{"gene":"MCOLN1","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":2008,"finding":"TRPML1 functions as a Fe2+-permeable channel in late endosomes and lysosomes, mediating iron release into the cytosol. ML4 disease mutations impair Fe2+ permeation at varying degrees correlating with disease severity. Loss of TRPML1 reduces cytosolic Fe2+ and increases intralysosomal Fe2+ accumulation.","method":"Radiolabelled iron uptake assays, cytosolic and intralysosomal iron monitoring, direct patch-clamping of late endosomal/lysosomal membrane, comparison of TRPML1-/- vs. control human fibroblasts","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct lysosomal patch-clamp electrophysiology combined with iron flux assays and disease-mutation functional correlation; multiple orthogonal methods in a single rigorous study","pmids":["18794901"],"is_preprint":false},{"year":2005,"finding":"TRP-ML1 can function as a H+ channel and its loss leads to lysosomal over-acidification in MLIV patient cells, reducing acidic lipase activity. Expression of TRP-ML1 rescues lipid hydrolysis, and dissipation of lysosomal pH reverses storage phenotype.","method":"Lysosomal pH measurement in TRP-ML1-/- patient cells, lipase activity assay with multiple substrates, cell fractionation, rescue by TRP-ML1 expression and pH-dissipating drugs","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — multiple orthogonal methods (pH measurement, lipase activity, genetic rescue, pharmacological rescue) in patient-derived cells; single lab","pmids":["16361256"],"is_preprint":false},{"year":2005,"finding":"TRP-ML1 is a lysosomal monovalent cation channel that undergoes proteolytic cleavage by cathepsin B at Arg200-Pro201; cleavage inhibits channel activity. N- and C-terminal fragments are co-immunoprecipitated. The R200H disease mutation alters this cleavage pattern.","method":"Electrophysiology (whole-lysosome/planar patch-clamp), co-immunoprecipitation, N-terminal sequencing of purified C-terminal fragment, cathepsin B inhibitor treatment, CatB-/- cell expression","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with protein sequencing, co-IP, and mutagenesis/pharmacological inhibition; single lab but multiple orthogonal methods","pmids":["16257972"],"is_preprint":false},{"year":2006,"finding":"TRPML1 forms homo- and heteromultimers with TRPML2 and TRPML3. TRPML1 and TRPML2 homomultimers are lysosomal, while TRPML3 homomultimers are in the ER. The presence of TRPML1 or TRPML2 specifically dictates lysosomal localization of TRPML3, but not vice versa.","method":"Co-immunoprecipitation, subcellular localization by fluorescence microscopy, co-expression studies with lysosomal targeting-disrupted mutants","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP and colocalization microscopy, single lab, two complementary methods","pmids":["16606612"],"is_preprint":false},{"year":2009,"finding":"Proline scanning mutagenesis revealed gain-of-function constitutive activating mutations in the S5 transmembrane domain of TRPML1 (e.g., V432P). TRPML1 is an inwardly rectifying, proton-impermeable, Ca2+ and Fe2+/Mn2+ permeable channel; constitutive Ca2+ release from lysosomes promotes lysosomal exocytosis and surface expression of LAMP-1.","method":"Systematic proline-substitution mutagenesis, whole-cell and lysosomal patch-clamp electrophysiology, LAMP-1 surface staining","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstitution via mutagenesis plus direct electrophysiology, single lab with multiple mutants and orthogonal readouts","pmids":["19638346"],"is_preprint":false},{"year":2011,"finding":"TRPML1 co-immunoprecipitates with TPC2 and shows near-complete colocalization with TPC2 on endolysosomes, but electrophysiology shows TPC1/TPC2 do not affect TRPML1 channel activity, and TRPML1 does not mediate NAADP-evoked Ca2+ signals — TRPML1 and TPCs are physically associated but functionally independent organellar ion channels.","method":"Co-immunoprecipitation, colocalization microscopy, whole-cell and whole-lysosome patch-clamp electrophysiology, Ca2+ imaging in TRP-ML1-/- cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — reciprocal co-IP plus direct electrophysiology plus knockout cell validation; single lab but three orthogonal methods","pmids":["21540176"],"is_preprint":false},{"year":2011,"finding":"CUP-5, the C. elegans ortholog of TRPML1, localizes to lysosomes and is required for proteolytic degradation in autolysosomes; cup-5 mutations cause accumulation of autophagy substrates in enlarged late endosomal/lysosomal vacuoles, and reduced autophagy activity partially suppresses cup-5 mutant defects.","method":"Genetic epistasis analysis, fluorescence microscopy with organelle markers, immunoprecipitation, genetic suppressor analysis in C. elegans","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis in a model organism, multiple complementary assays; ortholog of mammalian TRPML1","pmids":["21997367"],"is_preprint":false},{"year":2016,"finding":"ROS directly and specifically activate lysosomal TRPML1 channels, inducing lysosomal Ca2+ release. This Ca2+ release triggers calcineurin-dependent TFEB nuclear translocation, autophagy induction, and lysosome biogenesis. Genetic inactivation or pharmacological inhibition of TRPML1 blocks clearance of damaged mitochondria and removal of excess ROS.","method":"GCaMP3-ML1 Ca2+ imaging, pharmacological ROS manipulation, TRPML1 genetic knockout and inhibition, TFEB nuclear translocation assay, autophagy flux assays, mitochondrial damage assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods including live-cell Ca2+ imaging, genetic KO, pharmacological inhibition, TFEB assay; replicated across multiple stress conditions","pmids":["27357649"],"is_preprint":false},{"year":2017,"finding":"Cryo-EM structure of mouse TRPML1 in nanodiscs reveals that PtdIns(3,5)P2 binds to the N-terminus distal from the pore; an S2-S3 helix-turn-helix extension couples ligand binding to pore opening; the selectivity filter contains multiple ion-binding sites; conserved acidic residues form a luminal Ca2+-blocking site conferring pH and Ca2+ modulation; a luminal linker domain canopy creates a negative electrostatic trap for divalent cations.","method":"Single-particle cryo-EM structure determination, mutagenesis combined with electrophysiology","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution cryo-EM structure combined with mutagenesis and electrophysiological validation","pmids":["29019981"],"is_preprint":false},{"year":2017,"finding":"Cryo-EM structures of full-length human TRPML1 in apo (closed, pH 7.0) and agonist-bound (open, pH 6.0) states reveal that channel opening involves dilation of the lower gate and movement of pore helix 1; the agonist binds a hydrophobic cavity formed by S5, S6, and pore helix 1, distinct from TRPV1 agonist sites.","method":"Single-particle cryo-EM at 3.72 Å (closed) and 3.49 Å (open) resolution, structural comparison","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — near-atomic cryo-EM structures in two functional states with direct structural comparison","pmids":["29019983"],"is_preprint":false},{"year":2018,"finding":"Cryo-EM structures of human TRPML1 with PtdIns(3,5)P2 and PtdIns(4,5)P2 reveal a unique lipid-binding site on extended helices S1, S2, and S3. PtdIns(3,5)P2 induces Y355 to form a π-cation interaction with R403, moving the S4-S5 linker to allosterically activate the channel. PtdIns(4,5)P2 binds the same site but inhibits channel activity.","method":"Cryo-EM structure determination at pH 5.0 with bound lipids and ML-SA1, electrophysiological characterization","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM structures with bound ligands combined with electrophysiology; single lab, multiple ligand conditions","pmids":["30305615"],"is_preprint":false},{"year":2022,"finding":"Cryo-EM structures of mouse TRPML1 in apo-closed, PI(3,5)P2-bound closed, and PI(3,5)P2/temsirolimus(rapamycin analog)-bound open states reveal that PI(3,5)P2 and rapamycin bind distinct sites and work cooperatively; the structures elucidate the allosteric mechanism for synergistic channel activation.","method":"Cryo-EM structure determination in multiple states, electrophysiology","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — multiple cryo-EM structures in distinct functional states combined with electrophysiology; single lab","pmids":["35131932"],"is_preprint":false},{"year":2021,"finding":"Cryo-EM structure of human TRPML1 with antagonist ML-SI3 at 2.9 Å shows ML-SI3 binds the same hydrophobic cavity (S5, S6, PH1) as agonist ML-SA1; electrophysiology confirms ML-SI3 competes with ML-SA1 but does not inhibit PI(3,5)P2-dependent activation.","method":"Cryo-EM structure determination, whole-lysosome patch-clamp electrophysiology","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM structure plus functional electrophysiology; single lab, two orthogonal methods","pmids":["34171299"],"is_preprint":false},{"year":2015,"finding":"TRPML1 is a PtdIns(3,5)P2-gated lysosomal Ca2+ channel required for phagosome-lysosome fusion. Silencing TRPML1 causes lysosomes to dock but not fuse with phagosomes, impairing bactericidal capacity. PIKfyve generates PtdIns(3,5)P2 to activate TRPML1, raising cytosolic Ca2+ during phagocytosis; forced Ca2+ release rescues fusion in TRPML1-silenced cells.","method":"TRPML1 siRNA knockdown, phagocytosis assay with lysosomal marker acquisition, isolated phagosome analysis by electron microscopy, Ca2+ imaging, ionomycin rescue","journal":"Traffic","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic knockdown with defined phenotypic readout, Ca2+ imaging, pharmacological rescue, and mechanistic epistasis with PIKfyve; single lab but multiple orthogonal methods","pmids":["26010303"],"is_preprint":false},{"year":2015,"finding":"TOR kinase directly phosphorylates TRPML1, inactivating its channel activity and suppressing autophagy. Mutation of the TOR phosphorylation sites to unphosphorylatable residues blocks TOR regulation of TRPML1.","method":"In vitro kinase assay, phosphorylation site mutagenesis, channel activity recordings, autophagy flux assays","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct kinase phosphorylation assay combined with mutagenesis and functional channel assay; single lab","pmids":["26195823"],"is_preprint":false},{"year":2018,"finding":"Starvation activates MCOLN1 by relieving MTORC1's inhibition of the channel; activated MCOLN1 in turn facilitates MTORC1 reactivation through a calmodulin-dependent mechanism, constituting a negative feedback loop that prevents excessive MTORC1 inhibition during prolonged starvation.","method":"Pharmacological activation/inhibition of MCOLN1 and MTORC1, calmodulin inhibition, Ca2+ chelation, MTORC1 activity assays (S6K phosphorylation), MCOLN1 knockdown/knockout","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple pharmacological and genetic perturbations with defined signaling readout; single lab","pmids":["29460684"],"is_preprint":false},{"year":2019,"finding":"TRPML1 activation induces autophagosome biogenesis through a TFEB-independent pathway requiring CaMKKβ and AMPK, which activate ULK1 and VPS34 complexes and generate PI3P. MLIV patient cells show reduced PI3P-binding protein recruitment to phagophores.","method":"PI3P generation assay, phagophore recruitment of PI3P-binding proteins, CaMKKβ/AMPK inhibition, ULK1/VPS34 complex activation assays, TFEB knockout, patient cell analysis","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple pharmacological and genetic perturbations, patient cell validation, defined downstream signaling pathway; single lab but multiple orthogonal assays","pmids":["31822666"],"is_preprint":false},{"year":2014,"finding":"TRPML1 mutant isoforms (F465L, F408Δ) show strongly reduced activation by PtdIns(3,5)P2 but can be activated by synthetic ligands. F465L renders TRPML1 pH-insensitive; F408Δ impacts synthetic ligand binding. Small-molecule activators rescue trafficking defects and lysosomal zinc accumulation in MLIV patient fibroblasts.","method":"Whole-lysosome planar patch-clamp, pharmacological activation with synthetic ligands, trafficking assay, zinc accumulation assay in patient fibroblasts","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct lysosomal electrophysiology with disease mutants, pharmacological rescue in patient cells; single lab, multiple orthogonal methods","pmids":["25119295"],"is_preprint":false},{"year":2010,"finding":"TRPML1-deficient cells and Mcoln1-/- mouse brain show elevated chelatable zinc levels. siRNA knockdown of TRPML1 causes lysosomal zinc accumulation; TRPML1 loss delays zinc leak from lysosomes to cytoplasm and is associated with elevated MTF-1-dependent transcription. ZnT4 knockdown ameliorates the lysosomal enlargement phenotype in TRPML1-KD cells exposed to zinc.","method":"siRNA knockdown, fluorometric zinc quantification, ICP-MS of brain tissue, lysosomal zinc staining, MTF-1 and ZnT4 co-knockdown","journal":"The Biochemical journal / The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — multiple genetic and pharmacological perturbations with zinc quantification in cells and in vivo; findings from two independent labs (PMID 20864526 and 23368743)","pmids":["20864526","23368743"],"is_preprint":false},{"year":2014,"finding":"TMEM163 protein (a putative zinc transporter) is a novel interacting partner for TRPML1, confirmed by yeast two-hybrid, co-immunoprecipitation, mass spectrometry, and colocalization microscopy. Interaction requires part of TMEM163's N-terminus. Co-expression of TMEM163 does not alter TRPML1 channel activity, but TRPML1 co-expression reduces TMEM163 at the plasma membrane.","method":"Yeast two-hybrid, co-immunoprecipitation, mass spectrometry, confocal colocalization, subcellular localization analysis","journal":"Traffic","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — four orthogonal binding assays confirming interaction; single lab","pmids":["25130899"],"is_preprint":false},{"year":2020,"finding":"Mitochondria-lysosome contact sites facilitate Ca2+ transfer from lysosomes to mitochondria through TRPML1 lysosomal Ca2+ efflux. This transfer is mediated by tethering at contact sites and requires VDAC1 (outer mitochondrial membrane) and MCU (inner mitochondrial membrane). MLIV patient fibroblasts show altered contact dynamics and defective contact-dependent mitochondrial Ca2+ uptake.","method":"High spatial/temporal resolution live-cell microscopy, TRPML1 agonist stimulation, VDAC1/MCU inhibition, MLIV patient fibroblast analysis, contact site dynamics quantification","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Moderate — live-cell super-resolution microscopy with pharmacological and genetic perturbations, patient cell validation; single lab, multiple orthogonal approaches","pmids":["32703809"],"is_preprint":false},{"year":2017,"finding":"TRPML1-mediated lysosomal Ca2+ release activates calmodulin (CaM) to promote lysosome fission, reducing lysosomal size. TRPML1 activation suppresses enlarged vacuoles induced by vacuolin-1 or P2X4; effects are abolished by Ca2+ chelation or CaM inhibition.","method":"Pharmacological TRPML1 activation, Ca2+ chelation, CaM inhibition, lysosome size quantification by fluorescence microscopy","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — pharmacological manipulation with defined organelle phenotype readout; single lab, multiple perturbations","pmids":["28360104"],"is_preprint":false},{"year":2010,"finding":"Loss of TRPML1 in Trpml1-/- mice causes impaired gastric acid secretion associated with dynamic palmitoylation and dephosphorylation of Trpml1 in parietal cells upon histamine stimulation, mislocalization of the gastric proton pump, and enlarged/dysfunctional secretory canaliculi. TRPML1 is required for tubulovesicle formation and trafficking in parietal cells.","method":"Gene-targeted Trpml1-/- mouse model, gastric acid secretion measurement, biochemical analysis of palmitoylation/phosphorylation, immunohistochemistry, electron microscopy of parietal cells","journal":"Gastroenterology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic KO mouse with defined cellular phenotype, multiple biochemical readouts; single lab","pmids":["21111738"],"is_preprint":false},{"year":2017,"finding":"LAMTOR1, a subunit of the Ragulator complex, directly interacts with TRPML1 through its N-terminal domain and tonically inhibits TRPML1 channel activity independently of mTORC1. Disrupting LAMTOR1-TRPML1 binding increases TRPML1-mediated Ca2+ release, facilitates dynein-powered dendritic lysosomal trafficking, and alters synaptic plasticity and memory via calcineurin-dependent GluA1 dephosphorylation.","method":"Co-immunoprecipitation, LAMTOR1 deletion in hippocampal neurons, TRPML1 Ca2+ imaging, lysosomal trafficking assays, electrophysiology for synaptic plasticity, behavioral tests","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP with direct Ca2+ channel activity measurement, genetic KO with multiple cellular and in vivo readouts; single lab, multiple orthogonal methods","pmids":["35099830"],"is_preprint":false},{"year":2012,"finding":"Acute siRNA-mediated loss of TRPML1 causes lysosomal cathepsin B (CatB) leak into the cytoplasm, leading to apoptosis that is prevented by CatB inhibition. Bax inhibition prevents apoptosis but not cytosolic CatB release, placing TRPML1 upstream of CatB release and Bax-dependent apoptosis.","method":"siRNA knockdown of TRPML1, cathepsin B localization/activity assay, apoptosis assay, CatB inhibitor and Bax inhibitor treatment","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockdown with defined mechanistic pathway (cathepsin B leak, Bax), pharmacological dissection; single lab","pmids":["22262857"],"is_preprint":false},{"year":2019,"finding":"TRPML1 maintains oncogenic HRAS in signaling-competent nanoclusters at the plasma membrane by mediating cholesterol de-esterification and transport from endolysosomes. TRPML1 inhibition disrupts cholesterol distribution, reduces HRAS nanoclustering and plasma membrane abundance, and attenuates ERK phosphorylation and cell proliferation selectively in HRAS-mutant cancer cells.","method":"MCOLN1 knockdown, TRPML1 pharmacological inhibition, cholesterol distribution assay, HRAS nanoclustering analysis, ERK phosphorylation, cell proliferation assays in HRAS mutant vs. wild-type cells","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic and pharmacological perturbation with multiple downstream mechanistic readouts; single lab","pmids":["30787043"],"is_preprint":false},{"year":2019,"finding":"TRPML1 mediates lysosomal Ca2+ release that controls lysosome-multivesicular body (MVB) interaction and exosome release in podocytes. Acid ceramidase (AC)-generated sphingosine activates TRPML1-mediated Ca2+ release; AC inhibition or TRPML1 blockade suppresses lysosome-MVB interaction, increasing exosome release.","method":"GCaMP3 Ca2+ imaging, Port-a-Patch planar patch-clamp, pharmacological manipulation of sphingolipid pathway, structured illumination microscopy, nanoparticle tracking analysis","journal":"American journal of physiology. Cell physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct patch-clamp electrophysiology combined with Ca2+ imaging and functional membrane trafficking assay; single lab","pmids":["31268777"],"is_preprint":false},{"year":2020,"finding":"TRPML1 channels in late endosomes/lysosomes form stable nanoscale complexes with type 2 ryanodine receptors (RyR2) on the sarcoplasmic reticulum in vascular smooth muscle cells. TRPML1-mediated lysosomal Ca2+ release initiates Ca2+ sparks through RyR2 activation; loss of TRPML1 abolishes Ca2+ sparks, impairs Ca2+-activated K+ channel activity, causes vasoconstriction, and results in spontaneous hypertension in Mcoln1-/- mice.","method":"Superresolution nanoscale microscopy, TRPML1 KO mouse, live-cell confocal imaging, ex vivo pressure myography, radiotelemetry blood pressure measurement, Ca2+ spark imaging","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 2 / Strong — superresolution microscopy showing RyR2 complex, KO mouse with defined functional phenotype (Ca2+ sparks, vasomotor, blood pressure), multiple orthogonal methods; strongly replicated in lower urinary tract (PMID 33199609)","pmids":["32576680","33199609"],"is_preprint":false},{"year":2021,"finding":"TRPML1 activation inhibits autophagic flux by mediating lysosomal zinc release into the cytosol, which blocks the interaction between STX17 on autophagosomes and VAMP8 on lysosomes, thereby disrupting autophagosome-lysosome fusion.","method":"Co-immunoprecipitation of STX17 and VAMP8, lysosomal zinc measurement, TRPML1 agonist treatment, SNARE interaction assay, autophagy flux assay","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP of SNARE proteins plus zinc chelation/supplementation experiments with mechanistic rescue; single lab","pmids":["33890549"],"is_preprint":false},{"year":2017,"finding":"TRPML1 co-immunoprecipitates with ER Ca2+ sensor STIM1 in motor neurons and co-localizes with LAMP1 and ER. STIM1 is required for TRPML1-mediated Ca2+ release; in STIM1-deficient neurons, ML-SA1 and PI(3,5)P2 fail to induce lysosomal Ca2+ release. SERCA inhibition increases TRPML1-mediated Ca2+ efflux, indicating ER-lysosome Ca2+ interplay.","method":"Co-immunoprecipitation, GCaMP3-ML1 Ca2+ imaging, STIM1 knockdown, pharmacological (thapsigargin, ML-SA1), colocalization microscopy","journal":"Scientific reports / FASEB journal","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP and functional Ca2+ imaging with genetic knockdown; findings confirmed in two independent studies (PMID 31341250, PMID 33484198)","pmids":["31341250","33484198"],"is_preprint":false},{"year":2011,"finding":"In the C. elegans model, NAADP activates TRP-ML1 channel activity in reconstituted lysosomal preparations from wild-type but not TRPML1-/- cells; NAADP-induced Ca2+ release and enhanced endosome-lysosome interaction are abolished in TRPML1-/- cells and restored by TRPML1 gene rescue.","method":"Lysosomal channel reconstitution, Ca2+ fluorescence imaging, confocal microscopy of endosome-lysosome dynamics, TRPML1 gene rescue in knockout cells","journal":"American journal of physiology. Cell physiology","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — channel reconstitution in lysosomes, genetic rescue, and live-cell Ca2+ imaging; single lab; note that PMID 21540176 (different lab) shows TRPML1 does not mediate NAADP signaling in other cell types, creating a contradiction — confidence held at Medium","pmids":["21613607"],"is_preprint":false},{"year":2018,"finding":"TLR3 stimulation triggers lysosomal ATP release from astrocytes and RPE cells through TRPML1-mediated Ca2+ signaling; TRPML1 activation (ML-SA1) alone is sufficient to release lysosomal ATP and acid phosphatase; ATP release is abolished in TRPML1-/- cells and reduced by TBK-1 blockade.","method":"TRPML1-/- cells, ML-SA1 pharmacological activation, ATP release assay, lysosomal enzyme release assay, Ca2+ imaging","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO and pharmacological activation with defined secretory phenotype readout; single lab","pmids":["29636491"],"is_preprint":false},{"year":2022,"finding":"Oxidative stress-induced phosphorylation of JIP4 at T217 by CaMK2G in response to TRPML1-mediated Ca2+ fluxes promotes lysosomal retrograde transport (clustering around MTOC) via a JIP4-TRPML1-ALG2 pathway, enhancing autophagy as a defense mechanism against oxidative cytotoxicity.","method":"Phosphorylation site mutagenesis, CaMK2G inhibition, lysosomal positioning assay, JIP4 KO cell analysis, TRPML1 pharmacological manipulation, autophagy flux assay","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO and phosphosite mutagenesis with defined lysosomal positioning phenotype; single lab, multiple orthogonal methods","pmids":["36394115"],"is_preprint":false},{"year":2024,"finding":"AKT directly phosphorylates TRPML1 at Ser343 and inhibits K552 ubiquitination and proteasomal degradation of TRPML1. Stabilized TRPML1 binds ARL8B to trigger lysosomal exocytosis, reducing intracellular ferrous iron and enhancing membrane repair, thereby conferring ferroptosis resistance in AKT-hyperactivated cancer cells.","method":"In vitro AKT kinase assay, phosphosite mutagenesis, ubiquitination assay, co-immunoprecipitation of TRPML1-ARL8B, lysosomal exocytosis assay, ferroptosis assay, in vivo xenograft","journal":"Science translational medicine","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro kinase assay, mutagenesis, Co-IP, and functional rescue with in vivo validation; single lab but multiple orthogonal methods","pmids":["38424427"],"is_preprint":false},{"year":2017,"finding":"Lysosomal adenosine accumulation (caused by ADA deficiency) inhibits TRPML1 channel activity; overexpression of ENT3 (lysosomal adenosine transporter) rescues TRPML1 inhibition. TRPML1 inhibition leads to lysosome enlargement, alkalinization, and dysfunction; TRPML1 activation rescues ADA-deficient B-lymphocyte vulnerability to oxidative stress.","method":"ADA knockout, TRPML1 electrophysiology, ENT3 overexpression rescue, lysosomal pH and size measurement, oxidative stress assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO, electrophysiology, and genetic rescue; single lab","pmids":["28087698"],"is_preprint":false},{"year":2024,"finding":"TRPML1-mediated Ca2+ release promotes autophagosome-lysosome fusion and lysosome acidification within 10–20 min of activation, and increases transport of lysosomal SNARE proteins STX7 and VAMP7 via SNARE carrier vesicles. Incoming vesicle fusion is a prerequisite for lysosomal Ca2+ efflux leading to acidification and hydrolase activation; PI(3,5)P2 is proposed as the physiological TRPML1 activator generated by vesicle fusions.","method":"Pharmacological TRPML1 activation (ML-SA1), lysosomal pH measurement, autophagosome-lysosome fusion assay, SNARE protein trafficking assay, TRPML1 KO validation","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — acute pharmacological and genetic perturbations with defined Ca2+, SNARE, and fusion readouts; single lab","pmids":["39433126"],"is_preprint":false},{"year":2025,"finding":"TRPML1, activated secondarily to ROS upon inflammatory stimuli, mediates lysosomal Fe2+ release into the cytosol, activating prolyl hydroxylase domain enzymes (PHDs). PHDs repress NF-κB transcriptional activity, suppressing IL-1β transcription in macrophages as a negative feedback control of inflammation.","method":"TRPML1 agonist/antagonist treatment, Fe2+ measurement, PHD activity assay, NF-κB reporter assay, IL-1β ELISA, TRPML1 KO macrophages, in vivo colitis model","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple pharmacological and genetic perturbations with defined mechanistic pathway (Fe2+-PHD-NF-κB-IL1B), in vivo validation; single lab but rigorous multi-step mechanism","pmids":["39856099"],"is_preprint":false},{"year":2022,"finding":"TRPML1 localizes to lysosomes in NK cells; genetic deletion of TRPML1 causes mitochondrial fragmentation with collapsed cristae, loss of mitochondrial membrane potential, increased ROS, reduced ATP production, and Ca2+ overload in mitochondria. TRPML1 loss impedes autophagic flux and increases accumulation of dysfunctional mitochondria.","method":"TRPML1 genetic deletion in NK92 cells, organelle-specific Ca2+ probes, mitochondrial morphology analysis, mitochondrial membrane potential measurement, ROS assay, autophagic flux assay","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with multiple orthogonal organelle function readouts; single lab","pmids":["37737664"],"is_preprint":false}],"current_model":"TRPML1 (MCOLN1) is a lysosomal/late-endosomal non-selective cation channel (permeable to Ca2+, Fe2+, Zn2+, H+) gated by the phosphoinositide PI(3,5)P2 (which binds an allosteric site on S1-S3 helices) and modulated by luminal pH, Ca2+, ROS, TOR-mediated phosphorylation, LAMTOR1-mediated tonic inhibition, and the ceramide-sphingosine axis; its Ca2+ efflux into the cytosol triggers multiple downstream cascades including calcineurin-TFEB nuclear translocation (autophagy and lysosome biogenesis), CaMKKβ/AMPK/VPS34-dependent autophagosome biogenesis, calmodulin-dependent lysosome fission, MTORC1 feedback reactivation, RyR2-mediated sarcoplasmic reticulum Ca2+ spark initiation in smooth muscle, and SNARE-dependent autophagosome-lysosome fusion, while its Fe2+ and Zn2+ efflux controls iron homeostasis, ferroptosis susceptibility (via AKT-phosphorylation-stabilized TRPML1-ARL8B-lysosomal exocytosis), and NF-κB-dependent inflammatory gene transcription via PHD enzymes."},"narrative":{"mechanistic_narrative":"MCOLN1 (TRPML1) is a late-endosomal/lysosomal non-selective cation channel that couples lysosomal ion flux to membrane trafficking, autophagy, and organelle homeostasis [PMID:18794901, PMID:19638346]. It conducts Ca2+, Fe2+/Mn2+, Zn2+, and (in some studies) H+, and its loss perturbs lysosomal lipid hydrolysis, pH, iron, and zinc handling, defining the cellular basis of its trafficking and storage defects [PMID:18794901, PMID:16361256, PMID:25119295, PMID:20864526, PMID:23368743]. The channel is gated by the lysosomal phosphoinositide PtdIns(3,5)P2, which binds an allosteric site on extended S1–S3 helices and drives a Y355–R403 π-cation interaction to open the pore, whereas PtdIns(4,5)P2 binds the same site and inhibits; cryo-EM structures in closed and agonist-bound open states define a distinct hydrophobic agonist/antagonist cavity (S5/S6/PH1) and a luminal acidic Ca2+/pH-sensing region [PMID:29019981, PMID:29019983, PMID:30305615, PMID:34171299]. Activity is further set by multiple inputs: cathepsin B cleavage at Arg200–Pro201, TOR-mediated inhibitory phosphorylation, tonic LAMTOR1 binding, ROS, luminal adenosine, and AKT phosphorylation that stabilizes the channel against ubiquitin-dependent degradation [PMID:16257972, PMID:26195823, PMID:35099830, PMID:28087698, PMID:38424427, PMID:27357649]. TRPML1-mediated Ca2+ efflux is the central output, triggering calcineurin–TFEB-driven autophagy and lysosome biogenesis, CaMKKβ/AMPK/VPS34-dependent autophagosome biogenesis, calmodulin-dependent lysosome fission, MTORC1 reactivation, and SNARE-controlled autophagosome–lysosome and phagosome–lysosome fusion [PMID:27357649, PMID:31822666, PMID:28360104, PMID:29460684, PMID:26010303, PMID:33890549, PMID:39433126]. Through inter-organelle contacts it transfers Ca2+ to mitochondria via VDAC1/MCU and to the sarcoplasmic reticulum RyR2 to initiate Ca2+ sparks in vascular smooth muscle, and it functionally couples to ER STIM1 [PMID:32703809, PMID:32576680, PMID:33199609, PMID:31341250, PMID:33484198]. Its metal-ion output additionally governs iron homeostasis and ferroptosis resistance through an AKT–TRPML1–ARL8B lysosomal-exocytosis axis and restrains NF-κB-driven IL-1β transcription via Fe2+-dependent PHD activation [PMID:38424427, PMID:39856099]. Loss-of-function mutations that impair channel activity underlie mucolipidosis type IV, and small-molecule agonists rescue trafficking and lysosomal metal-accumulation defects in patient fibroblasts [PMID:18794901, PMID:25119295].","teleology":[{"year":2005,"claim":"Established the first functional and regulatory framework for the channel by showing it conducts H+ and monovalent cations and is post-translationally controlled, linking its activity directly to lysosomal acidification and lipid hydrolysis.","evidence":"Lysosomal pH and lipase assays plus genetic/pharmacological rescue in MLIV patient cells; whole-lysosome patch-clamp with cathepsin B cleavage mapping and co-IP","pmids":["16361256","16257972"],"confidence":"High","gaps":["H+ permeability later disputed by a proton-impermeable characterization","physiological trigger for cathepsin B cleavage in vivo not defined"]},{"year":2006,"claim":"Defined the channel's oligomeric behavior, showing it forms homo- and heteromultimers with TRPML2/3 and dictates the lysosomal targeting of TRPML3.","evidence":"Co-IP and colocalization microscopy with targeting-mutant co-expression","pmids":["16606612"],"confidence":"Medium","gaps":["functional consequence of heteromultimerization for channel gating untested","stoichiometry of native complexes unknown"]},{"year":2008,"claim":"Resolved a key permeant species by demonstrating Fe2+ conductance and linking disease-mutation severity to graded loss of iron permeation, explaining lysosomal iron storage.","evidence":"Direct lysosomal patch-clamp, radiolabelled iron flux, and cytosolic/intralysosomal iron monitoring in patient fibroblasts","pmids":["18794901"],"confidence":"High","gaps":["relative physiological contribution of Fe2+ vs Ca2+ flux not quantified","downstream iron-dependent signaling not addressed here"]},{"year":2009,"claim":"Defined permeation selectivity and gating by isolating constitutively active S5 mutants, showing Ca2+ release drives lysosomal exocytosis and surface LAMP-1.","evidence":"Proline-scanning mutagenesis with whole-cell/lysosomal patch-clamp and LAMP-1 surface staining","pmids":["19638346"],"confidence":"High","gaps":["proton impermeability contradicts earlier H+ channel report","endogenous gating trigger not identified in this study"]},{"year":2010,"claim":"Extended the channel's ion repertoire and physiology by linking it to lysosomal zinc handling and to gastric acid secretion via parietal-cell tubulovesicle trafficking.","evidence":"siRNA/KO with fluorometric zinc and ICP-MS; Trpml1-/- mouse gastric secretion, palmitoylation/phosphorylation analysis and EM","pmids":["20864526","23368743","21111738"],"confidence":"Medium","gaps":["direct Zn2+ permeation through the channel vs indirect transporter effects not fully separated","mechanism coupling palmitoylation to channel function unresolved"]},{"year":2011,"claim":"Clarified relationships with other endolysosomal channels, showing physical but functionally independent association with TPCs while ortholog studies linked the channel to NAADP-evoked Ca2+ release and autophagic degradation.","evidence":"Reciprocal co-IP, colocalization, patch-clamp and Ca2+ imaging in KO cells; C. elegans CUP-5 genetics and lysosomal reconstitution","pmids":["21540176","21997367","21613607"],"confidence":"Medium","gaps":["NAADP responsiveness is contradictory between cell types/labs","molecular basis of TRPML1-TPC association not defined"]},{"year":2012,"claim":"Connected channel loss to cell death by placing acute TRPML1 loss upstream of cathepsin B leak and Bax-dependent apoptosis.","evidence":"siRNA knockdown with cathepsin B localization/activity assays and CatB/Bax inhibitor dissection","pmids":["22262857"],"confidence":"Medium","gaps":["mechanism by which channel loss triggers membrane permeabilization unclear","acute knockdown phenotype may differ from chronic disease state"]},{"year":2015,"claim":"Established PtdIns(3,5)P2 as the activating ligand controlling membrane fusion and showed TOR phosphorylation as a direct inhibitory input regulating autophagy.","evidence":"PIKfyve epistasis with phagosome-lysosome fusion and Ca2+ rescue; in vitro kinase assay with phosphosite mutagenesis and channel/autophagy readouts","pmids":["26010303","26195823"],"confidence":"High","gaps":["TOR phosphosite study is Medium confidence single-lab","precise residues and kinase specificity for TOR phosphorylation not structurally mapped"]},{"year":2017,"claim":"Defined multiple physiological activators and partners, establishing ROS-triggered Ca2+ release driving TFEB/autophagy, LAMTOR1 as a tonic inhibitor, sphingosine/ER-STIM1 coupling, and calmodulin-dependent lysosome fission.","evidence":"GCaMP3-ML1 Ca2+ imaging, genetic KO/knockdown, co-IP, and pharmacological dissection across cell types","pmids":["27357649","35099830","31341250","33484198","28360104","28087698"],"confidence":"High","gaps":["several upstream activators tested in distinct systems; integration in a single cell unclear","how ROS chemically modifies the channel not defined"]},{"year":2017,"claim":"Provided the first atomic-resolution framework by solving cryo-EM structures in closed and agonist-bound open states and locating the PtdIns(3,5)P2 site distal to the pore.","evidence":"Single-particle cryo-EM of mouse and human channel with mutagenesis/electrophysiology","pmids":["29019981","29019983"],"confidence":"High","gaps":["physiological gating transitions in native lysosomal membranes not directly observed","structural basis of Fe2+/Zn2+ selectivity not resolved"]},{"year":2018,"claim":"Refined the lipid-gating mechanism (Y355–R403 π-cation, opposing PtdIns(4,5)P2 inhibition) and embedded the channel in an MTORC1 negative-feedback loop activated by starvation.","evidence":"Ligand-bound cryo-EM with electrophysiology; pharmacological/genetic perturbation with calmodulin inhibition and S6K readout","pmids":["30305615","29460684"],"confidence":"High","gaps":["how PtdIns(4,5)P2 reaches the lysosomal-facing site physiologically unclear","feedback-loop study is Medium confidence single-lab"]},{"year":2019,"claim":"Diversified the channel's downstream outputs, defining a TFEB-independent CaMKKβ/AMPK/VPS34 autophagosome-biogenesis pathway, MVB/exosome control via acid ceramidase, and a cholesterol-transport role sustaining oncogenic HRAS nanoclustering.","evidence":"PI3P and phagophore-recruitment assays with kinase inhibition; Ca2+ imaging with sphingolipid manipulation; cholesterol and HRAS nanoclustering analysis in mutant cells","pmids":["31822666","31268777","30787043"],"confidence":"Medium","gaps":["cholesterol and exosome roles single-lab Medium confidence","directness of TRPML1 in cholesterol de-esterification not established"]},{"year":2020,"claim":"Established inter-organelle Ca2+ signaling roles by showing channel-mediated Ca2+ transfer to mitochondria (VDAC1/MCU) and SR RyR2-dependent Ca2+ spark initiation controlling vascular tone and blood pressure.","evidence":"Super-resolution live-cell microscopy, KO mice, pressure myography and radiotelemetry; patient-fibroblast contact dynamics","pmids":["32703809","32576680","33199609"],"confidence":"High","gaps":["tethering machinery at mitochondria-lysosome contacts not fully identified","tissue-specificity of RyR2 coupling beyond smooth muscle untested"]},{"year":2021,"claim":"Resolved the antagonist binding mode and uncovered a divalent-ion output controlling SNARE-mediated fusion, showing ML-SI3 occupies the agonist cavity and Zn2+ release blocks STX17-VAMP8 pairing.","evidence":"Cryo-EM with ML-SI3 plus patch-clamp; SNARE co-IP with lysosomal zinc measurement and autophagy flux","pmids":["34171299","33890549"],"confidence":"High","gaps":["antagonist study High confidence; zinc-SNARE mechanism Medium single-lab","apparent opposing effects of activation on fusion across studies need reconciliation"]},{"year":2022,"claim":"Defined cooperative dual-ligand gating and additional Ca2+-dependent outputs, showing PI(3,5)P2 and rapamycin synergize at distinct sites and that ROS/CaMK2G-JIP4 signaling drives retrograde lysosome transport.","evidence":"Multi-state cryo-EM with electrophysiology; phosphosite mutagenesis, CaMK2G inhibition and lysosomal positioning/autophagy assays; NK-cell KO mitochondrial phenotyping","pmids":["35131932","36394115","37737664"],"confidence":"High","gaps":["physiological relevance of rapamycin-analog synergy unclear","JIP4 and NK-cell roles Medium confidence single-lab"]},{"year":2024,"claim":"Connected channel regulation to ferroptosis and membrane repair, showing AKT phosphorylation at Ser343 stabilizes the channel and ARL8B-dependent lysosomal exocytosis lowers iron and confers ferroptosis resistance.","evidence":"In vitro AKT kinase assay, phosphosite/ubiquitination mutagenesis, TRPML1-ARL8B co-IP, exocytosis and ferroptosis assays with xenografts","pmids":["38424427"],"confidence":"High","gaps":["generality beyond AKT-hyperactivated cancers untested","competition between Ser343 and other regulatory inputs in vivo unknown"]},{"year":2025,"claim":"Extended the channel's iron output to inflammatory control, showing Fe2+ release activates PHD enzymes that repress NF-κB and suppress IL-1β transcription in macrophages.","evidence":"Agonist/antagonist and KO macrophages with Fe2+/PHD/NF-κB readouts and an in vivo colitis model","pmids":["39856099"],"confidence":"High","gaps":["direct PHD substrate engagement by cytosolic Fe2+ not structurally shown","balance between pro- and anti-inflammatory iron effects in different cells unclear"]},{"year":null,"claim":"How the diverse, sometimes opposing outputs of a single channel (Ca2+, Fe2+, Zn2+, H+ flux promoting versus inhibiting autophagosome-lysosome fusion) are integrated, prioritized, and spatially partitioned in native lysosomes remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["conflicting reports on whether activation promotes or blocks autophagic flux","no unified model linking ion-species selection to specific downstream cascade","physiological vs pharmacological activation regimes not reconciled"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[0,4,17,18]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[8,10]},{"term_id":"GO:0140299","term_label":"molecular sensor activity","supporting_discovery_ids":[7]}],"localization":[{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[0,1,2,4,17]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[0,3,5]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[3,29]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[4,25]}],"pathway":[{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[6,7,16,35]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[13,28,35]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[0,18,36]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[15,20,27]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[31,36,37]}],"complexes":["TRPML1/TRPML2/TRPML3 heteromultimer","TRPML1-RyR2 ER/SR-lysosome complex"],"partners":["TPC2","TMEM163","LAMTOR1","STIM1","RYR2","VDAC1","MCU","ARL8B"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9GZU1","full_name":"Mucolipin-1","aliases":["MG-2","Mucolipidin","Transient receptor potential channel mucolipin 1","TRPML1"],"length_aa":580,"mass_kda":65.0,"function":"Nonselective cation channel probably playing a role in the regulation of membrane trafficking events and of metal homeostasis (PubMed:11013137, PubMed:12459486, PubMed:14749347, PubMed:15336987, PubMed:18794901, PubMed:25720963, PubMed:27623384, PubMed:29019983). Acts as a Ca(2+)-permeable cation channel with inwardly rectifying activity (PubMed:25720963, PubMed:29019983). Proposed to play a major role in Ca(2+) release from late endosome and lysosome vesicles to the cytoplasm, which is important for many lysosome-dependent cellular events, including the fusion and trafficking of these organelles, exocytosis and autophagy (PubMed:11013137, PubMed:12459486, PubMed:14749347, PubMed:15336987, PubMed:25720963, PubMed:27623384, PubMed:29019983). Required for efficient uptake of large particles in macrophages in which Ca(2+) release from the lysosomes triggers lysosomal exocytosis. May also play a role in phagosome-lysosome fusion (By similarity). Involved in lactosylceramide trafficking indicative for a role in the regulation of late endocytic membrane fusion/fission events (PubMed:16978393). By mediating lysosomal Ca(2+) release is involved in regulation of mTORC1 signaling and in mTOR/TFEB-dependent lysosomal adaptation to environmental cues such as nutrient levels (PubMed:25720963, PubMed:25733853, PubMed:27787197). Seems to act as lysosomal active oxygen species (ROS) sensor involved in ROS-induced TFEB activation and autophagy (PubMed:27357649). Also functions as a Fe(2+) permeable channel in late endosomes and lysosomes (PubMed:18794901). Also permeable to Mg(2+), Na(+). K(+) and Cs(+) (By similarity). Proposed to play a role in zinc homeostasis probably implicating its association with TMEM163 (PubMed:25130899) In adaptive immunity, TRPML2 and TRPML1 may play redundant roles in the function of the specialized lysosomes of B cells (By similarity) May contribute to cellular lipase activity within the late endosomal pathway or at the cell surface which may be involved in processes of membrane reshaping and vesiculation, especially the growth of tubular structures. However, it is not known, whether it conveys the enzymatic activity directly, or merely facilitates the activity of an associated phospholipase","subcellular_location":"Late endosome membrane; Lysosome membrane; Cytoplasmic vesicle membrane; Cell projection, phagocytic cup; Cytoplasmic vesicle, phagosome membrane; Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q9GZU1/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MCOLN1","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000090674","cell_line_id":"CID001658","localizations":[{"compartment":"vesicles","grade":3}],"interactors":[{"gene":"MCOLN2","stoichiometry":0.2},{"gene":"C6ORF106","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001658","total_profiled":1310},"omim":[{"mim_id":"620763","title":"CORNEAL DYSTROPHY, LISCH EPITHELIAL; LECD","url":"https://www.omim.org/entry/620763"},{"mim_id":"618978","title":"TRANSMEMBRANE PROTEIN 163; TMEM163","url":"https://www.omim.org/entry/618978"},{"mim_id":"608179","title":"CAYTAXIN; ATCAY","url":"https://www.omim.org/entry/608179"},{"mim_id":"607400","title":"MUCOLIPIN 3; MCOLN3","url":"https://www.omim.org/entry/607400"},{"mim_id":"607399","title":"MUCOLIPIN 2; MCOLN2","url":"https://www.omim.org/entry/607399"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/MCOLN1"},"hgnc":{"alias_symbol":["TRPML1","ML4","MLIV","MST080","MSTP080","TRPM-L1"],"prev_symbol":[]},"alphafold":{"accession":"Q9GZU1","domains":[{"cath_id":"-","chopping":"106-198_220-282","consensus_level":"high","plddt":90.6236,"start":106,"end":282},{"cath_id":"1.20.120","chopping":"63-83_294-407","consensus_level":"high","plddt":89.3101,"start":63,"end":407},{"cath_id":"1.10.287","chopping":"414-527","consensus_level":"high","plddt":91.4214,"start":414,"end":527}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9GZU1","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9GZU1-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9GZU1-F1-predicted_aligned_error_v6.png","plddt_mean":81.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MCOLN1","jax_strain_url":"https://www.jax.org/strain/search?query=MCOLN1"},"sequence":{"accession":"Q9GZU1","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9GZU1.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9GZU1/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9GZU1"}},"corpus_meta":[{"pmid":"18794901","id":"PMC_18794901","title":"The 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ML4 disease mutations impair Fe2+ permeation at varying degrees correlating with disease severity. Loss of TRPML1 reduces cytosolic Fe2+ and increases intralysosomal Fe2+ accumulation.\",\n      \"method\": \"Radiolabelled iron uptake assays, cytosolic and intralysosomal iron monitoring, direct patch-clamping of late endosomal/lysosomal membrane, comparison of TRPML1-/- vs. control human fibroblasts\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct lysosomal patch-clamp electrophysiology combined with iron flux assays and disease-mutation functional correlation; multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"18794901\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"TRP-ML1 can function as a H+ channel and its loss leads to lysosomal over-acidification in MLIV patient cells, reducing acidic lipase activity. Expression of TRP-ML1 rescues lipid hydrolysis, and dissipation of lysosomal pH reverses storage phenotype.\",\n      \"method\": \"Lysosomal pH measurement in TRP-ML1-/- patient cells, lipase activity assay with multiple substrates, cell fractionation, rescue by TRP-ML1 expression and pH-dissipating drugs\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — multiple orthogonal methods (pH measurement, lipase activity, genetic rescue, pharmacological rescue) in patient-derived cells; single lab\",\n      \"pmids\": [\"16361256\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"TRP-ML1 is a lysosomal monovalent cation channel that undergoes proteolytic cleavage by cathepsin B at Arg200-Pro201; cleavage inhibits channel activity. N- and C-terminal fragments are co-immunoprecipitated. The R200H disease mutation alters this cleavage pattern.\",\n      \"method\": \"Electrophysiology (whole-lysosome/planar patch-clamp), co-immunoprecipitation, N-terminal sequencing of purified C-terminal fragment, cathepsin B inhibitor treatment, CatB-/- cell expression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with protein sequencing, co-IP, and mutagenesis/pharmacological inhibition; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"16257972\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"TRPML1 forms homo- and heteromultimers with TRPML2 and TRPML3. TRPML1 and TRPML2 homomultimers are lysosomal, while TRPML3 homomultimers are in the ER. The presence of TRPML1 or TRPML2 specifically dictates lysosomal localization of TRPML3, but not vice versa.\",\n      \"method\": \"Co-immunoprecipitation, subcellular localization by fluorescence microscopy, co-expression studies with lysosomal targeting-disrupted mutants\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP and colocalization microscopy, single lab, two complementary methods\",\n      \"pmids\": [\"16606612\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Proline scanning mutagenesis revealed gain-of-function constitutive activating mutations in the S5 transmembrane domain of TRPML1 (e.g., V432P). TRPML1 is an inwardly rectifying, proton-impermeable, Ca2+ and Fe2+/Mn2+ permeable channel; constitutive Ca2+ release from lysosomes promotes lysosomal exocytosis and surface expression of LAMP-1.\",\n      \"method\": \"Systematic proline-substitution mutagenesis, whole-cell and lysosomal patch-clamp electrophysiology, LAMP-1 surface staining\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstitution via mutagenesis plus direct electrophysiology, single lab with multiple mutants and orthogonal readouts\",\n      \"pmids\": [\"19638346\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"TRPML1 co-immunoprecipitates with TPC2 and shows near-complete colocalization with TPC2 on endolysosomes, but electrophysiology shows TPC1/TPC2 do not affect TRPML1 channel activity, and TRPML1 does not mediate NAADP-evoked Ca2+ signals — TRPML1 and TPCs are physically associated but functionally independent organellar ion channels.\",\n      \"method\": \"Co-immunoprecipitation, colocalization microscopy, whole-cell and whole-lysosome patch-clamp electrophysiology, Ca2+ imaging in TRP-ML1-/- cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — reciprocal co-IP plus direct electrophysiology plus knockout cell validation; single lab but three orthogonal methods\",\n      \"pmids\": [\"21540176\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"CUP-5, the C. elegans ortholog of TRPML1, localizes to lysosomes and is required for proteolytic degradation in autolysosomes; cup-5 mutations cause accumulation of autophagy substrates in enlarged late endosomal/lysosomal vacuoles, and reduced autophagy activity partially suppresses cup-5 mutant defects.\",\n      \"method\": \"Genetic epistasis analysis, fluorescence microscopy with organelle markers, immunoprecipitation, genetic suppressor analysis in C. elegans\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis in a model organism, multiple complementary assays; ortholog of mammalian TRPML1\",\n      \"pmids\": [\"21997367\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ROS directly and specifically activate lysosomal TRPML1 channels, inducing lysosomal Ca2+ release. This Ca2+ release triggers calcineurin-dependent TFEB nuclear translocation, autophagy induction, and lysosome biogenesis. Genetic inactivation or pharmacological inhibition of TRPML1 blocks clearance of damaged mitochondria and removal of excess ROS.\",\n      \"method\": \"GCaMP3-ML1 Ca2+ imaging, pharmacological ROS manipulation, TRPML1 genetic knockout and inhibition, TFEB nuclear translocation assay, autophagy flux assays, mitochondrial damage assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods including live-cell Ca2+ imaging, genetic KO, pharmacological inhibition, TFEB assay; replicated across multiple stress conditions\",\n      \"pmids\": [\"27357649\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Cryo-EM structure of mouse TRPML1 in nanodiscs reveals that PtdIns(3,5)P2 binds to the N-terminus distal from the pore; an S2-S3 helix-turn-helix extension couples ligand binding to pore opening; the selectivity filter contains multiple ion-binding sites; conserved acidic residues form a luminal Ca2+-blocking site conferring pH and Ca2+ modulation; a luminal linker domain canopy creates a negative electrostatic trap for divalent cations.\",\n      \"method\": \"Single-particle cryo-EM structure determination, mutagenesis combined with electrophysiology\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution cryo-EM structure combined with mutagenesis and electrophysiological validation\",\n      \"pmids\": [\"29019981\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Cryo-EM structures of full-length human TRPML1 in apo (closed, pH 7.0) and agonist-bound (open, pH 6.0) states reveal that channel opening involves dilation of the lower gate and movement of pore helix 1; the agonist binds a hydrophobic cavity formed by S5, S6, and pore helix 1, distinct from TRPV1 agonist sites.\",\n      \"method\": \"Single-particle cryo-EM at 3.72 Å (closed) and 3.49 Å (open) resolution, structural comparison\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — near-atomic cryo-EM structures in two functional states with direct structural comparison\",\n      \"pmids\": [\"29019983\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Cryo-EM structures of human TRPML1 with PtdIns(3,5)P2 and PtdIns(4,5)P2 reveal a unique lipid-binding site on extended helices S1, S2, and S3. PtdIns(3,5)P2 induces Y355 to form a π-cation interaction with R403, moving the S4-S5 linker to allosterically activate the channel. PtdIns(4,5)P2 binds the same site but inhibits channel activity.\",\n      \"method\": \"Cryo-EM structure determination at pH 5.0 with bound lipids and ML-SA1, electrophysiological characterization\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structures with bound ligands combined with electrophysiology; single lab, multiple ligand conditions\",\n      \"pmids\": [\"30305615\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Cryo-EM structures of mouse TRPML1 in apo-closed, PI(3,5)P2-bound closed, and PI(3,5)P2/temsirolimus(rapamycin analog)-bound open states reveal that PI(3,5)P2 and rapamycin bind distinct sites and work cooperatively; the structures elucidate the allosteric mechanism for synergistic channel activation.\",\n      \"method\": \"Cryo-EM structure determination in multiple states, electrophysiology\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple cryo-EM structures in distinct functional states combined with electrophysiology; single lab\",\n      \"pmids\": [\"35131932\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Cryo-EM structure of human TRPML1 with antagonist ML-SI3 at 2.9 Å shows ML-SI3 binds the same hydrophobic cavity (S5, S6, PH1) as agonist ML-SA1; electrophysiology confirms ML-SI3 competes with ML-SA1 but does not inhibit PI(3,5)P2-dependent activation.\",\n      \"method\": \"Cryo-EM structure determination, whole-lysosome patch-clamp electrophysiology\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structure plus functional electrophysiology; single lab, two orthogonal methods\",\n      \"pmids\": [\"34171299\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TRPML1 is a PtdIns(3,5)P2-gated lysosomal Ca2+ channel required for phagosome-lysosome fusion. Silencing TRPML1 causes lysosomes to dock but not fuse with phagosomes, impairing bactericidal capacity. PIKfyve generates PtdIns(3,5)P2 to activate TRPML1, raising cytosolic Ca2+ during phagocytosis; forced Ca2+ release rescues fusion in TRPML1-silenced cells.\",\n      \"method\": \"TRPML1 siRNA knockdown, phagocytosis assay with lysosomal marker acquisition, isolated phagosome analysis by electron microscopy, Ca2+ imaging, ionomycin rescue\",\n      \"journal\": \"Traffic\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockdown with defined phenotypic readout, Ca2+ imaging, pharmacological rescue, and mechanistic epistasis with PIKfyve; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"26010303\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TOR kinase directly phosphorylates TRPML1, inactivating its channel activity and suppressing autophagy. Mutation of the TOR phosphorylation sites to unphosphorylatable residues blocks TOR regulation of TRPML1.\",\n      \"method\": \"In vitro kinase assay, phosphorylation site mutagenesis, channel activity recordings, autophagy flux assays\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct kinase phosphorylation assay combined with mutagenesis and functional channel assay; single lab\",\n      \"pmids\": [\"26195823\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Starvation activates MCOLN1 by relieving MTORC1's inhibition of the channel; activated MCOLN1 in turn facilitates MTORC1 reactivation through a calmodulin-dependent mechanism, constituting a negative feedback loop that prevents excessive MTORC1 inhibition during prolonged starvation.\",\n      \"method\": \"Pharmacological activation/inhibition of MCOLN1 and MTORC1, calmodulin inhibition, Ca2+ chelation, MTORC1 activity assays (S6K phosphorylation), MCOLN1 knockdown/knockout\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple pharmacological and genetic perturbations with defined signaling readout; single lab\",\n      \"pmids\": [\"29460684\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TRPML1 activation induces autophagosome biogenesis through a TFEB-independent pathway requiring CaMKKβ and AMPK, which activate ULK1 and VPS34 complexes and generate PI3P. MLIV patient cells show reduced PI3P-binding protein recruitment to phagophores.\",\n      \"method\": \"PI3P generation assay, phagophore recruitment of PI3P-binding proteins, CaMKKβ/AMPK inhibition, ULK1/VPS34 complex activation assays, TFEB knockout, patient cell analysis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple pharmacological and genetic perturbations, patient cell validation, defined downstream signaling pathway; single lab but multiple orthogonal assays\",\n      \"pmids\": [\"31822666\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TRPML1 mutant isoforms (F465L, F408Δ) show strongly reduced activation by PtdIns(3,5)P2 but can be activated by synthetic ligands. F465L renders TRPML1 pH-insensitive; F408Δ impacts synthetic ligand binding. Small-molecule activators rescue trafficking defects and lysosomal zinc accumulation in MLIV patient fibroblasts.\",\n      \"method\": \"Whole-lysosome planar patch-clamp, pharmacological activation with synthetic ligands, trafficking assay, zinc accumulation assay in patient fibroblasts\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct lysosomal electrophysiology with disease mutants, pharmacological rescue in patient cells; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"25119295\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"TRPML1-deficient cells and Mcoln1-/- mouse brain show elevated chelatable zinc levels. siRNA knockdown of TRPML1 causes lysosomal zinc accumulation; TRPML1 loss delays zinc leak from lysosomes to cytoplasm and is associated with elevated MTF-1-dependent transcription. ZnT4 knockdown ameliorates the lysosomal enlargement phenotype in TRPML1-KD cells exposed to zinc.\",\n      \"method\": \"siRNA knockdown, fluorometric zinc quantification, ICP-MS of brain tissue, lysosomal zinc staining, MTF-1 and ZnT4 co-knockdown\",\n      \"journal\": \"The Biochemical journal / The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — multiple genetic and pharmacological perturbations with zinc quantification in cells and in vivo; findings from two independent labs (PMID 20864526 and 23368743)\",\n      \"pmids\": [\"20864526\", \"23368743\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TMEM163 protein (a putative zinc transporter) is a novel interacting partner for TRPML1, confirmed by yeast two-hybrid, co-immunoprecipitation, mass spectrometry, and colocalization microscopy. Interaction requires part of TMEM163's N-terminus. Co-expression of TMEM163 does not alter TRPML1 channel activity, but TRPML1 co-expression reduces TMEM163 at the plasma membrane.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, mass spectrometry, confocal colocalization, subcellular localization analysis\",\n      \"journal\": \"Traffic\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — four orthogonal binding assays confirming interaction; single lab\",\n      \"pmids\": [\"25130899\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Mitochondria-lysosome contact sites facilitate Ca2+ transfer from lysosomes to mitochondria through TRPML1 lysosomal Ca2+ efflux. This transfer is mediated by tethering at contact sites and requires VDAC1 (outer mitochondrial membrane) and MCU (inner mitochondrial membrane). MLIV patient fibroblasts show altered contact dynamics and defective contact-dependent mitochondrial Ca2+ uptake.\",\n      \"method\": \"High spatial/temporal resolution live-cell microscopy, TRPML1 agonist stimulation, VDAC1/MCU inhibition, MLIV patient fibroblast analysis, contact site dynamics quantification\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live-cell super-resolution microscopy with pharmacological and genetic perturbations, patient cell validation; single lab, multiple orthogonal approaches\",\n      \"pmids\": [\"32703809\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TRPML1-mediated lysosomal Ca2+ release activates calmodulin (CaM) to promote lysosome fission, reducing lysosomal size. TRPML1 activation suppresses enlarged vacuoles induced by vacuolin-1 or P2X4; effects are abolished by Ca2+ chelation or CaM inhibition.\",\n      \"method\": \"Pharmacological TRPML1 activation, Ca2+ chelation, CaM inhibition, lysosome size quantification by fluorescence microscopy\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — pharmacological manipulation with defined organelle phenotype readout; single lab, multiple perturbations\",\n      \"pmids\": [\"28360104\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Loss of TRPML1 in Trpml1-/- mice causes impaired gastric acid secretion associated with dynamic palmitoylation and dephosphorylation of Trpml1 in parietal cells upon histamine stimulation, mislocalization of the gastric proton pump, and enlarged/dysfunctional secretory canaliculi. TRPML1 is required for tubulovesicle formation and trafficking in parietal cells.\",\n      \"method\": \"Gene-targeted Trpml1-/- mouse model, gastric acid secretion measurement, biochemical analysis of palmitoylation/phosphorylation, immunohistochemistry, electron microscopy of parietal cells\",\n      \"journal\": \"Gastroenterology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO mouse with defined cellular phenotype, multiple biochemical readouts; single lab\",\n      \"pmids\": [\"21111738\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"LAMTOR1, a subunit of the Ragulator complex, directly interacts with TRPML1 through its N-terminal domain and tonically inhibits TRPML1 channel activity independently of mTORC1. Disrupting LAMTOR1-TRPML1 binding increases TRPML1-mediated Ca2+ release, facilitates dynein-powered dendritic lysosomal trafficking, and alters synaptic plasticity and memory via calcineurin-dependent GluA1 dephosphorylation.\",\n      \"method\": \"Co-immunoprecipitation, LAMTOR1 deletion in hippocampal neurons, TRPML1 Ca2+ imaging, lysosomal trafficking assays, electrophysiology for synaptic plasticity, behavioral tests\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP with direct Ca2+ channel activity measurement, genetic KO with multiple cellular and in vivo readouts; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"35099830\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Acute siRNA-mediated loss of TRPML1 causes lysosomal cathepsin B (CatB) leak into the cytoplasm, leading to apoptosis that is prevented by CatB inhibition. Bax inhibition prevents apoptosis but not cytosolic CatB release, placing TRPML1 upstream of CatB release and Bax-dependent apoptosis.\",\n      \"method\": \"siRNA knockdown of TRPML1, cathepsin B localization/activity assay, apoptosis assay, CatB inhibitor and Bax inhibitor treatment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockdown with defined mechanistic pathway (cathepsin B leak, Bax), pharmacological dissection; single lab\",\n      \"pmids\": [\"22262857\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TRPML1 maintains oncogenic HRAS in signaling-competent nanoclusters at the plasma membrane by mediating cholesterol de-esterification and transport from endolysosomes. TRPML1 inhibition disrupts cholesterol distribution, reduces HRAS nanoclustering and plasma membrane abundance, and attenuates ERK phosphorylation and cell proliferation selectively in HRAS-mutant cancer cells.\",\n      \"method\": \"MCOLN1 knockdown, TRPML1 pharmacological inhibition, cholesterol distribution assay, HRAS nanoclustering analysis, ERK phosphorylation, cell proliferation assays in HRAS mutant vs. wild-type cells\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic and pharmacological perturbation with multiple downstream mechanistic readouts; single lab\",\n      \"pmids\": [\"30787043\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TRPML1 mediates lysosomal Ca2+ release that controls lysosome-multivesicular body (MVB) interaction and exosome release in podocytes. Acid ceramidase (AC)-generated sphingosine activates TRPML1-mediated Ca2+ release; AC inhibition or TRPML1 blockade suppresses lysosome-MVB interaction, increasing exosome release.\",\n      \"method\": \"GCaMP3 Ca2+ imaging, Port-a-Patch planar patch-clamp, pharmacological manipulation of sphingolipid pathway, structured illumination microscopy, nanoparticle tracking analysis\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct patch-clamp electrophysiology combined with Ca2+ imaging and functional membrane trafficking assay; single lab\",\n      \"pmids\": [\"31268777\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TRPML1 channels in late endosomes/lysosomes form stable nanoscale complexes with type 2 ryanodine receptors (RyR2) on the sarcoplasmic reticulum in vascular smooth muscle cells. TRPML1-mediated lysosomal Ca2+ release initiates Ca2+ sparks through RyR2 activation; loss of TRPML1 abolishes Ca2+ sparks, impairs Ca2+-activated K+ channel activity, causes vasoconstriction, and results in spontaneous hypertension in Mcoln1-/- mice.\",\n      \"method\": \"Superresolution nanoscale microscopy, TRPML1 KO mouse, live-cell confocal imaging, ex vivo pressure myography, radiotelemetry blood pressure measurement, Ca2+ spark imaging\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — superresolution microscopy showing RyR2 complex, KO mouse with defined functional phenotype (Ca2+ sparks, vasomotor, blood pressure), multiple orthogonal methods; strongly replicated in lower urinary tract (PMID 33199609)\",\n      \"pmids\": [\"32576680\", \"33199609\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TRPML1 activation inhibits autophagic flux by mediating lysosomal zinc release into the cytosol, which blocks the interaction between STX17 on autophagosomes and VAMP8 on lysosomes, thereby disrupting autophagosome-lysosome fusion.\",\n      \"method\": \"Co-immunoprecipitation of STX17 and VAMP8, lysosomal zinc measurement, TRPML1 agonist treatment, SNARE interaction assay, autophagy flux assay\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP of SNARE proteins plus zinc chelation/supplementation experiments with mechanistic rescue; single lab\",\n      \"pmids\": [\"33890549\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TRPML1 co-immunoprecipitates with ER Ca2+ sensor STIM1 in motor neurons and co-localizes with LAMP1 and ER. STIM1 is required for TRPML1-mediated Ca2+ release; in STIM1-deficient neurons, ML-SA1 and PI(3,5)P2 fail to induce lysosomal Ca2+ release. SERCA inhibition increases TRPML1-mediated Ca2+ efflux, indicating ER-lysosome Ca2+ interplay.\",\n      \"method\": \"Co-immunoprecipitation, GCaMP3-ML1 Ca2+ imaging, STIM1 knockdown, pharmacological (thapsigargin, ML-SA1), colocalization microscopy\",\n      \"journal\": \"Scientific reports / FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP and functional Ca2+ imaging with genetic knockdown; findings confirmed in two independent studies (PMID 31341250, PMID 33484198)\",\n      \"pmids\": [\"31341250\", \"33484198\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"In the C. elegans model, NAADP activates TRP-ML1 channel activity in reconstituted lysosomal preparations from wild-type but not TRPML1-/- cells; NAADP-induced Ca2+ release and enhanced endosome-lysosome interaction are abolished in TRPML1-/- cells and restored by TRPML1 gene rescue.\",\n      \"method\": \"Lysosomal channel reconstitution, Ca2+ fluorescence imaging, confocal microscopy of endosome-lysosome dynamics, TRPML1 gene rescue in knockout cells\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — channel reconstitution in lysosomes, genetic rescue, and live-cell Ca2+ imaging; single lab; note that PMID 21540176 (different lab) shows TRPML1 does not mediate NAADP signaling in other cell types, creating a contradiction — confidence held at Medium\",\n      \"pmids\": [\"21613607\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TLR3 stimulation triggers lysosomal ATP release from astrocytes and RPE cells through TRPML1-mediated Ca2+ signaling; TRPML1 activation (ML-SA1) alone is sufficient to release lysosomal ATP and acid phosphatase; ATP release is abolished in TRPML1-/- cells and reduced by TBK-1 blockade.\",\n      \"method\": \"TRPML1-/- cells, ML-SA1 pharmacological activation, ATP release assay, lysosomal enzyme release assay, Ca2+ imaging\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO and pharmacological activation with defined secretory phenotype readout; single lab\",\n      \"pmids\": [\"29636491\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Oxidative stress-induced phosphorylation of JIP4 at T217 by CaMK2G in response to TRPML1-mediated Ca2+ fluxes promotes lysosomal retrograde transport (clustering around MTOC) via a JIP4-TRPML1-ALG2 pathway, enhancing autophagy as a defense mechanism against oxidative cytotoxicity.\",\n      \"method\": \"Phosphorylation site mutagenesis, CaMK2G inhibition, lysosomal positioning assay, JIP4 KO cell analysis, TRPML1 pharmacological manipulation, autophagy flux assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO and phosphosite mutagenesis with defined lysosomal positioning phenotype; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"36394115\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"AKT directly phosphorylates TRPML1 at Ser343 and inhibits K552 ubiquitination and proteasomal degradation of TRPML1. Stabilized TRPML1 binds ARL8B to trigger lysosomal exocytosis, reducing intracellular ferrous iron and enhancing membrane repair, thereby conferring ferroptosis resistance in AKT-hyperactivated cancer cells.\",\n      \"method\": \"In vitro AKT kinase assay, phosphosite mutagenesis, ubiquitination assay, co-immunoprecipitation of TRPML1-ARL8B, lysosomal exocytosis assay, ferroptosis assay, in vivo xenograft\",\n      \"journal\": \"Science translational medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro kinase assay, mutagenesis, Co-IP, and functional rescue with in vivo validation; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"38424427\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Lysosomal adenosine accumulation (caused by ADA deficiency) inhibits TRPML1 channel activity; overexpression of ENT3 (lysosomal adenosine transporter) rescues TRPML1 inhibition. TRPML1 inhibition leads to lysosome enlargement, alkalinization, and dysfunction; TRPML1 activation rescues ADA-deficient B-lymphocyte vulnerability to oxidative stress.\",\n      \"method\": \"ADA knockout, TRPML1 electrophysiology, ENT3 overexpression rescue, lysosomal pH and size measurement, oxidative stress assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO, electrophysiology, and genetic rescue; single lab\",\n      \"pmids\": [\"28087698\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TRPML1-mediated Ca2+ release promotes autophagosome-lysosome fusion and lysosome acidification within 10–20 min of activation, and increases transport of lysosomal SNARE proteins STX7 and VAMP7 via SNARE carrier vesicles. Incoming vesicle fusion is a prerequisite for lysosomal Ca2+ efflux leading to acidification and hydrolase activation; PI(3,5)P2 is proposed as the physiological TRPML1 activator generated by vesicle fusions.\",\n      \"method\": \"Pharmacological TRPML1 activation (ML-SA1), lysosomal pH measurement, autophagosome-lysosome fusion assay, SNARE protein trafficking assay, TRPML1 KO validation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — acute pharmacological and genetic perturbations with defined Ca2+, SNARE, and fusion readouts; single lab\",\n      \"pmids\": [\"39433126\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TRPML1, activated secondarily to ROS upon inflammatory stimuli, mediates lysosomal Fe2+ release into the cytosol, activating prolyl hydroxylase domain enzymes (PHDs). PHDs repress NF-κB transcriptional activity, suppressing IL-1β transcription in macrophages as a negative feedback control of inflammation.\",\n      \"method\": \"TRPML1 agonist/antagonist treatment, Fe2+ measurement, PHD activity assay, NF-κB reporter assay, IL-1β ELISA, TRPML1 KO macrophages, in vivo colitis model\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple pharmacological and genetic perturbations with defined mechanistic pathway (Fe2+-PHD-NF-κB-IL1B), in vivo validation; single lab but rigorous multi-step mechanism\",\n      \"pmids\": [\"39856099\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TRPML1 localizes to lysosomes in NK cells; genetic deletion of TRPML1 causes mitochondrial fragmentation with collapsed cristae, loss of mitochondrial membrane potential, increased ROS, reduced ATP production, and Ca2+ overload in mitochondria. TRPML1 loss impedes autophagic flux and increases accumulation of dysfunctional mitochondria.\",\n      \"method\": \"TRPML1 genetic deletion in NK92 cells, organelle-specific Ca2+ probes, mitochondrial morphology analysis, mitochondrial membrane potential measurement, ROS assay, autophagic flux assay\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with multiple orthogonal organelle function readouts; single lab\",\n      \"pmids\": [\"37737664\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TRPML1 (MCOLN1) is a lysosomal/late-endosomal non-selective cation channel (permeable to Ca2+, Fe2+, Zn2+, H+) gated by the phosphoinositide PI(3,5)P2 (which binds an allosteric site on S1-S3 helices) and modulated by luminal pH, Ca2+, ROS, TOR-mediated phosphorylation, LAMTOR1-mediated tonic inhibition, and the ceramide-sphingosine axis; its Ca2+ efflux into the cytosol triggers multiple downstream cascades including calcineurin-TFEB nuclear translocation (autophagy and lysosome biogenesis), CaMKKβ/AMPK/VPS34-dependent autophagosome biogenesis, calmodulin-dependent lysosome fission, MTORC1 feedback reactivation, RyR2-mediated sarcoplasmic reticulum Ca2+ spark initiation in smooth muscle, and SNARE-dependent autophagosome-lysosome fusion, while its Fe2+ and Zn2+ efflux controls iron homeostasis, ferroptosis susceptibility (via AKT-phosphorylation-stabilized TRPML1-ARL8B-lysosomal exocytosis), and NF-κB-dependent inflammatory gene transcription via PHD enzymes.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MCOLN1 (TRPML1) is a late-endosomal/lysosomal non-selective cation channel that couples lysosomal ion flux to membrane trafficking, autophagy, and organelle homeostasis [#0, #4]. It conducts Ca2+, Fe2+/Mn2+, Zn2+, and (in some studies) H+, and its loss perturbs lysosomal lipid hydrolysis, pH, iron, and zinc handling, defining the cellular basis of its trafficking and storage defects [#0, #1, #17, #18]. The channel is gated by the lysosomal phosphoinositide PtdIns(3,5)P2, which binds an allosteric site on extended S1–S3 helices and drives a Y355–R403 π-cation interaction to open the pore, whereas PtdIns(4,5)P2 binds the same site and inhibits; cryo-EM structures in closed and agonist-bound open states define a distinct hydrophobic agonist/antagonist cavity (S5/S6/PH1) and a luminal acidic Ca2+/pH-sensing region [#8, #9, #10, #12]. Activity is further set by multiple inputs: cathepsin B cleavage at Arg200–Pro201, TOR-mediated inhibitory phosphorylation, tonic LAMTOR1 binding, ROS, luminal adenosine, and AKT phosphorylation that stabilizes the channel against ubiquitin-dependent degradation [#2, #14, #23, #34, #33, #7]. TRPML1-mediated Ca2+ efflux is the central output, triggering calcineurin–TFEB-driven autophagy and lysosome biogenesis, CaMKKβ/AMPK/VPS34-dependent autophagosome biogenesis, calmodulin-dependent lysosome fission, MTORC1 reactivation, and SNARE-controlled autophagosome–lysosome and phagosome–lysosome fusion [#7, #16, #21, #15, #13, #28, #35]. Through inter-organelle contacts it transfers Ca2+ to mitochondria via VDAC1/MCU and to the sarcoplasmic reticulum RyR2 to initiate Ca2+ sparks in vascular smooth muscle, and it functionally couples to ER STIM1 [#20, #27, #29]. Its metal-ion output additionally governs iron homeostasis and ferroptosis resistance through an AKT–TRPML1–ARL8B lysosomal-exocytosis axis and restrains NF-κB-driven IL-1β transcription via Fe2+-dependent PHD activation [#33, #36]. Loss-of-function mutations that impair channel activity underlie mucolipidosis type IV, and small-molecule agonists rescue trafficking and lysosomal metal-accumulation defects in patient fibroblasts [#0, #17].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Established the first functional and regulatory framework for the channel by showing it conducts H+ and monovalent cations and is post-translationally controlled, linking its activity directly to lysosomal acidification and lipid hydrolysis.\",\n      \"evidence\": \"Lysosomal pH and lipase assays plus genetic/pharmacological rescue in MLIV patient cells; whole-lysosome patch-clamp with cathepsin B cleavage mapping and co-IP\",\n      \"pmids\": [\"16361256\", \"16257972\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"H+ permeability later disputed by a proton-impermeable characterization\", \"physiological trigger for cathepsin B cleavage in vivo not defined\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Defined the channel's oligomeric behavior, showing it forms homo- and heteromultimers with TRPML2/3 and dictates the lysosomal targeting of TRPML3.\",\n      \"evidence\": \"Co-IP and colocalization microscopy with targeting-mutant co-expression\",\n      \"pmids\": [\"16606612\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"functional consequence of heteromultimerization for channel gating untested\", \"stoichiometry of native complexes unknown\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Resolved a key permeant species by demonstrating Fe2+ conductance and linking disease-mutation severity to graded loss of iron permeation, explaining lysosomal iron storage.\",\n      \"evidence\": \"Direct lysosomal patch-clamp, radiolabelled iron flux, and cytosolic/intralysosomal iron monitoring in patient fibroblasts\",\n      \"pmids\": [\"18794901\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"relative physiological contribution of Fe2+ vs Ca2+ flux not quantified\", \"downstream iron-dependent signaling not addressed here\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Defined permeation selectivity and gating by isolating constitutively active S5 mutants, showing Ca2+ release drives lysosomal exocytosis and surface LAMP-1.\",\n      \"evidence\": \"Proline-scanning mutagenesis with whole-cell/lysosomal patch-clamp and LAMP-1 surface staining\",\n      \"pmids\": [\"19638346\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"proton impermeability contradicts earlier H+ channel report\", \"endogenous gating trigger not identified in this study\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Extended the channel's ion repertoire and physiology by linking it to lysosomal zinc handling and to gastric acid secretion via parietal-cell tubulovesicle trafficking.\",\n      \"evidence\": \"siRNA/KO with fluorometric zinc and ICP-MS; Trpml1-/- mouse gastric secretion, palmitoylation/phosphorylation analysis and EM\",\n      \"pmids\": [\"20864526\", \"23368743\", \"21111738\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"direct Zn2+ permeation through the channel vs indirect transporter effects not fully separated\", \"mechanism coupling palmitoylation to channel function unresolved\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Clarified relationships with other endolysosomal channels, showing physical but functionally independent association with TPCs while ortholog studies linked the channel to NAADP-evoked Ca2+ release and autophagic degradation.\",\n      \"evidence\": \"Reciprocal co-IP, colocalization, patch-clamp and Ca2+ imaging in KO cells; C. elegans CUP-5 genetics and lysosomal reconstitution\",\n      \"pmids\": [\"21540176\", \"21997367\", \"21613607\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"NAADP responsiveness is contradictory between cell types/labs\", \"molecular basis of TRPML1-TPC association not defined\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Connected channel loss to cell death by placing acute TRPML1 loss upstream of cathepsin B leak and Bax-dependent apoptosis.\",\n      \"evidence\": \"siRNA knockdown with cathepsin B localization/activity assays and CatB/Bax inhibitor dissection\",\n      \"pmids\": [\"22262857\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"mechanism by which channel loss triggers membrane permeabilization unclear\", \"acute knockdown phenotype may differ from chronic disease state\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Established PtdIns(3,5)P2 as the activating ligand controlling membrane fusion and showed TOR phosphorylation as a direct inhibitory input regulating autophagy.\",\n      \"evidence\": \"PIKfyve epistasis with phagosome-lysosome fusion and Ca2+ rescue; in vitro kinase assay with phosphosite mutagenesis and channel/autophagy readouts\",\n      \"pmids\": [\"26010303\", \"26195823\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"TOR phosphosite study is Medium confidence single-lab\", \"precise residues and kinase specificity for TOR phosphorylation not structurally mapped\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Defined multiple physiological activators and partners, establishing ROS-triggered Ca2+ release driving TFEB/autophagy, LAMTOR1 as a tonic inhibitor, sphingosine/ER-STIM1 coupling, and calmodulin-dependent lysosome fission.\",\n      \"evidence\": \"GCaMP3-ML1 Ca2+ imaging, genetic KO/knockdown, co-IP, and pharmacological dissection across cell types\",\n      \"pmids\": [\"27357649\", \"35099830\", \"31341250\", \"33484198\", \"28360104\", \"28087698\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"several upstream activators tested in distinct systems; integration in a single cell unclear\", \"how ROS chemically modifies the channel not defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Provided the first atomic-resolution framework by solving cryo-EM structures in closed and agonist-bound open states and locating the PtdIns(3,5)P2 site distal to the pore.\",\n      \"evidence\": \"Single-particle cryo-EM of mouse and human channel with mutagenesis/electrophysiology\",\n      \"pmids\": [\"29019981\", \"29019983\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"physiological gating transitions in native lysosomal membranes not directly observed\", \"structural basis of Fe2+/Zn2+ selectivity not resolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Refined the lipid-gating mechanism (Y355–R403 π-cation, opposing PtdIns(4,5)P2 inhibition) and embedded the channel in an MTORC1 negative-feedback loop activated by starvation.\",\n      \"evidence\": \"Ligand-bound cryo-EM with electrophysiology; pharmacological/genetic perturbation with calmodulin inhibition and S6K readout\",\n      \"pmids\": [\"30305615\", \"29460684\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"how PtdIns(4,5)P2 reaches the lysosomal-facing site physiologically unclear\", \"feedback-loop study is Medium confidence single-lab\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Diversified the channel's downstream outputs, defining a TFEB-independent CaMKKβ/AMPK/VPS34 autophagosome-biogenesis pathway, MVB/exosome control via acid ceramidase, and a cholesterol-transport role sustaining oncogenic HRAS nanoclustering.\",\n      \"evidence\": \"PI3P and phagophore-recruitment assays with kinase inhibition; Ca2+ imaging with sphingolipid manipulation; cholesterol and HRAS nanoclustering analysis in mutant cells\",\n      \"pmids\": [\"31822666\", \"31268777\", \"30787043\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"cholesterol and exosome roles single-lab Medium confidence\", \"directness of TRPML1 in cholesterol de-esterification not established\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Established inter-organelle Ca2+ signaling roles by showing channel-mediated Ca2+ transfer to mitochondria (VDAC1/MCU) and SR RyR2-dependent Ca2+ spark initiation controlling vascular tone and blood pressure.\",\n      \"evidence\": \"Super-resolution live-cell microscopy, KO mice, pressure myography and radiotelemetry; patient-fibroblast contact dynamics\",\n      \"pmids\": [\"32703809\", \"32576680\", \"33199609\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"tethering machinery at mitochondria-lysosome contacts not fully identified\", \"tissue-specificity of RyR2 coupling beyond smooth muscle untested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Resolved the antagonist binding mode and uncovered a divalent-ion output controlling SNARE-mediated fusion, showing ML-SI3 occupies the agonist cavity and Zn2+ release blocks STX17-VAMP8 pairing.\",\n      \"evidence\": \"Cryo-EM with ML-SI3 plus patch-clamp; SNARE co-IP with lysosomal zinc measurement and autophagy flux\",\n      \"pmids\": [\"34171299\", \"33890549\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"antagonist study High confidence; zinc-SNARE mechanism Medium single-lab\", \"apparent opposing effects of activation on fusion across studies need reconciliation\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined cooperative dual-ligand gating and additional Ca2+-dependent outputs, showing PI(3,5)P2 and rapamycin synergize at distinct sites and that ROS/CaMK2G-JIP4 signaling drives retrograde lysosome transport.\",\n      \"evidence\": \"Multi-state cryo-EM with electrophysiology; phosphosite mutagenesis, CaMK2G inhibition and lysosomal positioning/autophagy assays; NK-cell KO mitochondrial phenotyping\",\n      \"pmids\": [\"35131932\", \"36394115\", \"37737664\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"physiological relevance of rapamycin-analog synergy unclear\", \"JIP4 and NK-cell roles Medium confidence single-lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Connected channel regulation to ferroptosis and membrane repair, showing AKT phosphorylation at Ser343 stabilizes the channel and ARL8B-dependent lysosomal exocytosis lowers iron and confers ferroptosis resistance.\",\n      \"evidence\": \"In vitro AKT kinase assay, phosphosite/ubiquitination mutagenesis, TRPML1-ARL8B co-IP, exocytosis and ferroptosis assays with xenografts\",\n      \"pmids\": [\"38424427\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"generality beyond AKT-hyperactivated cancers untested\", \"competition between Ser343 and other regulatory inputs in vivo unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extended the channel's iron output to inflammatory control, showing Fe2+ release activates PHD enzymes that repress NF-κB and suppress IL-1β transcription in macrophages.\",\n      \"evidence\": \"Agonist/antagonist and KO macrophages with Fe2+/PHD/NF-κB readouts and an in vivo colitis model\",\n      \"pmids\": [\"39856099\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"direct PHD substrate engagement by cytosolic Fe2+ not structurally shown\", \"balance between pro- and anti-inflammatory iron effects in different cells unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the diverse, sometimes opposing outputs of a single channel (Ca2+, Fe2+, Zn2+, H+ flux promoting versus inhibiting autophagosome-lysosome fusion) are integrated, prioritized, and spatially partitioned in native lysosomes remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"conflicting reports on whether activation promotes or blocks autophagic flux\", \"no unified model linking ion-species selection to specific downstream cascade\", \"physiological vs pharmacological activation regimes not reconciled\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [0, 4, 17, 18]},\n      {\"term_id\": \"GO:0005262\", \"supporting_discovery_ids\": [4, 7]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [8, 10]},\n      {\"term_id\": \"GO:0140299\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [0, 1, 2, 4, 17]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [0, 3, 5]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [3, 29]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [4, 25]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [6, 7, 16, 35]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [13, 28, 35]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 18, 36]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [15, 20, 27]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [31, 36, 37]}\n    ],\n    \"complexes\": [\n      \"TRPML1/TRPML2/TRPML3 heteromultimer\",\n      \"TRPML1-RyR2 ER/SR-lysosome complex\"\n    ],\n    \"partners\": [\n      \"TPC2\",\n      \"TMEM163\",\n      \"LAMTOR1\",\n      \"STIM1\",\n      \"RyR2\",\n      \"VDAC1\",\n      \"MCU\",\n      \"ARL8B\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":{"gene":"MCOLN1","tier":"GROUNDING","verdict":"Evidence-grounding concern","subtype":"fabrication","uniprot_band":"rich","rules_fired":"R7","issue":"R7: fabricated (no corpus paper): 38424427"},"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}