative stress, metabolic, and inflammatory signals into diverse cellular outcomes including cell death, autophagy inhibition, inflammasome activation, and endothelial barrier regulation. The channel is gated by ADPR binding to the MHR1/2 cleft and by Ca2+ binding to transmembrane sites; cryo-EM structures show that ADPR rotates MHR1/2 and disrupts intersubunit NUDT9-H contacts while Ca2+ opens the lower gate, and additional agonists including 2′-deoxy-ADPR (a superagonist), cADPR, and bilirubin (an extracellular agonist acting independently of ADPR) expand the activation repertoire [PMID:30250252, PMID:30467180, PMID:28671679, PMID:36921602]. TRPM2-mediated Ca2+ influx drives downstream signaling through CAMK2-dependent BECN1 phosphorylation to inhibit autophagy, calcineurin-dependent GSK-3β dephosphorylation relevant to bipolar disorder-like phenotypes, caspase/PARP-mediated apoptosis, NLRP3 inflammasome priming, and MCU/ALOX12-dependent ferroptosis [PMID:27245989, PMID:26311765, PMID:16306129, PMID:37275121]. Beyond plasma-membrane Ca2+ entry, TRPM2 functions as a lysosomal Ca2+/Zn2+ release channel—lysosomal Zn2+ mobilization drives mitochondrial fission and β-cell death, while lysosomal TRPM2 supports dendritic cell chemotaxis—and physically associates with extrasynaptic NMDARs to promote their surface expression and excitotoxicity, and with CD36 to amplify oxLDL uptake and atherogenesis [PMID:25562606, PMID:28753206, PMID:21753080, PMID:35421327].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"The foundational question of TRPM2's ligand identity and ion selectivity was answered: ADPR directly gates TRPM2 as a Ca2+-permeable nonselective cation channel via a C-terminal NUDT9 homology domain, establishing ADPR as a novel intracellular second messenger for Ca2+ entry.\",\n      \"evidence\": \"Whole-cell and single-channel patch-clamp in HEK-293 cells and native U937 monocytes/immunocytes, biochemical identification of NUDT9-H domain\",\n      \"pmids\": [\"11385575\", \"11509734\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether NUDT9-H retains enzymatic ADPRase activity in vertebrate TRPM2 was unknown\", \"Structural basis of ADPR recognition unresolved\", \"Physiological source of ADPR in vivo not established\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"TRPM2 was shown to be a sensor of oxidative stress: H2O2 and ROS activate the channel through a mechanism partially independent of the ADPR-binding Nudix domain, as demonstrated by a splice variant lacking this region, linking TRPM2 to redox-sensitive cell death.\",\n      \"evidence\": \"Patch-clamp and Ca2+ imaging with H2O2/TNFα stimulation; antisense knockdown; functional characterization of NUDT9-H-deleted splice variant in HEK-293 cells\",\n      \"pmids\": [\"11804595\", \"11960981\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Exact ROS-sensing mechanism (direct oxidation vs. intermediate metabolite generation) unresolved\", \"Whether PARP-dependent ADPR generation is required for all ROS-mediated activation unclear\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Discovery of the dominant-negative splice isoform TRPM2-S demonstrated that channel oligomerization is functionally regulatable: TRPM2-S directly interacts with full-length TRPM2-L and suppresses H2O2-induced Ca2+ influx and cell death.\",\n      \"evidence\": \"Co-immunoprecipitation, confocal localization, Ca2+ influx and cell death assays in HEK-293T cells\",\n      \"pmids\": [\"12594222\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of TRPM2-S/L heteromers unknown\", \"Physiological regulation of TRPM2-S expression not defined\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"The downstream cell-death mechanism was mapped: TRPM2-dependent Ca2+ entry activates caspases-8, -9, -3, -7 and PARP cleavage, and intracellular Ca2+ chelation fully blocks this cascade, establishing Ca2+ as the critical intermediary between channel opening and apoptosis.\",\n      \"evidence\": \"Retroviral overexpression/knockdown of TRPM2 isoforms in U937 cells; BAPTA-AM chelation; caspase/PARP western blot\",\n      \"pmids\": [\"16306129\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Zn2+ contributes alongside Ca2+ was not yet explored\", \"Cell-type specificity of the apoptotic pathway not addressed\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"TRPM2 was placed in endothelial barrier function: H2O2-stimulated ADPR formation opens TRPM2 in endothelial cells, causing Ca2+ entry and increased permeability; siRNA or dominant-negative TRPM2-S suppresses this response.\",\n      \"evidence\": \"Patch-clamp, fura-2 imaging, transendothelial electrical resistance in primary endothelial cells with siRNA and dominant-negative approaches\",\n      \"pmids\": [\"18048770\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream targets linking TRPM2 Ca2+ to junctional disassembly (e.g., VE-cadherin phosphorylation) not yet identified\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"An ADPR-independent gating mechanism was uncovered: intracellular Ca2+ alone activates TRPM2 via an N-terminal calmodulin-binding domain, explaining how ADPR-insensitive splice variants retain channel function and establishing Ca2+ as both a co-agonist and a standalone gating signal.\",\n      \"evidence\": \"Patch-clamp with ADPR-binding-site mutations; CaM-binding domain identification; intact cell activation assays\",\n      \"pmids\": [\"19372375\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"CaM-TRPM2 interface not structurally resolved\", \"Relative contribution of CaM-dependent vs. transmembrane Ca2+-binding-site gating unknown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"TRPM2 was shown to function as an intracellular (endolysosomal) Ca2+ release channel in dendritic cells, expanding its role beyond plasma membrane Ca2+ entry and linking it to immune cell chemotaxis and maturation.\",\n      \"evidence\": \"TRPM2-KO mice and siRNA knockdown; endolysosomal confocal localization; Ca2+ signaling and in vitro/in vivo chemotaxis assays in DCs\",\n      \"pmids\": [\"21753080\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How TRPM2 is targeted to lysosomes vs. plasma membrane not defined\", \"Whether lysosomal TRPM2 gating uses the same ADPR/Ca2+ mechanism unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Three key downstream signaling axes were mapped: (1) lysosomal Zn2+ release (not Ca2+ alone) mediates β-cell apoptosis; (2) Ca2+-dependent calcineurin activation dephosphorylates GSK-3β (relevant to bipolar disorder); (3) TRPM2-mediated Ca2+ maintains cardiac mitochondrial function during ischemia-reperfusion, as confirmed by loss-of-function mutant rescue in KO cardiomyocytes.\",\n      \"evidence\": \"TRPM2-KO mice, Zn2+ chelation/imaging in islets, calcineurin inhibitor experiments plus D543E mutant analysis, and adenoviral rescue with E960D/P1018L mutants in KO hearts\",\n      \"pmids\": [\"25562606\", \"26311765\", \"25576627\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of TRPM2-dependent lysosomal Zn2+ permeation not structurally resolved\", \"Whether GSK-3β regulation is cell-type-specific beyond neurons unclear\", \"Identity of cardiac TRPM2 agonist in ischemia-reperfusion not determined\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"TRPM2 was positioned as an autophagy regulator: TRPM2-mediated Ca2+ activates CAMK2, which phosphorylates BECN1 at Ser295 to disrupt BECN1-PIK3C3 association and promote BECN1-BCL2 binding, thereby inhibiting autophagy induction during oxidative stress.\",\n      \"evidence\": \"TRPM2 KO/inhibition; CAMK2 phosphorylation assays; BECN1 Co-IP with PIK3C3/BCL2; S295 mutagenesis; autophagy flux assays; acetaminophen liver injury model\",\n      \"pmids\": [\"27245989\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether autophagy inhibition is a general consequence of TRPM2 activation across tissues not tested\", \"CAMK2 isoform specificity not established\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Multiple advances refined TRPM2 pharmacology, structure-activity relationships, and tissue-specific signaling: 2′-deoxy-ADPR was identified as an endogenous superagonist; Arg1433/Tyr1349 in NUDT9-H were identified as critical ADPR-binding residues; lysosomal Zn2+ was linked to palmitate-induced mitochondrial fission via Drp-1; neutrophil ROS-TRPM2-VE-cadherin phosphorylation was established as the mechanism for endothelial barrier opening and transendothelial migration; and a PKC/NOX/PARP-1→TRPM2→PYK2/MEK/ERK positive feedback loop was mapped in microglia.\",\n      \"evidence\": \"Electrophysiology with synthetic ADPR analogues; mass spectrometry for endogenous 2′-deoxy-ADPR; conditional EC-TRPM2-KO mice with VE-cadherin phosphorylation assays; TRPM2-KO microglia with pharmacological inhibitor panels; Zn2+/Ca2+ imaging with Drp-1 recruitment in human islets\",\n      \"pmids\": [\"28671679\", \"28515263\", \"28753206\", \"28790198\", \"28322340\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Binding site for 2′-deoxy-ADPR not structurally resolved\", \"Relative physiological concentrations of ADPR vs. 2′-deoxy-ADPR in most tissues unknown\", \"PYK2-TRPM2 physical interaction not demonstrated\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Cryo-EM structures of zebrafish and human TRPM2 in multiple ligand-bound states revealed a two-step gating mechanism: ADPR binds the MHR1/2 cleft (not solely NUDT9-H), rotating MHR1/2 and disrupting intersubunit NUDT9-H contacts, while Ca2+ binding at a transmembrane site engages the TRP helix to open the lower gate.\",\n      \"evidence\": \"Single-particle cryo-EM at near-atomic resolution of zebrafish and human TRPM2 in apo, ADPR-bound, and ADPR+Ca2+-bound states; functional validation by electrophysiology\",\n      \"pmids\": [\"30250252\", \"30467180\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Open-state structure at true atomic resolution still lacking\", \"CaM-binding domain and N-terminal gating contributions not resolved in structures\", \"Mechanism of inactivation not captured\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"The evolutionary trajectory of TRPM2 was clarified: invertebrate TRPM2 retains active ADPRase catalysis in the Nudix domain, but this activity was lost in early vertebrate evolution concurrently with loss of pore stability, explaining why vertebrate NUDT9-H is vestigial for hydrolysis but critical for ligand-dependent gating.\",\n      \"evidence\": \"Comparative in vitro ADPRase assays with isolated domains from Nematostella, Salpingoeca, zebrafish, and human; patch-clamp of orthologues; pore mutagenesis\",\n      \"pmids\": [\"30938679\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Selective pressure driving loss of catalysis not established\", \"Whether vestigial NUDT9-H has any residual product release function unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Two new functional partnerships were defined: TRPM2 is ubiquitinated by the E3 ligase TRIM21 at K1218 (regulated by the antisense lncRNA TRPM2-AS), and TRPM2 functionally couples with CD36 to amplify oxLDL uptake and foam cell formation, promoting atherogenesis in macrophages.\",\n      \"evidence\": \"TRIM21-TRPM2 Co-IP, K1218 mutation, ubiquitination assays, lncRNA-immunoprecipitation; global and macrophage-specific TRPM2-KO in ApoE−/− atherosclerosis model with CD36 signaling assays\",\n      \"pmids\": [\"33903714\", \"35421327\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether TRIM21-mediated degradation is regulated by oxidative stress not tested\", \"Structural basis of TRPM2-CD36 functional coupling unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"TRPM2 was shown to physically associate with extrasynaptic NMDARs, promoting their surface expression and enhancing excitotoxic neuronal death during ischemia; a peptide disrupting this complex is neuroprotective in vivo.\",\n      \"evidence\": \"Co-immunoprecipitation of TRPM2-NMDAR complex; surface biotinylation; electrophysiology; therapeutic peptide in MCAO stroke model\",\n      \"pmids\": [\"35421327\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Interaction interface between TRPM2 and specific NMDAR subunits not structurally defined\", \"Whether TRPM2-NMDAR coupling occurs in non-neuronal cells unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Bilirubin was identified as a direct extracellular agonist of TRPM2, gating the channel independently of ADPR/Ca2+ through a specific cavity (D1066), establishing a new non-canonical activation mode that amplifies ischemic brain injury in a feed-forward loop; additionally, TRPM2-mediated Ca2+ was linked to ferroptosis via an MCU-ALOX12 axis in hepatic ischemia-reperfusion.\",\n      \"evidence\": \"D1066A knock-in mice; TRPM2-KO; electrophysiology with bilirubin; competitive binding assays; MCAO model; RNA-seq with ALOX12 upregulation; MCU-dependent mitochondrial Ca2+ measurement; ferroptosis assays in vivo\",\n      \"pmids\": [\"36921602\", \"37275121\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Bilirubin binding site not structurally resolved at atomic level\", \"Whether bilirubin-TRPM2 signaling operates in non-neuronal tissues not tested\", \"Relative contribution of ferroptosis vs. apoptosis in TRPM2-dependent cell death not quantified\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include: the structural basis of CaM-dependent gating, the mechanism by which TRPM2 is differentially sorted to plasma membrane vs. lysosomes, how bilirubin and ADPR gating interact when both are present, and whether distinct downstream death programs (apoptosis, ferroptosis, necrosis) are independently tunable by different TRPM2 activation modes.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"CaM-TRPM2 structural interface unresolved\", \"Lysosomal vs. plasma membrane sorting mechanism unknown\", \"Integrated model of multi-agonist gating lacking\", \"Cell-type-specific wiring of downstream death programs not systematically mapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [0, 1, 2, 22, 23]},\n      {\"term_id\": \"GO:0140299\", \"supporting_discovery_ids\": [2, 8, 28]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 1, 4, 7, 12, 19]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [9, 14]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [5, 14, 16, 30]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [8, 15, 16, 26]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [9, 19, 25]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [16]},\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [0, 1, 22, 23]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [2, 13, 30]}\n    ],\n    \"complexes\": [\n      \"TRPM2 homotetramer\"\n    ],\n    \"partners\": [\n      \"EFHC1\",\n      \"RAC1\",\n      \"TRIM21\",\n      \"CD36\",\n      \"GRIN2B\",\n      \"CaM\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}