{"gene":"TRPV6","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2003,"finding":"TRPV6 forms homotetramers and can form heterotetramers with TRPV5; the tetrameric stoichiometry was confirmed by sucrose gradient sedimentation (~400 kDa), co-immunoprecipitation from oocytes, and electrophysiological analysis of concatemeric channels. Heterotetrameric TRPV5/TRPV6 complexes show distinct Ca2+-dependent inactivation, Ba2+ selectivity, and pharmacological block compared to homotetramers.","method":"Sucrose gradient sedimentation, co-immunoprecipitation, concatemeric channel electrophysiology in HEK293 cells","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (biochemical + electrophysiological reconstitution) in single rigorous study","pmids":["12574114"],"is_preprint":false},{"year":2003,"finding":"S100A10 binds specifically to the conserved C-terminal VATTV motif of TRPV6 (with the first threonine, T600, being critical); S100A10 forms a heterotetrameric complex with annexin 2, and the S100A10–annexin 2 complex is required for routing TRPV6 to the plasma membrane. Mutation T600A abolishes S100A10 binding, redistributes TRPV6 to a sub-plasma-membrane compartment, and eliminates channel activity. Knockdown of annexin 2 by siRNA inhibits TRPV6-mediated currents.","method":"Yeast two-hybrid, GST pull-down, co-immunoprecipitation, site-directed mutagenesis, siRNA knockdown, electrophysiology in HEK293 cells, immunofluorescence","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (Y2H, GST pulldown, co-IP, mutagenesis, siRNA + electrophysiology) in single rigorous study","pmids":["12660155"],"is_preprint":false},{"year":2004,"finding":"Calmodulin (CaM) binds TRPV6 in a Ca2+-dependent manner and positively regulates TRPV6 channel activity. CaM binding sites were localized to the transmembrane domain, an N-terminal 1-5-10 motif (residues 88–97), and a C-terminal 1-8-14 motif (residues 643–656). Overexpression of Ca2+-insensitive CaM mutants (CaM1234, CaM34) significantly reduced Ca2+ and Na+ currents of TRPV6-expressing HEK293 cells but not TRPV5 cells, implicating EF-hands 3 and 4 of CaM's C-lobe.","method":"GST pull-down, co-immunoprecipitation, whole-cell patch clamp, chimeric channel analysis in HEK293 cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — reciprocal biochemical binding assays + mutagenesis + electrophysiology, multiple orthogonal methods","pmids":["15123711"],"is_preprint":false},{"year":2016,"finding":"Crystal structure of rat TRPV6 at 3.25 Å resolution revealed: tetrameric assembly with an intracellular 'skirt' involved in allosteric modulation; Ca2+ selectivity determined by a ring of aspartate side chains in the selectivity filter; multiple cation-binding sites along the pore axis and extracellular vestibule defining a Ca2+ permeation mechanism.","method":"X-ray crystallography at 3.25 Å resolution","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — atomic-resolution crystal structure with multiple functional implications validated structurally","pmids":["27296226"],"is_preprint":false},{"year":2017,"finding":"Cryo-EM structures of human TRPV6 in open and closed states revealed that channel opening involves an α-to-π helical transition in the pore-lining S6 helix at an alanine hinge below the selectivity filter, causing S6 helices to bend and rotate (iris-like gating). The selectivity filter adopts similar conformations in both states. Open probability is increased by PIP2.","method":"Cryo-electron microscopy, electrophysiology","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structures of both open and closed states with corroborating electrophysiology","pmids":["29258289"],"is_preprint":false},{"year":2018,"finding":"2-APB binds to TRPV6 in a pocket formed by the cytoplasmic half of the S1–S4 transmembrane helix bundle. 2-APB induces channel closure by modulating protein–lipid interactions, as shown by comparing wild-type and high-affinity Y467A mutant structures. Mutagenesis and functional analysis confirmed the binding site.","method":"Crystal and cryo-EM structures of human and rat TRPV6 bound to 2-APB, mutagenesis, functional assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — atomic structures plus mutagenesis plus functional validation in single study","pmids":["29941865"],"is_preprint":false},{"year":2008,"finding":"Crystal structure of the TRPV6 ankyrin repeat domain (ARD) at 1.7 Å revealed a conserved large twist between repeats 4 and 5 and a variable third finger loop with putative regulatory phosphorylation sites. Pull-down assays showed the TRPV6 ARD does not bind ATP or calmodulin, contrasting with TRPV1 ARD, indicating a distinct regulatory role for the TRPV6 ARD.","method":"X-ray crystallography (1.7 Å), ATP-agarose and calmodulin-agarose pull-down assays, size exclusion chromatography","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — high-resolution crystal structure plus orthogonal biochemical assays in single study","pmids":["18232717"],"is_preprint":false},{"year":2006,"finding":"GDP-bound Rab11a directly and specifically interacts with TRPV5 and TRPV6 via a conserved stretch in their C-termini. Rab11a colocalizes with TRPV6 in vesicular structures beneath the apical membrane. Co-expression of GDP-locked Rab11a significantly decreased TRPV6-mediated Ca2+ uptake by reducing channel surface expression. Rab11a thus mediates TRPV6 trafficking to the plasma membrane in a novel GDP-bound, direct cargo-interaction mode.","method":"Co-immunoprecipitation, confocal colocalization, Ca2+ uptake assays, surface expression analysis in epithelial cells and oocytes","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP plus functional Ca2+ uptake assays plus localization data","pmids":["16354700"],"is_preprint":false},{"year":2008,"finding":"Klotho (acting as a β-glucuronidase) and β-glucuronidase specifically increase TRPV6 (and TRPV5) activity but not TRPV4 or TRPM6, suggesting activation via N-oligosaccharide hydrolysis on the channel. Deglycosylation by endoglycosidase-F also stimulated TRPV6 activity.","method":"Ca2+-influx measurements in transfected HEK293 cells, enzymatic deglycosylation","journal":"Nephrology, dialysis, transplantation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional Ca2+ influx assays with pharmacological specificity controls, single lab","pmids":["18495742"],"is_preprint":false},{"year":2006,"finding":"RGS2 interacts with the N-terminal domain of TRPV6 in a Ca2+-independent manner (identified by yeast two-hybrid and GST pull-down). RGS2 overexpression reduces Na+ and Ca2+ currents of TRPV6 but not TRPV5. The inhibitory effect is not due to altered channel trafficking (cell-surface biotinylation was unchanged), suggesting direct effects on gating. DeltaN-RGS2 lacking the N-terminal domain does not inhibit TRPV6, and the effect is GPCR-independent.","method":"Yeast two-hybrid, GST pull-down, whole-cell electrophysiology, cell-surface biotinylation in HEK293 cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — Y2H + GST pulldown + electrophysiology + surface biotinylation, multiple orthogonal methods, single lab","pmids":["16895908"],"is_preprint":false},{"year":2008,"finding":"Ca2+ influx through TRPV6 activates phospholipase C, causing depletion of PIP2, which contributes to Ca2+-induced inactivation of TRPV6. Ba2+ (which does not activate PLC) does not cause inactivation. Dialysis of DiC8-PIP2 inhibited Ca2+-dependent inactivation; PIP2 activated TRPV6 in excised patches; rapamycin-inducible PI(4,5)P2 5-phosphatase inhibited TRPV6 currents. PI(4)P had no significant effect.","method":"Whole-cell and excised patch-clamp, rapamycin-inducible phosphatase system, Ca2+ imaging","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — multiple electrophysiology approaches plus pharmacological tools, orthogonal methods, single lab","pmids":["18390907"],"is_preprint":false},{"year":2011,"finding":"PIP2 is a direct activator of TRPV6: reconstituted purified TRPV6 in planar lipid bilayers showed high activity in presence of PIP2 but not PI(4)P. Intracellular ATP supports TRPV6 activity indirectly by serving as substrate for type III PI4-kinases to resynthesize PIP2; MgATP reactivated TRPV6 after rundown in excised patches, an effect blocked by PI4K inhibitors.","method":"Excised inside-out patch clamp, planar lipid bilayer reconstitution with purified channel, pharmacological inhibition of PI4K","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — channel reconstitution in lipid bilayers plus excised patch clamp; direct activation by PIP2 established","pmids":["21810903"],"is_preprint":false},{"year":2008,"finding":"Cyclophilin B (CypB) co-purifies with TRPV6 from human placenta (also with annexin A2) and co-expression of CypB with TRPV6 in Xenopus oocytes significantly increases TRPV6-mediated Ca2+ uptake. This stimulatory effect is reversed by cyclosporin A (which inhibits CypB's peptidyl-prolyl cis/trans isomerase activity), suggesting CypB activates TRPV6 through its isomerase activity.","method":"Protein enrichment/co-purification from human placenta, Xenopus oocyte Ca2+ uptake assay, pharmacological inhibition with cyclosporin A","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — endogenous co-purification plus functional oocyte assay, single lab","pmids":["18445599"],"is_preprint":false},{"year":2009,"finding":"ATP binds directly to TRPV6 (at sites within the ankyrin repeat domain and C-terminus), reduces whole-cell current increments, and prevents channel rundown (EC50 ~380 µM). PKCβII-mediated phosphorylation at defined ARD and C-terminal sites counteracts ATP-dependent stabilization, constituting a metabolic switch for Ca2+ influx.","method":"Biochemical ATP-binding studies, patch-clamp electrophysiology, site-directed mutagenesis of ARD and C-terminal residues","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding assays plus electrophysiology plus mutagenesis, single lab","pmids":["19805577"],"is_preprint":false},{"year":2010,"finding":"Nedd4-2 (HECT ubiquitin E3 ligase) down-regulates TRPV6 protein abundance and Ca2+ influx by increasing TRPV6 ubiquitination and decreasing its stability (partially via proteasomal but not lysosomal pathway). The HECT domain is essential for both inhibition and association. WW1 and WW2 domains interact with TRPV6 terminal regions and their integrity limits the degree of TRPV6 ubiquitination.","method":"Xenopus oocyte co-expression, Ca2+ influx assays, ubiquitination assays, proteasome/lysosome inhibitors, mutagenesis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple functional assays and mutagenesis in oocyte system, single lab","pmids":["20843805"],"is_preprint":false},{"year":2008,"finding":"The annexin 2–S100A10 complex association with TRPV6 is regulated by cAMP/PKA/calcineurin A (CnA) signaling: forskolin-stimulated cAMP increases annexin 2–S100A10 complex formation (blocked by PKA or CnA inhibitors), and CnA-dependent dephosphorylation of annexin 2 mediates the interaction with TRPV6. PKA and CnA inhibitors attenuated 45Ca uptake in Caco-2 cells.","method":"Co-immunoprecipitation, 45Ca uptake assays, pharmacological inhibitors (PKA, CnA), cell biology in 16HBE14o- and Caco-2 cells","journal":"Cell calcium","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — co-IP plus functional Ca2+ transport assays, single lab","pmids":["18187190"],"is_preprint":false},{"year":2006,"finding":"NHERF4 (PDZ protein) interacts with TRPV6 via the fourth PDZ domain binding a conserved region in the TRPV6 C-terminus, distinct from the NHERF2 binding site. Interaction confirmed by yeast two-hybrid, GST pull-down, and co-immunoprecipitation.","method":"Yeast two-hybrid, GST pull-down, co-immunoprecipitation in HEK293 cells and oocytes","journal":"Pflugers Archiv","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Y2H plus GST pulldown plus co-IP, multiple binding assays, single lab","pmids":["16565876"],"is_preprint":false},{"year":2008,"finding":"Nipsnap1 was identified as a novel TRPV6-interacting protein (by GST pull-down/bioinformatics) that abolishes TRPV6 currents without reducing plasma membrane expression of TRPV6, suggesting direct inhibition of channel gating rather than trafficking.","method":"GST pull-down, electrophysiology, cell-surface biotinylation, RT-PCR, immunohistochemistry","journal":"Pflugers Archiv","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — GST pulldown plus electrophysiology plus surface biotinylation, single lab","pmids":["18392847"],"is_preprint":false},{"year":2011,"finding":"Male mice carrying the TRPV6-D541A pore mutation (rendering the channel nonfunctional) show severely impaired fertility due to failure of Ca2+ absorption by epididymal epithelium, resulting in ~10-fold higher Ca2+ in cauda epididymis luminal fluid. TRPV6 was localized to the apical membrane of epididymal epithelium (not in spermatozoa), establishing that TRPV6-mediated Ca2+ absorption in the epididymis is essential for sperm motility and viability.","method":"Knock-in mouse model (D541A), Ca2+ concentration measurements, Ca2+ absorption assays, immunolocalization, sperm functional assays","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo knock-in model with defined biochemical and functional phenotype, replicated by TRPV6-null mouse study","pmids":["21540454"],"is_preprint":false},{"year":2012,"finding":"Trpv6 knockout mice (deletion of transmembrane/pore-forming and C-terminal regions) phenocopy the TRPV6-D541A pore mutant regarding epididymal Ca2+ handling and male fertility defects, arguing against residual channel-independent functions of the D541A mutant.","method":"Trpv6 knockout mouse (exon deletion), comparison with D541A knock-in, Ca2+ uptake assays, sperm function tests","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic null compared to pore mutant with multiple biochemical and functional endpoints; independent replication of epididymal phenotype","pmids":["22427671"],"is_preprint":false},{"year":2008,"finding":"TRPV6 is required for maternal-fetal Ca2+ transport: Trpv6 knockout fetuses had significantly lower fetal blood Ca2+, lower amniotic fluid Ca2+, ~40% lower 45Ca transport from mother to fetus, and lower ash weight (bone mineralization). TRPV6 mRNA and protein were localized in intraplacental and extraplacental yolk sac, co-localizing with calbindin-D9K.","method":"Trpv6 knockout mice, 45Ca transport assays, immunohistochemistry, qRT-PCR","journal":"Journal of bone and mineral research","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo knockout with isotope transport assay and localization, well-replicated finding","pmids":["18348695"],"is_preprint":false},{"year":2012,"finding":"TRPV6-D541A knockin mice (non-functional pore) show significantly impaired intestinal Ca2+ uptake under Ca2+-deficient diet conditions, establishing that functional TRPV6 channels mediate transepithelial Ca2+ absorption in the intestine, particularly under low-Ca2+ dietary challenge.","method":"TRPV6-D541A knockin mouse model, 45Ca intestinal absorption assay, metabolic cage measurements, protein expression analysis","journal":"American journal of physiology. Gastrointestinal and liver physiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo knockin model with isotope-based absorption assay","pmids":["22878123"],"is_preprint":false},{"year":2004,"finding":"Under physiological intracellular Ca2+ buffering conditions, TRPV6 does not form constitutively open channels in HEK293 cells; channel activity requires chelation of intracellular Ca2+ (with EGTA). Monovalent cations permeate TRPV6 when Ca2+ permeation is absent, indicating extracellular divalent cation block. The C-terminal domain required for calmodulin binding is not necessary for this regulation; the pore residue D542 is essential for channel function.","method":"Whole-cell and perforated patch clamp, Fura-2 microfluorimetry, site-directed mutagenesis (D542A, truncation mutant) in HEK293 cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — patch clamp with mutagenesis defining pore and gating requirements, multiple conditions tested","pmids":["15184369"],"is_preprint":false},{"year":2013,"finding":"The in vivo TRPV6 protein has an extended N-terminus: translation initiates at a non-AUG codon (ACG, decoded as methionine) upstream of the annotated AUG (which is not used). The extended full-length protein has similar channel properties to the shorter form in vitro, but increased trafficking to the plasma membrane and provides an additional scaffold for channel assembly.","method":"Mass spectrometry of endogenous placenta protein, cell biology/trafficking assays, electrophysiology","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — endogenous protein identification plus functional trafficking assay, single lab","pmids":["23612980"],"is_preprint":false},{"year":2011,"finding":"G protein-coupled receptor PSGR activates endogenous TRPV6 channels in prostate cells via Src kinase. PSGR stimulation enhances cytosolic Ca2+ through TRPV6; the Ca2+ signal has biophysical characteristics of TRPV6 currents; Src kinase activation occurs independently of G-protein activation, presumably through direct PSGR–Src interaction. siRNA knockdown of TRPV6 confirmed channel identity.","method":"Electrophysiology, live-cell Ca2+ imaging, siRNA knockdown, biochemical co-immunoprecipitation, Src kinase inhibition","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods including siRNA confirmation and electrophysiology, single lab","pmids":["21349844"],"is_preprint":false},{"year":2009,"finding":"WNK3 positively regulates TRPV6 activity in a kinase-dependent manner; the kinase-inactive WNK3-D294A mutation abolishes the effect. WNK3 increases Ca2+ influx and Na+ current mediated by TRPV6, and enhances delivery of mature (glycosylated) TRPV6 to the plasma membrane via the secretory pathway; the effect is abolished by the microtubule inhibitor colchicine.","method":"Xenopus oocyte co-expression, Ca2+ uptake assays, voltage clamp, surface biotinylation, exocytosis assays, kinase-dead mutagenesis","journal":"American journal of physiology. Renal physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional assays plus surface expression plus mutagenesis, single lab","pmids":["18768590"],"is_preprint":false},{"year":2009,"finding":"SGK1 and PKB/Akt increase TRPV6 activity and plasma membrane abundance. PIKfyve, phosphorylated by SGK1 at S318, further augments TRPV6 activity when co-expressed with active SGK1; the S318A-PIKfyve mutant lacking the SGK1 phosphorylation site fails to enhance TRPV6 activity.","method":"Xenopus oocyte co-expression, Ca2+-activated Cl- current recording as indirect readout of TRPV6-mediated Ca2+ influx, immunohistochemistry, Western blotting","journal":"The Journal of membrane biology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — indirect readout (endogenous Cl- current), single lab, single oocyte system","pmids":["20041238"],"is_preprint":false},{"year":2014,"finding":"TRPV6 translocates to the plasma membrane in prostate cancer cells via an Orai1/TRPC1-mediated Ca2+/Annexin I/S100A11 pathway. TRPV6-mediated Ca2+ entry increases cell survival (proliferation and apoptosis resistance). Xenograft models confirmed increased aggressiveness of TRPV6-overexpressing tumors.","method":"Co-immunoprecipitation, Ca2+ imaging, siRNA knockdown, xenograft mouse models, immunohistochemistry","journal":"Proceedings of the National Academy of Sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus functional assays plus in vivo xenograft, single lab","pmids":["25172921"],"is_preprint":false},{"year":2007,"finding":"TRPV6 mediates Ca2+ entry in human keratinocytes and is required for Ca2+-induced differentiation: siRNA-mediated TRPV6 silencing impairs differentiated phenotype, reduces differentiation markers (involucrin, transglutaminase-1, cytokeratin-10), disrupts cell morphology and stratification. 1,25-Dihydroxyvitamin D3 dose-dependently increases TRPV6 expression and Ca2+ uptake, promoting differentiation.","method":"siRNA knockdown, Ca2+ uptake assays (Fura-2), RT-PCR, immunoblotting in primary human keratinocytes","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA knockdown with defined differentiation phenotype plus Ca2+ functional assays, single lab","pmids":["17550901"],"is_preprint":false},{"year":2007,"finding":"TRPV6 controls prostate cancer cell (LNCaP) proliferation: siRNA knockdown of TRPV6 decreases proliferation rate, reduces S-phase accumulation, and decreases PCNA expression. TRPV6-mediated Ca2+ entry activates NFAT transcription factor downstream. TRPV6 also contributes to apoptosis resistance.","method":"siRNA knockdown, cell cycle analysis, Ca2+ imaging, NFAT reporter assays in LNCaP cells","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA knockdown with multiple cellular phenotype readouts plus pathway confirmation, single lab","pmids":["17533368"],"is_preprint":false},{"year":2019,"finding":"Activation of the Ca2+-sensing receptor (CaSR) on the basolateral membrane of intestinal epithelium directly attenuates Ca2+ absorption via TRPV6: cinacalcet (calcimimetic) decreased net Ca2+ flux in Ussing chambers in a TRPV6-dependent manner (absent in TRPV6-D541A mice), and inhibited Ca2+ flux through TRPV6 when co-expressed with CaSR in oocytes. PLC inhibitor U73122 prevented cinacalcet-mediated TRPV6 inhibition.","method":"Ussing chamber Ca2+ flux assays, TRPV6-D541A knockin mice, Xenopus oocyte co-expression, pharmacological inhibition","journal":"JCI insight","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple model systems (Ussing chamber, knockin mouse, oocyte) with pharmacological and genetic epistasis","pmids":["31013259"],"is_preprint":false},{"year":2010,"finding":"TRPV6 permeates heavy metal cations including Zn2+, Cd2+, Ba2+, Sr2+, Mn2+, La3+, and Gd3+ in addition to Ca2+, as shown by live-cell ion imaging (Fura-2, Newport Green DCF) and patch clamp in hTRPV6-expressing HEK293 cells. At higher concentrations, Cd2+, La3+, and Gd3+ are efficient inhibitors of TRPV6-mediated Ca2+ influx.","method":"Live-cell ion imaging (Fura-2, Newport Green DCF), whole-cell patch clamp, 45Ca uptake assays in HEK293 cells","journal":"Cell calcium","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal electrophysiology and imaging methods, single lab","pmids":["21146870"],"is_preprint":false},{"year":2012,"finding":"Numb1 isoform interacts with TRPV6 via an aspartate at residue 716 (TRPV6) and arginine at residue 434 (Numb1), as identified by co-immunoprecipitation, FRET, and C-terminal truncation mutagenesis. Numb1 negatively regulates TRPV6 activity: its expression decreases cytosolic Ca2+ in TRPV6-transfected cells, and a Numb1 mutant lacking the TRPV6-binding site fails to inhibit TRPV6.","method":"Co-immunoprecipitation, FRET, C-terminal truncation mutagenesis, Ca2+ measurements in HEK293 and cancer cells","journal":"Cell calcium","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — co-IP plus FRET plus mutagenesis defining binding residues, single lab","pmids":["23140583"],"is_preprint":false},{"year":2020,"finding":"TRPV6 autoinhibition is maintained by two intramolecular interactions: S4–S5 linker to C-terminal TRP helix (L/C; mediated by Arg470:Trp593) and N-terminal pre-S1 to TRP helix (N/C; mediated by Trp321:Ile597). Disruption of either interaction by mutations or blocking peptides activates TRPV6. The N/C interaction depends on L/C but not vice versa. PIP2 binds three cationic residues in S5/C-terminus, suppresses both interactions, and activates TRPV6.","method":"Site-directed mutagenesis, blocking peptides, patch-clamp electrophysiology, Ca2+ imaging","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis plus peptide competition plus electrophysiology defining intramolecular regulatory interactions, single lab","pmids":["32829285"],"is_preprint":false},{"year":2016,"finding":"In rat cauda epididymal principal cells, a constitutively active TRPV6-like Ca2+ current is coupled to a calcium-activated chloride conductance (TMEM16A). TRPV6 and TMEM16A proteins co-localize at the apical membrane. Removal of extracellular Ca2+ attenuates both currents; lanthanide block inhibits the TRPV6-like component first, then CaCC. In vivo perfusion showed substantial Ca2+ reabsorption from epididymal lumen suppressed by ruthenium red.","method":"Patch clamp in single epididymal cells, pharmacological inhibition, in vivo luminal perfusion, immunofluorescence colocalization, mRNA detection","journal":"The Journal of general physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — electrophysiology plus colocalization plus in vivo perfusion, single lab","pmids":["27481714"],"is_preprint":false},{"year":2019,"finding":"In zebrafish, Trpv6 functions as a cell-autonomous regulator of epithelial quiescence: Trpv6-mediated constitutive Ca2+ influx activates PP2A, which suppresses IGF-mediated Akt-Tor and Erk signaling to maintain cellular quiescence. Genetic deletion or pharmacological blockade of Trpv6 caused epithelial cells to exit quiescence and re-enter the cell cycle; re-introduction of Trpv6 but not a channel-dead mutant restored quiescence.","method":"Zebrafish genetic deletion, pharmacological inhibition, cell cycle analysis, Ca2+ imaging, PP2A activity assays, IGF signaling readouts, human colon cancer cell experiments","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic and pharmacological loss-of-function with channel-dead rescue, pathway mechanistic follow-up, conserved in human cells, single lab","pmids":["31526479"],"is_preprint":false},{"year":2022,"finding":"TRPV6 is required for alcohol-induced intestinal barrier dysfunction: ethanol and acetaldehyde activate TRPV6 ionic currents in Caco-2 cells; TRPV6 deficiency (Trpv6-/- mice and organoids) attenuates ethanol/acetaldehyde-induced Ca2+ influx, tight junction disruption, and barrier dysfunction. Photoaffinity labeling of 3-azibutanol identified a histidine as a putative alcohol-binding site; substitution of this histidine (and a nearby arginine) reduces ethanol-activated TRPV6 currents.","method":"Patch clamp electrophysiology, Ca2+ imaging, Trpv6-/- mouse model, intestinal organoids, Caco-2 monolayers, photoaffinity labeling, site-directed mutagenesis","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — multiple orthogonal methods including genetic null mouse, mutagenesis of binding site, and organoid/cell systems; single lab","pmids":["35705057"],"is_preprint":false},{"year":2021,"finding":"TRPV6 promotes breast cancer metastasis via NFATC2IP phosphorylation at Ser204 (with CDK5 as candidate kinase), which activates NFATC2 transcription factor, leading to upregulation of ADAMTS6 and increased cell migration. TRPV6 overexpression accelerates migration; TRPV6 suppression decreases it.","method":"Overexpression and siRNA knockdown, phosphorylation analysis, reporter assays, migration assays in breast cancer cells","journal":"Cancer letters","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, indirect mechanistic pathway, CDK5 as kinase is proposed but not definitively established","pmids":["34265397"],"is_preprint":false},{"year":2020,"finding":"TRPV6 suppresses osteoclastogenesis via the IGF-PI3K-AKT signaling pathway: TRPV6 knockout mice develop osteoporosis with enhanced osteoclast differentiation and bone resorption. TRPV6 located on the osteoclast cell membrane decreases p-IGF/total-IGF, p-PI3K/total-PI3K, and p-AKT/total-AKT ratios. Blocking IGF-PI3K-AKT with inhibitors relieved the inhibitory effect of TRPV6 on osteoclasts.","method":"Trpv6 knockout mice, lentiviral overexpression/silencing in osteoclasts, micro-CT, TRAP staining, pit formation assay, Western blot for pathway components","journal":"Cell proliferation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO plus pathway inhibitor rescue plus overexpression/silencing in vitro, single lab","pmids":["33159483"],"is_preprint":false}],"current_model":"TRPV6 is a highly Ca2+-selective tetrameric ion channel that mediates constitutive and regulated Ca2+ entry at the apical membrane of epithelia; its Ca2+ selectivity is determined by a ring of aspartate residues in the selectivity filter, channel opening involves an α-to-π helical transition in S6 at an alanine hinge (iris-like gating), and channel activity is directly activated by PIP2 and inhibited by autoinhibitory intramolecular interactions (S4-S5 linker–TRP helix and pre-S1–TRP helix contacts); it is positively regulated by calmodulin (Ca2+-dependent), CypB (prolyl-isomerase-dependent), Rab11a-mediated trafficking, WNK3, SGK1/Akt, and klotho-mediated deglycosylation, and negatively regulated by PLC-driven PIP2 depletion (Ca2+-induced inactivation), RGS2 (direct gating inhibition), Nedd4-2-mediated ubiquitination, and Nipsnap1; its obligate plasma membrane localization requires the S100A10–annexin 2 complex binding to the C-terminal VATTV motif; the channel is physiologically essential for intestinal Ca2+ absorption, renal/epididymal Ca2+ reabsorption, maternal-fetal Ca2+ transport via the placenta (critical for fetal bone mineralization), and maintenance of low luminal Ca2+ in the epididymis for male fertility, and it also controls keratinocyte differentiation and epithelial cell quiescence via Ca2+-dependent suppression of IGF/Akt/PP2A signaling."},"narrative":{"mechanistic_narrative":"TRPV6 is a highly Ca2+-selective cation channel that mediates apical Ca2+ entry into epithelia and is physiologically essential for transepithelial Ca2+ transport [PMID:21540454, PMID:22878123]. It assembles as a homotetramer (and can form heterotetramers with TRPV5) with an intracellular skirt for allosteric modulation, and its Ca2+ selectivity is set by a ring of aspartate side chains in the selectivity filter, with the pore residue D541/D542 essential for permeation [PMID:12574114, PMID:27296226, PMID:15184369]. Channel opening proceeds through an α-to-π helical transition in the pore-lining S6 helix at an alanine hinge, producing an iris-like gating motion [PMID:29258289]. The basal state is autoinhibited by two intramolecular contacts—an S4–S5 linker to TRP-helix interaction and a pre-S1 to TRP-helix interaction—both of which are relieved by direct binding of PIP2, the principal activating lipid demonstrated using purified channel reconstituted in lipid bilayers [PMID:21810903, PMID:32829285]; conversely, Ca2+ influx drives phospholipase C-mediated PIP2 depletion that underlies Ca2+-induced inactivation [PMID:18390907]. Activity is further tuned by Ca2+-dependent calmodulin binding and by direct gating inhibitors RGS2, Nipsnap1, and Numb1, while small molecules such as 2-APB close the channel through a defined S1–S4 pocket [PMID:15123711, PMID:29941865, PMID:16895908, PMID:18392847, PMID:23140583]. Surface delivery and stability are governed by the S100A10–annexin 2 complex binding the C-terminal VATTV motif (required for plasma-membrane targeting), GDP-bound Rab11a-mediated trafficking, kinase inputs (WNK3, SGK1/Akt), klotho/β-glucuronidase-mediated deglycosylation, and Nedd4-2-mediated ubiquitination and degradation [PMID:12660155, PMID:16354700, PMID:18495742, PMID:20843805, PMID:18768590]. In vivo, functional TRPV6 channels are required for intestinal Ca2+ absorption, maternal-fetal placental Ca2+ transport supporting fetal bone mineralization, and epididymal Ca2+ reabsorption essential for male fertility, with basolateral CaSR signaling negatively regulating intestinal TRPV6 via PLC [PMID:21540454, PMID:18348695, PMID:22878123, PMID:31013259]. Beyond bulk Ca2+ handling, TRPV6-mediated Ca2+ entry controls keratinocyte differentiation and, through Ca2+-dependent activation of PP2A, suppresses IGF/Akt signaling to maintain epithelial cell quiescence [PMID:17550901, PMID:31526479].","teleology":[{"year":2003,"claim":"Establishing the channel's quaternary architecture was needed to interpret all later functional and structural work; TRPV6 was shown to be a tetramer that can mix with TRPV5 to create functionally distinct channels.","evidence":"Sucrose gradient sedimentation, co-IP, and concatemeric channel electrophysiology in HEK293 cells","pmids":["12574114"],"confidence":"High","gaps":["Native epithelial stoichiometry of homo- versus heterotetramers not quantified","No atomic structure at this stage"]},{"year":2003,"claim":"How TRPV6 reaches the plasma membrane was unknown; the S100A10–annexin 2 complex was identified as binding the C-terminal VATTV motif and being required for surface targeting and channel activity.","evidence":"Yeast two-hybrid, GST pull-down, co-IP, T600A mutagenesis, annexin 2 siRNA, and electrophysiology in HEK293 cells","pmids":["12660155"],"confidence":"High","gaps":["Step in the trafficking itinerary controlled by the complex not resolved","Regulation of complex assembly addressed only later"]},{"year":2004,"claim":"The basis of Ca2+-dependent feedback regulation was addressed by showing calmodulin binds TRPV6 in a Ca2+-dependent manner at defined N- and C-terminal motifs and positively modulates current.","evidence":"GST pull-down, co-IP, whole-cell patch clamp, and chimeric channel analysis in HEK293 cells","pmids":["15123711"],"confidence":"High","gaps":["Structural coupling of CaM binding to gating not defined","Relationship to PIP2-dependent inactivation not integrated"]},{"year":2004,"claim":"Whether TRPV6 is constitutively open was tested; under physiological Ca2+ buffering it is not, and divalent block plus the pore aspartate D542 govern permeation behavior.","evidence":"Whole-cell/perforated patch clamp, Fura-2, and pore/truncation mutagenesis in HEK293 cells","pmids":["15184369"],"confidence":"High","gaps":["Molecular trigger for physiological opening in epithelia not defined here"]},{"year":2006,"claim":"Mechanisms of surface regulation and direct gating control were expanded by identifying GDP-Rab11a as a trafficking partner and RGS2 as a direct, trafficking-independent gating inhibitor, with NHERF4 as an additional C-terminal PDZ interactor.","evidence":"Co-IP, confocal colocalization, Ca2+ uptake/surface biotinylation, yeast two-hybrid, and GST pull-down in epithelial cells and oocytes","pmids":["16354700","16895908","16565876"],"confidence":"High","gaps":["Functional consequence of NHERF4 binding not established","How RGS2 alters gating structurally unknown"]},{"year":2008,"claim":"Multiple inputs converging on TRPV6 were defined: cAMP/PKA/calcineurin control of the annexin 2–S100A10 association, CypB isomerase-dependent activation, klotho/β-glucuronidase deglycosylation activation, and Nipsnap1 as a direct gating inhibitor.","evidence":"Co-IP, 45Ca uptake, oocyte Ca2+ assays, enzymatic deglycosylation, GST pull-down, and biotinylation across HEK293, Caco-2, oocyte and placental systems","pmids":["18187190","18445599","18495742","18392847"],"confidence":"Medium","gaps":["Several findings rest on single-lab functional assays","Direct enzymatic action of klotho on the channel inferred from specificity not structurally shown"]},{"year":2008,"claim":"The physiological necessity of TRPV6 was established in vivo, showing it mediates maternal-fetal placental Ca2+ transport critical for fetal bone mineralization.","evidence":"Trpv6 knockout mice with 45Ca transport assays, immunohistochemistry, and qRT-PCR","pmids":["18348695"],"confidence":"High","gaps":["Relative contribution of other Ca2+ pathways in placenta not quantified"]},{"year":2008,"claim":"The lipid and metabolic regulation of TRPV6 was clarified by showing Ca2+ influx activates PLC to deplete PIP2, driving Ca2+-induced inactivation.","evidence":"Whole-cell and excised patch clamp with rapamycin-inducible phosphatase and Ca2+ imaging","pmids":["18390907"],"confidence":"High","gaps":["Identity of the PLC isoform in native epithelia not defined"]},{"year":2009,"claim":"Kinase and nucleotide inputs were defined: WNK3 and SGK1/Akt increase activity and surface abundance, while ATP binding to the ARD/C-terminus prevents rundown and PKCβII phosphorylation opposes it.","evidence":"Oocyte co-expression, Ca2+/current assays, surface biotinylation, ATP-binding studies, and mutagenesis","pmids":["18768590","19805577","20041238"],"confidence":"Medium","gaps":["SGK1/PIKfyve effect relies on indirect Cl- current readout (Low confidence)","Direct phosphosites on TRPV6 versus partners not fully resolved"]},{"year":2008,"claim":"The structural basis of the regulatory cytoplasmic domain was probed by solving the ankyrin repeat domain, showing it does not bind ATP or CaM unlike TRPV1, implying a distinct regulatory role.","evidence":"X-ray crystallography at 1.7 Å with ATP- and CaM-agarose pull-downs and SEC","pmids":["18232717"],"confidence":"High","gaps":["Functional role of the variable third-finger loop and its phosphosites not tested in vivo"]},{"year":2010,"claim":"Negative protein-stability control was defined by showing Nedd4-2 ubiquitinates TRPV6 and reduces its abundance, predominantly via the proteasome.","evidence":"Oocyte co-expression, Ca2+ influx, ubiquitination assays, proteasome/lysosome inhibitors, and HECT/WW domain mutagenesis","pmids":["20843805"],"confidence":"Medium","gaps":["Physiological context regulating Nedd4-2 action on TRPV6 not established"]},{"year":2011,"claim":"PIP2 was demonstrated to be a direct activator using purified, reconstituted channel, decoupling lipid activation from cellular machinery and explaining ATP-dependent recovery via PIP2 resynthesis.","evidence":"Planar lipid bilayer reconstitution of purified TRPV6 plus excised inside-out patch clamp and PI4K inhibition","pmids":["21810903"],"confidence":"High","gaps":["Lipid-binding site not resolved structurally until later mutational work"]},{"year":2011,"claim":"In vivo significance for fertility was established by showing a pore-dead D541A channel impairs epididymal Ca2+ absorption and male fertility, localizing TRPV6 to apical epididymal epithelium.","evidence":"D541A knock-in mice with luminal Ca2+ measurements, absorption assays, immunolocalization, and sperm assays","pmids":["21540454"],"confidence":"High","gaps":["Downstream coupling to sperm physiology mechanistic detail limited"]},{"year":2011,"claim":"A receptor-driven mode of TRPV6 activation was identified in prostate cells, with PSGR activating TRPV6 through Src kinase independent of G-protein signaling.","evidence":"Electrophysiology, Ca2+ imaging, TRPV6 siRNA, co-IP, and Src inhibition","pmids":["21349844"],"confidence":"Medium","gaps":["Direct Src target residues on TRPV6 not mapped","Single-lab cancer-cell context"]},{"year":2012,"claim":"Genetic null mice confirmed that epididymal and fertility phenotypes arise from loss of channel function rather than channel-independent effects of the pore mutant, and established intestinal Ca2+ absorption requires functional channels; an extended non-AUG-initiated N-terminus enhances trafficking; Numb1 was identified as a direct inhibitor.","evidence":"Trpv6 knockout and D541A knock-in mice, 45Ca absorption assays, mass spectrometry of placental protein, and co-IP/FRET/mutagenesis","pmids":["22427671","22878123","23612980","23140583"],"confidence":"High","gaps":["Tissue distribution and regulation of the extended isoform not fully mapped","Numb1 inhibition mechanism on gating not structurally resolved"]},{"year":2016,"claim":"Atomic structure of TRPV6 revealed the tetrameric assembly, the aspartate ring selectivity filter, and a multi-site Ca2+ permeation pathway, providing the framework for all mechanistic interpretation.","evidence":"X-ray crystallography of rat TRPV6 at 3.25 Å","pmids":["27296226"],"confidence":"High","gaps":["Single conformational state; gating transition not yet captured at this stage"]},{"year":2016,"claim":"The native epithelial output of TRPV6 Ca2+ entry was linked to a calcium-activated chloride conductance (TMEM16A) coupling in epididymal principal cells, connecting Ca2+ entry to downstream transport.","evidence":"Single-cell patch clamp, pharmacology, in vivo luminal perfusion, and immunofluorescence colocalization","pmids":["27481714"],"confidence":"Medium","gaps":["Direct physical coupling between TRPV6 and TMEM16A not demonstrated"]},{"year":2017,"claim":"Open and closed cryo-EM structures defined the gating mechanism as an α-to-π S6 transition at an alanine hinge with iris-like opening, and confirmed PIP2 increases open probability.","evidence":"Cryo-EM of human TRPV6 in two states plus electrophysiology","pmids":["29258289"],"confidence":"High","gaps":["PIP2 binding pose not resolved in these maps"]},{"year":2018,"claim":"The pharmacological inhibitor 2-APB was localized to a defined S1–S4 cytoplasmic pocket, showing closure occurs by modulating protein–lipid interactions.","evidence":"Crystal/cryo-EM of human and rat TRPV6 with 2-APB plus Y467A mutagenesis and functional assays","pmids":["29941865"],"confidence":"High","gaps":["Selectivity of this pocket relative to other TRPV channels not addressed"]},{"year":2019,"claim":"Two regulatory questions were answered: basolateral CaSR signaling restrains intestinal TRPV6 Ca2+ absorption via PLC, and TRPV6-mediated constitutive Ca2+ influx enforces epithelial quiescence by activating PP2A to suppress IGF-Akt/Erk signaling.","evidence":"Ussing chambers with D541A mice and oocyte co-expression; zebrafish genetic deletion with channel-dead rescue, PP2A assays, and human colon cancer cells","pmids":["31013259","31526479"],"confidence":"High","gaps":["Direct CaSR-PLC-TRPV6 molecular coupling not resolved","Quiescence pathway demonstrated in single-lab models"]},{"year":2020,"claim":"The autoinhibitory architecture of resting TRPV6 was defined as two intramolecular TRP-helix interactions relieved by PIP2 binding at S5/C-terminal cationic residues, unifying lipid activation with gating control.","evidence":"Site-directed mutagenesis, blocking peptides, patch clamp, and Ca2+ imaging","pmids":["32829285"],"confidence":"Medium","gaps":["Interactions inferred functionally without a corresponding apo/PIP2-bound structure in this study"]},{"year":2020,"claim":"A skeletal role beyond Ca2+ absorption was proposed, with TRPV6 suppressing osteoclastogenesis through IGF-PI3K-AKT signaling, paralleling its quiescence-promoting role.","evidence":"Trpv6 knockout mice, micro-CT, TRAP/pit assays, lentiviral manipulation, and pathway inhibitor rescue in osteoclasts","pmids":["33159483"],"confidence":"Medium","gaps":["Cell-autonomous versus systemic Ca2+ contributions not fully separated"]},{"year":2022,"claim":"A pathological gating modality was identified: ethanol and acetaldehyde directly activate TRPV6 via a histidine-containing site, driving Ca2+ influx that disrupts intestinal tight junctions.","evidence":"Patch clamp, Ca2+ imaging, Trpv6-/- mice and organoids, Caco-2 monolayers, photoaffinity labeling, and mutagenesis","pmids":["35705057"],"confidence":"High","gaps":["Structural definition of the alcohol-binding pocket not resolved"]},{"year":null,"claim":"How the diverse regulatory inputs (CaM, PIP2 autoinhibition, kinases, trafficking partners, ubiquitination) are integrated dynamically within a single epithelial cell to set TRPV6 set-point in vivo remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified quantitative model linking lipid, Ca2+, and post-translational regulation","Structural states bound to physiological protein regulators (CaM, RGS2, S100A10) not solved","Native channel composition and regulation across tissues incompletely mapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[0,3,22,31]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[10,11,33]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[4,22,30]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[1,7,18,34]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[7]}],"pathway":[{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[18,20,21,30]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[10,33,35]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[1,7,25]}],"complexes":["TRPV6 homotetramer","TRPV5/TRPV6 heterotetramer","S100A10-annexin 2 complex"],"partners":["TRPV5","S100A10","ANXA2","CALM1","RAB11A","RGS2","NEDD4L","NUMB"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9H1D0","full_name":"Transient receptor potential cation channel subfamily V member 6","aliases":["CaT-like","CaT-L","Calcium transport protein 1","CaT1","Epithelial calcium channel 2","ECaC2"],"length_aa":765,"mass_kda":87.3,"function":"Calcium selective cation channel that mediates Ca(2+) uptake in various tissues, including the intestine (PubMed:11097838, PubMed:11248124, PubMed:11278579, PubMed:15184369, PubMed:23612980, PubMed:29258289). Important for normal Ca(2+) ion homeostasis in the body, including bone and skin (By similarity). The channel is activated by low internal calcium level, probably including intracellular calcium store depletion, and the current exhibits an inward rectification (PubMed:15184369). Inactivation includes both a rapid Ca(2+)-dependent and a slower Ca(2+)-calmodulin-dependent mechanism; the latter may be regulated by phosphorylation. In vitro, is slowly inhibited by Mg(2+) in a voltage-independent manner. Heteromeric assembly with TRPV5 seems to modify channel properties. TRPV5-TRPV6 heteromultimeric concatemers exhibit voltage-dependent gating","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q9H1D0/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TRPV6","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/TRPV6","total_profiled":1310},"omim":[{"mim_id":"618188","title":"HYPERPARATHYROIDISM, TRANSIENT NEONATAL; HRPTTN","url":"https://www.omim.org/entry/618188"},{"mim_id":"616252","title":"TRPM8 CHANNEL-ASSOCIATED FACTOR 2; TCAF2","url":"https://www.omim.org/entry/616252"},{"mim_id":"616251","title":"TRPM8 CHANNEL-ASSOCIATED FACTOR 1; TCAF1","url":"https://www.omim.org/entry/616251"},{"mim_id":"606680","title":"TRANSIENT RECEPTOR POTENTIAL CATION CHANNEL, SUBFAMILY V, MEMBER 6; TRPV6","url":"https://www.omim.org/entry/606680"},{"mim_id":"606679","title":"TRANSIENT RECEPTOR POTENTIAL CATION CHANNEL, SUBFAMILY V, MEMBER 5; TRPV5","url":"https://www.omim.org/entry/606679"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"pancreas","ntpm":36.4},{"tissue":"prostate","ntpm":22.1},{"tissue":"salivary gland","ntpm":65.3}],"url":"https://www.proteinatlas.org/search/TRPV6"},"hgnc":{"alias_symbol":["CaT1"],"prev_symbol":["ECAC2"]},"alphafold":{"accession":"Q9H1D0","domains":[{"cath_id":"1.25.40.20","chopping":"76-276","consensus_level":"medium","plddt":92.9338,"start":76,"end":276},{"cath_id":"1.10.287.70","chopping":"522-644","consensus_level":"high","plddt":89.785,"start":522,"end":644},{"cath_id":"1.20.120","chopping":"354-396_410-510","consensus_level":"high","plddt":90.6865,"start":354,"end":510}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H1D0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H1D0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H1D0-F1-predicted_aligned_error_v6.png","plddt_mean":80.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TRPV6","jax_strain_url":"https://www.jax.org/strain/search?query=TRPV6"},"sequence":{"accession":"Q9H1D0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9H1D0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9H1D0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H1D0"}},"corpus_meta":[{"pmid":"12574114","id":"PMC_12574114","title":"Homo- and heterotetrameric architecture of the epithelial Ca2+ channels TRPV5 and TRPV6.","date":"2003","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/12574114","citation_count":255,"is_preprint":false},{"pmid":"12660155","id":"PMC_12660155","title":"Functional expression of the epithelial Ca(2+) channels (TRPV5 and TRPV6) requires association of the S100A10-annexin 2 complex.","date":"2003","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/12660155","citation_count":228,"is_preprint":false},{"pmid":"17533368","id":"PMC_17533368","title":"TRPV6 channel controls prostate cancer cell proliferation via Ca(2+)/NFAT-dependent pathways.","date":"2007","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/17533368","citation_count":200,"is_preprint":false},{"pmid":"27296226","id":"PMC_27296226","title":"Crystal structure of the epithelial calcium channel 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electrophysiological analysis of concatemeric channels. Heterotetrameric TRPV5/TRPV6 complexes show distinct Ca2+-dependent inactivation, Ba2+ selectivity, and pharmacological block compared to homotetramers.\",\n      \"method\": \"Sucrose gradient sedimentation, co-immunoprecipitation, concatemeric channel electrophysiology in HEK293 cells\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (biochemical + electrophysiological reconstitution) in single rigorous study\",\n      \"pmids\": [\"12574114\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"S100A10 binds specifically to the conserved C-terminal VATTV motif of TRPV6 (with the first threonine, T600, being critical); S100A10 forms a heterotetrameric complex with annexin 2, and the S100A10–annexin 2 complex is required for routing TRPV6 to the plasma membrane. Mutation T600A abolishes S100A10 binding, redistributes TRPV6 to a sub-plasma-membrane compartment, and eliminates channel activity. Knockdown of annexin 2 by siRNA inhibits TRPV6-mediated currents.\",\n      \"method\": \"Yeast two-hybrid, GST pull-down, co-immunoprecipitation, site-directed mutagenesis, siRNA knockdown, electrophysiology in HEK293 cells, immunofluorescence\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (Y2H, GST pulldown, co-IP, mutagenesis, siRNA + electrophysiology) in single rigorous study\",\n      \"pmids\": [\"12660155\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Calmodulin (CaM) binds TRPV6 in a Ca2+-dependent manner and positively regulates TRPV6 channel activity. CaM binding sites were localized to the transmembrane domain, an N-terminal 1-5-10 motif (residues 88–97), and a C-terminal 1-8-14 motif (residues 643–656). Overexpression of Ca2+-insensitive CaM mutants (CaM1234, CaM34) significantly reduced Ca2+ and Na+ currents of TRPV6-expressing HEK293 cells but not TRPV5 cells, implicating EF-hands 3 and 4 of CaM's C-lobe.\",\n      \"method\": \"GST pull-down, co-immunoprecipitation, whole-cell patch clamp, chimeric channel analysis in HEK293 cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — reciprocal biochemical binding assays + mutagenesis + electrophysiology, multiple orthogonal methods\",\n      \"pmids\": [\"15123711\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Crystal structure of rat TRPV6 at 3.25 Å resolution revealed: tetrameric assembly with an intracellular 'skirt' involved in allosteric modulation; Ca2+ selectivity determined by a ring of aspartate side chains in the selectivity filter; multiple cation-binding sites along the pore axis and extracellular vestibule defining a Ca2+ permeation mechanism.\",\n      \"method\": \"X-ray crystallography at 3.25 Å resolution\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — atomic-resolution crystal structure with multiple functional implications validated structurally\",\n      \"pmids\": [\"27296226\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Cryo-EM structures of human TRPV6 in open and closed states revealed that channel opening involves an α-to-π helical transition in the pore-lining S6 helix at an alanine hinge below the selectivity filter, causing S6 helices to bend and rotate (iris-like gating). The selectivity filter adopts similar conformations in both states. Open probability is increased by PIP2.\",\n      \"method\": \"Cryo-electron microscopy, electrophysiology\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structures of both open and closed states with corroborating electrophysiology\",\n      \"pmids\": [\"29258289\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"2-APB binds to TRPV6 in a pocket formed by the cytoplasmic half of the S1–S4 transmembrane helix bundle. 2-APB induces channel closure by modulating protein–lipid interactions, as shown by comparing wild-type and high-affinity Y467A mutant structures. Mutagenesis and functional analysis confirmed the binding site.\",\n      \"method\": \"Crystal and cryo-EM structures of human and rat TRPV6 bound to 2-APB, mutagenesis, functional assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — atomic structures plus mutagenesis plus functional validation in single study\",\n      \"pmids\": [\"29941865\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Crystal structure of the TRPV6 ankyrin repeat domain (ARD) at 1.7 Å revealed a conserved large twist between repeats 4 and 5 and a variable third finger loop with putative regulatory phosphorylation sites. Pull-down assays showed the TRPV6 ARD does not bind ATP or calmodulin, contrasting with TRPV1 ARD, indicating a distinct regulatory role for the TRPV6 ARD.\",\n      \"method\": \"X-ray crystallography (1.7 Å), ATP-agarose and calmodulin-agarose pull-down assays, size exclusion chromatography\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — high-resolution crystal structure plus orthogonal biochemical assays in single study\",\n      \"pmids\": [\"18232717\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"GDP-bound Rab11a directly and specifically interacts with TRPV5 and TRPV6 via a conserved stretch in their C-termini. Rab11a colocalizes with TRPV6 in vesicular structures beneath the apical membrane. Co-expression of GDP-locked Rab11a significantly decreased TRPV6-mediated Ca2+ uptake by reducing channel surface expression. Rab11a thus mediates TRPV6 trafficking to the plasma membrane in a novel GDP-bound, direct cargo-interaction mode.\",\n      \"method\": \"Co-immunoprecipitation, confocal colocalization, Ca2+ uptake assays, surface expression analysis in epithelial cells and oocytes\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP plus functional Ca2+ uptake assays plus localization data\",\n      \"pmids\": [\"16354700\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Klotho (acting as a β-glucuronidase) and β-glucuronidase specifically increase TRPV6 (and TRPV5) activity but not TRPV4 or TRPM6, suggesting activation via N-oligosaccharide hydrolysis on the channel. Deglycosylation by endoglycosidase-F also stimulated TRPV6 activity.\",\n      \"method\": \"Ca2+-influx measurements in transfected HEK293 cells, enzymatic deglycosylation\",\n      \"journal\": \"Nephrology, dialysis, transplantation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional Ca2+ influx assays with pharmacological specificity controls, single lab\",\n      \"pmids\": [\"18495742\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"RGS2 interacts with the N-terminal domain of TRPV6 in a Ca2+-independent manner (identified by yeast two-hybrid and GST pull-down). RGS2 overexpression reduces Na+ and Ca2+ currents of TRPV6 but not TRPV5. The inhibitory effect is not due to altered channel trafficking (cell-surface biotinylation was unchanged), suggesting direct effects on gating. DeltaN-RGS2 lacking the N-terminal domain does not inhibit TRPV6, and the effect is GPCR-independent.\",\n      \"method\": \"Yeast two-hybrid, GST pull-down, whole-cell electrophysiology, cell-surface biotinylation in HEK293 cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — Y2H + GST pulldown + electrophysiology + surface biotinylation, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"16895908\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Ca2+ influx through TRPV6 activates phospholipase C, causing depletion of PIP2, which contributes to Ca2+-induced inactivation of TRPV6. Ba2+ (which does not activate PLC) does not cause inactivation. Dialysis of DiC8-PIP2 inhibited Ca2+-dependent inactivation; PIP2 activated TRPV6 in excised patches; rapamycin-inducible PI(4,5)P2 5-phosphatase inhibited TRPV6 currents. PI(4)P had no significant effect.\",\n      \"method\": \"Whole-cell and excised patch-clamp, rapamycin-inducible phosphatase system, Ca2+ imaging\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — multiple electrophysiology approaches plus pharmacological tools, orthogonal methods, single lab\",\n      \"pmids\": [\"18390907\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"PIP2 is a direct activator of TRPV6: reconstituted purified TRPV6 in planar lipid bilayers showed high activity in presence of PIP2 but not PI(4)P. Intracellular ATP supports TRPV6 activity indirectly by serving as substrate for type III PI4-kinases to resynthesize PIP2; MgATP reactivated TRPV6 after rundown in excised patches, an effect blocked by PI4K inhibitors.\",\n      \"method\": \"Excised inside-out patch clamp, planar lipid bilayer reconstitution with purified channel, pharmacological inhibition of PI4K\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — channel reconstitution in lipid bilayers plus excised patch clamp; direct activation by PIP2 established\",\n      \"pmids\": [\"21810903\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Cyclophilin B (CypB) co-purifies with TRPV6 from human placenta (also with annexin A2) and co-expression of CypB with TRPV6 in Xenopus oocytes significantly increases TRPV6-mediated Ca2+ uptake. This stimulatory effect is reversed by cyclosporin A (which inhibits CypB's peptidyl-prolyl cis/trans isomerase activity), suggesting CypB activates TRPV6 through its isomerase activity.\",\n      \"method\": \"Protein enrichment/co-purification from human placenta, Xenopus oocyte Ca2+ uptake assay, pharmacological inhibition with cyclosporin A\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — endogenous co-purification plus functional oocyte assay, single lab\",\n      \"pmids\": [\"18445599\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"ATP binds directly to TRPV6 (at sites within the ankyrin repeat domain and C-terminus), reduces whole-cell current increments, and prevents channel rundown (EC50 ~380 µM). PKCβII-mediated phosphorylation at defined ARD and C-terminal sites counteracts ATP-dependent stabilization, constituting a metabolic switch for Ca2+ influx.\",\n      \"method\": \"Biochemical ATP-binding studies, patch-clamp electrophysiology, site-directed mutagenesis of ARD and C-terminal residues\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding assays plus electrophysiology plus mutagenesis, single lab\",\n      \"pmids\": [\"19805577\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Nedd4-2 (HECT ubiquitin E3 ligase) down-regulates TRPV6 protein abundance and Ca2+ influx by increasing TRPV6 ubiquitination and decreasing its stability (partially via proteasomal but not lysosomal pathway). The HECT domain is essential for both inhibition and association. WW1 and WW2 domains interact with TRPV6 terminal regions and their integrity limits the degree of TRPV6 ubiquitination.\",\n      \"method\": \"Xenopus oocyte co-expression, Ca2+ influx assays, ubiquitination assays, proteasome/lysosome inhibitors, mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple functional assays and mutagenesis in oocyte system, single lab\",\n      \"pmids\": [\"20843805\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The annexin 2–S100A10 complex association with TRPV6 is regulated by cAMP/PKA/calcineurin A (CnA) signaling: forskolin-stimulated cAMP increases annexin 2–S100A10 complex formation (blocked by PKA or CnA inhibitors), and CnA-dependent dephosphorylation of annexin 2 mediates the interaction with TRPV6. PKA and CnA inhibitors attenuated 45Ca uptake in Caco-2 cells.\",\n      \"method\": \"Co-immunoprecipitation, 45Ca uptake assays, pharmacological inhibitors (PKA, CnA), cell biology in 16HBE14o- and Caco-2 cells\",\n      \"journal\": \"Cell calcium\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — co-IP plus functional Ca2+ transport assays, single lab\",\n      \"pmids\": [\"18187190\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"NHERF4 (PDZ protein) interacts with TRPV6 via the fourth PDZ domain binding a conserved region in the TRPV6 C-terminus, distinct from the NHERF2 binding site. Interaction confirmed by yeast two-hybrid, GST pull-down, and co-immunoprecipitation.\",\n      \"method\": \"Yeast two-hybrid, GST pull-down, co-immunoprecipitation in HEK293 cells and oocytes\",\n      \"journal\": \"Pflugers Archiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Y2H plus GST pulldown plus co-IP, multiple binding assays, single lab\",\n      \"pmids\": [\"16565876\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Nipsnap1 was identified as a novel TRPV6-interacting protein (by GST pull-down/bioinformatics) that abolishes TRPV6 currents without reducing plasma membrane expression of TRPV6, suggesting direct inhibition of channel gating rather than trafficking.\",\n      \"method\": \"GST pull-down, electrophysiology, cell-surface biotinylation, RT-PCR, immunohistochemistry\",\n      \"journal\": \"Pflugers Archiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — GST pulldown plus electrophysiology plus surface biotinylation, single lab\",\n      \"pmids\": [\"18392847\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Male mice carrying the TRPV6-D541A pore mutation (rendering the channel nonfunctional) show severely impaired fertility due to failure of Ca2+ absorption by epididymal epithelium, resulting in ~10-fold higher Ca2+ in cauda epididymis luminal fluid. TRPV6 was localized to the apical membrane of epididymal epithelium (not in spermatozoa), establishing that TRPV6-mediated Ca2+ absorption in the epididymis is essential for sperm motility and viability.\",\n      \"method\": \"Knock-in mouse model (D541A), Ca2+ concentration measurements, Ca2+ absorption assays, immunolocalization, sperm functional assays\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo knock-in model with defined biochemical and functional phenotype, replicated by TRPV6-null mouse study\",\n      \"pmids\": [\"21540454\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Trpv6 knockout mice (deletion of transmembrane/pore-forming and C-terminal regions) phenocopy the TRPV6-D541A pore mutant regarding epididymal Ca2+ handling and male fertility defects, arguing against residual channel-independent functions of the D541A mutant.\",\n      \"method\": \"Trpv6 knockout mouse (exon deletion), comparison with D541A knock-in, Ca2+ uptake assays, sperm function tests\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic null compared to pore mutant with multiple biochemical and functional endpoints; independent replication of epididymal phenotype\",\n      \"pmids\": [\"22427671\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"TRPV6 is required for maternal-fetal Ca2+ transport: Trpv6 knockout fetuses had significantly lower fetal blood Ca2+, lower amniotic fluid Ca2+, ~40% lower 45Ca transport from mother to fetus, and lower ash weight (bone mineralization). TRPV6 mRNA and protein were localized in intraplacental and extraplacental yolk sac, co-localizing with calbindin-D9K.\",\n      \"method\": \"Trpv6 knockout mice, 45Ca transport assays, immunohistochemistry, qRT-PCR\",\n      \"journal\": \"Journal of bone and mineral research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo knockout with isotope transport assay and localization, well-replicated finding\",\n      \"pmids\": [\"18348695\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"TRPV6-D541A knockin mice (non-functional pore) show significantly impaired intestinal Ca2+ uptake under Ca2+-deficient diet conditions, establishing that functional TRPV6 channels mediate transepithelial Ca2+ absorption in the intestine, particularly under low-Ca2+ dietary challenge.\",\n      \"method\": \"TRPV6-D541A knockin mouse model, 45Ca intestinal absorption assay, metabolic cage measurements, protein expression analysis\",\n      \"journal\": \"American journal of physiology. Gastrointestinal and liver physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo knockin model with isotope-based absorption assay\",\n      \"pmids\": [\"22878123\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Under physiological intracellular Ca2+ buffering conditions, TRPV6 does not form constitutively open channels in HEK293 cells; channel activity requires chelation of intracellular Ca2+ (with EGTA). Monovalent cations permeate TRPV6 when Ca2+ permeation is absent, indicating extracellular divalent cation block. The C-terminal domain required for calmodulin binding is not necessary for this regulation; the pore residue D542 is essential for channel function.\",\n      \"method\": \"Whole-cell and perforated patch clamp, Fura-2 microfluorimetry, site-directed mutagenesis (D542A, truncation mutant) in HEK293 cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — patch clamp with mutagenesis defining pore and gating requirements, multiple conditions tested\",\n      \"pmids\": [\"15184369\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The in vivo TRPV6 protein has an extended N-terminus: translation initiates at a non-AUG codon (ACG, decoded as methionine) upstream of the annotated AUG (which is not used). The extended full-length protein has similar channel properties to the shorter form in vitro, but increased trafficking to the plasma membrane and provides an additional scaffold for channel assembly.\",\n      \"method\": \"Mass spectrometry of endogenous placenta protein, cell biology/trafficking assays, electrophysiology\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — endogenous protein identification plus functional trafficking assay, single lab\",\n      \"pmids\": [\"23612980\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"G protein-coupled receptor PSGR activates endogenous TRPV6 channels in prostate cells via Src kinase. PSGR stimulation enhances cytosolic Ca2+ through TRPV6; the Ca2+ signal has biophysical characteristics of TRPV6 currents; Src kinase activation occurs independently of G-protein activation, presumably through direct PSGR–Src interaction. siRNA knockdown of TRPV6 confirmed channel identity.\",\n      \"method\": \"Electrophysiology, live-cell Ca2+ imaging, siRNA knockdown, biochemical co-immunoprecipitation, Src kinase inhibition\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods including siRNA confirmation and electrophysiology, single lab\",\n      \"pmids\": [\"21349844\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"WNK3 positively regulates TRPV6 activity in a kinase-dependent manner; the kinase-inactive WNK3-D294A mutation abolishes the effect. WNK3 increases Ca2+ influx and Na+ current mediated by TRPV6, and enhances delivery of mature (glycosylated) TRPV6 to the plasma membrane via the secretory pathway; the effect is abolished by the microtubule inhibitor colchicine.\",\n      \"method\": \"Xenopus oocyte co-expression, Ca2+ uptake assays, voltage clamp, surface biotinylation, exocytosis assays, kinase-dead mutagenesis\",\n      \"journal\": \"American journal of physiology. Renal physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional assays plus surface expression plus mutagenesis, single lab\",\n      \"pmids\": [\"18768590\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"SGK1 and PKB/Akt increase TRPV6 activity and plasma membrane abundance. PIKfyve, phosphorylated by SGK1 at S318, further augments TRPV6 activity when co-expressed with active SGK1; the S318A-PIKfyve mutant lacking the SGK1 phosphorylation site fails to enhance TRPV6 activity.\",\n      \"method\": \"Xenopus oocyte co-expression, Ca2+-activated Cl- current recording as indirect readout of TRPV6-mediated Ca2+ influx, immunohistochemistry, Western blotting\",\n      \"journal\": \"The Journal of membrane biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — indirect readout (endogenous Cl- current), single lab, single oocyte system\",\n      \"pmids\": [\"20041238\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TRPV6 translocates to the plasma membrane in prostate cancer cells via an Orai1/TRPC1-mediated Ca2+/Annexin I/S100A11 pathway. TRPV6-mediated Ca2+ entry increases cell survival (proliferation and apoptosis resistance). Xenograft models confirmed increased aggressiveness of TRPV6-overexpressing tumors.\",\n      \"method\": \"Co-immunoprecipitation, Ca2+ imaging, siRNA knockdown, xenograft mouse models, immunohistochemistry\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus functional assays plus in vivo xenograft, single lab\",\n      \"pmids\": [\"25172921\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"TRPV6 mediates Ca2+ entry in human keratinocytes and is required for Ca2+-induced differentiation: siRNA-mediated TRPV6 silencing impairs differentiated phenotype, reduces differentiation markers (involucrin, transglutaminase-1, cytokeratin-10), disrupts cell morphology and stratification. 1,25-Dihydroxyvitamin D3 dose-dependently increases TRPV6 expression and Ca2+ uptake, promoting differentiation.\",\n      \"method\": \"siRNA knockdown, Ca2+ uptake assays (Fura-2), RT-PCR, immunoblotting in primary human keratinocytes\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA knockdown with defined differentiation phenotype plus Ca2+ functional assays, single lab\",\n      \"pmids\": [\"17550901\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"TRPV6 controls prostate cancer cell (LNCaP) proliferation: siRNA knockdown of TRPV6 decreases proliferation rate, reduces S-phase accumulation, and decreases PCNA expression. TRPV6-mediated Ca2+ entry activates NFAT transcription factor downstream. TRPV6 also contributes to apoptosis resistance.\",\n      \"method\": \"siRNA knockdown, cell cycle analysis, Ca2+ imaging, NFAT reporter assays in LNCaP cells\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA knockdown with multiple cellular phenotype readouts plus pathway confirmation, single lab\",\n      \"pmids\": [\"17533368\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Activation of the Ca2+-sensing receptor (CaSR) on the basolateral membrane of intestinal epithelium directly attenuates Ca2+ absorption via TRPV6: cinacalcet (calcimimetic) decreased net Ca2+ flux in Ussing chambers in a TRPV6-dependent manner (absent in TRPV6-D541A mice), and inhibited Ca2+ flux through TRPV6 when co-expressed with CaSR in oocytes. PLC inhibitor U73122 prevented cinacalcet-mediated TRPV6 inhibition.\",\n      \"method\": \"Ussing chamber Ca2+ flux assays, TRPV6-D541A knockin mice, Xenopus oocyte co-expression, pharmacological inhibition\",\n      \"journal\": \"JCI insight\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple model systems (Ussing chamber, knockin mouse, oocyte) with pharmacological and genetic epistasis\",\n      \"pmids\": [\"31013259\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"TRPV6 permeates heavy metal cations including Zn2+, Cd2+, Ba2+, Sr2+, Mn2+, La3+, and Gd3+ in addition to Ca2+, as shown by live-cell ion imaging (Fura-2, Newport Green DCF) and patch clamp in hTRPV6-expressing HEK293 cells. At higher concentrations, Cd2+, La3+, and Gd3+ are efficient inhibitors of TRPV6-mediated Ca2+ influx.\",\n      \"method\": \"Live-cell ion imaging (Fura-2, Newport Green DCF), whole-cell patch clamp, 45Ca uptake assays in HEK293 cells\",\n      \"journal\": \"Cell calcium\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal electrophysiology and imaging methods, single lab\",\n      \"pmids\": [\"21146870\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Numb1 isoform interacts with TRPV6 via an aspartate at residue 716 (TRPV6) and arginine at residue 434 (Numb1), as identified by co-immunoprecipitation, FRET, and C-terminal truncation mutagenesis. Numb1 negatively regulates TRPV6 activity: its expression decreases cytosolic Ca2+ in TRPV6-transfected cells, and a Numb1 mutant lacking the TRPV6-binding site fails to inhibit TRPV6.\",\n      \"method\": \"Co-immunoprecipitation, FRET, C-terminal truncation mutagenesis, Ca2+ measurements in HEK293 and cancer cells\",\n      \"journal\": \"Cell calcium\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — co-IP plus FRET plus mutagenesis defining binding residues, single lab\",\n      \"pmids\": [\"23140583\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TRPV6 autoinhibition is maintained by two intramolecular interactions: S4–S5 linker to C-terminal TRP helix (L/C; mediated by Arg470:Trp593) and N-terminal pre-S1 to TRP helix (N/C; mediated by Trp321:Ile597). Disruption of either interaction by mutations or blocking peptides activates TRPV6. The N/C interaction depends on L/C but not vice versa. PIP2 binds three cationic residues in S5/C-terminus, suppresses both interactions, and activates TRPV6.\",\n      \"method\": \"Site-directed mutagenesis, blocking peptides, patch-clamp electrophysiology, Ca2+ imaging\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis plus peptide competition plus electrophysiology defining intramolecular regulatory interactions, single lab\",\n      \"pmids\": [\"32829285\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In rat cauda epididymal principal cells, a constitutively active TRPV6-like Ca2+ current is coupled to a calcium-activated chloride conductance (TMEM16A). TRPV6 and TMEM16A proteins co-localize at the apical membrane. Removal of extracellular Ca2+ attenuates both currents; lanthanide block inhibits the TRPV6-like component first, then CaCC. In vivo perfusion showed substantial Ca2+ reabsorption from epididymal lumen suppressed by ruthenium red.\",\n      \"method\": \"Patch clamp in single epididymal cells, pharmacological inhibition, in vivo luminal perfusion, immunofluorescence colocalization, mRNA detection\",\n      \"journal\": \"The Journal of general physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — electrophysiology plus colocalization plus in vivo perfusion, single lab\",\n      \"pmids\": [\"27481714\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"In zebrafish, Trpv6 functions as a cell-autonomous regulator of epithelial quiescence: Trpv6-mediated constitutive Ca2+ influx activates PP2A, which suppresses IGF-mediated Akt-Tor and Erk signaling to maintain cellular quiescence. Genetic deletion or pharmacological blockade of Trpv6 caused epithelial cells to exit quiescence and re-enter the cell cycle; re-introduction of Trpv6 but not a channel-dead mutant restored quiescence.\",\n      \"method\": \"Zebrafish genetic deletion, pharmacological inhibition, cell cycle analysis, Ca2+ imaging, PP2A activity assays, IGF signaling readouts, human colon cancer cell experiments\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic and pharmacological loss-of-function with channel-dead rescue, pathway mechanistic follow-up, conserved in human cells, single lab\",\n      \"pmids\": [\"31526479\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TRPV6 is required for alcohol-induced intestinal barrier dysfunction: ethanol and acetaldehyde activate TRPV6 ionic currents in Caco-2 cells; TRPV6 deficiency (Trpv6-/- mice and organoids) attenuates ethanol/acetaldehyde-induced Ca2+ influx, tight junction disruption, and barrier dysfunction. Photoaffinity labeling of 3-azibutanol identified a histidine as a putative alcohol-binding site; substitution of this histidine (and a nearby arginine) reduces ethanol-activated TRPV6 currents.\",\n      \"method\": \"Patch clamp electrophysiology, Ca2+ imaging, Trpv6-/- mouse model, intestinal organoids, Caco-2 monolayers, photoaffinity labeling, site-directed mutagenesis\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — multiple orthogonal methods including genetic null mouse, mutagenesis of binding site, and organoid/cell systems; single lab\",\n      \"pmids\": [\"35705057\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TRPV6 promotes breast cancer metastasis via NFATC2IP phosphorylation at Ser204 (with CDK5 as candidate kinase), which activates NFATC2 transcription factor, leading to upregulation of ADAMTS6 and increased cell migration. TRPV6 overexpression accelerates migration; TRPV6 suppression decreases it.\",\n      \"method\": \"Overexpression and siRNA knockdown, phosphorylation analysis, reporter assays, migration assays in breast cancer cells\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, indirect mechanistic pathway, CDK5 as kinase is proposed but not definitively established\",\n      \"pmids\": [\"34265397\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TRPV6 suppresses osteoclastogenesis via the IGF-PI3K-AKT signaling pathway: TRPV6 knockout mice develop osteoporosis with enhanced osteoclast differentiation and bone resorption. TRPV6 located on the osteoclast cell membrane decreases p-IGF/total-IGF, p-PI3K/total-PI3K, and p-AKT/total-AKT ratios. Blocking IGF-PI3K-AKT with inhibitors relieved the inhibitory effect of TRPV6 on osteoclasts.\",\n      \"method\": \"Trpv6 knockout mice, lentiviral overexpression/silencing in osteoclasts, micro-CT, TRAP staining, pit formation assay, Western blot for pathway components\",\n      \"journal\": \"Cell proliferation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO plus pathway inhibitor rescue plus overexpression/silencing in vitro, single lab\",\n      \"pmids\": [\"33159483\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TRPV6 is a highly Ca2+-selective tetrameric ion channel that mediates constitutive and regulated Ca2+ entry at the apical membrane of epithelia; its Ca2+ selectivity is determined by a ring of aspartate residues in the selectivity filter, channel opening involves an α-to-π helical transition in S6 at an alanine hinge (iris-like gating), and channel activity is directly activated by PIP2 and inhibited by autoinhibitory intramolecular interactions (S4-S5 linker–TRP helix and pre-S1–TRP helix contacts); it is positively regulated by calmodulin (Ca2+-dependent), CypB (prolyl-isomerase-dependent), Rab11a-mediated trafficking, WNK3, SGK1/Akt, and klotho-mediated deglycosylation, and negatively regulated by PLC-driven PIP2 depletion (Ca2+-induced inactivation), RGS2 (direct gating inhibition), Nedd4-2-mediated ubiquitination, and Nipsnap1; its obligate plasma membrane localization requires the S100A10–annexin 2 complex binding to the C-terminal VATTV motif; the channel is physiologically essential for intestinal Ca2+ absorption, renal/epididymal Ca2+ reabsorption, maternal-fetal Ca2+ transport via the placenta (critical for fetal bone mineralization), and maintenance of low luminal Ca2+ in the epididymis for male fertility, and it also controls keratinocyte differentiation and epithelial cell quiescence via Ca2+-dependent suppression of IGF/Akt/PP2A signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TRPV6 is a highly Ca2+-selective cation channel that mediates apical Ca2+ entry into epithelia and is physiologically essential for transepithelial Ca2+ transport [#18, #21]. It assembles as a homotetramer (and can form heterotetramers with TRPV5) with an intracellular skirt for allosteric modulation, and its Ca2+ selectivity is set by a ring of aspartate side chains in the selectivity filter, with the pore residue D541/D542 essential for permeation [#0, #3, #22]. Channel opening proceeds through an α-to-π helical transition in the pore-lining S6 helix at an alanine hinge, producing an iris-like gating motion [#4]. The basal state is autoinhibited by two intramolecular contacts—an S4–S5 linker to TRP-helix interaction and a pre-S1 to TRP-helix interaction—both of which are relieved by direct binding of PIP2, the principal activating lipid demonstrated using purified channel reconstituted in lipid bilayers [#11, #33]; conversely, Ca2+ influx drives phospholipase C-mediated PIP2 depletion that underlies Ca2+-induced inactivation [#10]. Activity is further tuned by Ca2+-dependent calmodulin binding and by direct gating inhibitors RGS2, Nipsnap1, and Numb1, while small molecules such as 2-APB close the channel through a defined S1–S4 pocket [#2, #5, #9, #17, #32]. Surface delivery and stability are governed by the S100A10–annexin 2 complex binding the C-terminal VATTV motif (required for plasma-membrane targeting), GDP-bound Rab11a-mediated trafficking, kinase inputs (WNK3, SGK1/Akt), klotho/β-glucuronidase-mediated deglycosylation, and Nedd4-2-mediated ubiquitination and degradation [#1, #7, #8, #14, #25]. In vivo, functional TRPV6 channels are required for intestinal Ca2+ absorption, maternal-fetal placental Ca2+ transport supporting fetal bone mineralization, and epididymal Ca2+ reabsorption essential for male fertility, with basolateral CaSR signaling negatively regulating intestinal TRPV6 via PLC [#18, #20, #21, #30]. Beyond bulk Ca2+ handling, TRPV6-mediated Ca2+ entry controls keratinocyte differentiation and, through Ca2+-dependent activation of PP2A, suppresses IGF/Akt signaling to maintain epithelial cell quiescence [#28, #35].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Establishing the channel's quaternary architecture was needed to interpret all later functional and structural work; TRPV6 was shown to be a tetramer that can mix with TRPV5 to create functionally distinct channels.\",\n      \"evidence\": \"Sucrose gradient sedimentation, co-IP, and concatemeric channel electrophysiology in HEK293 cells\",\n      \"pmids\": [\"12574114\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Native epithelial stoichiometry of homo- versus heterotetramers not quantified\", \"No atomic structure at this stage\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"How TRPV6 reaches the plasma membrane was unknown; the S100A10–annexin 2 complex was identified as binding the C-terminal VATTV motif and being required for surface targeting and channel activity.\",\n      \"evidence\": \"Yeast two-hybrid, GST pull-down, co-IP, T600A mutagenesis, annexin 2 siRNA, and electrophysiology in HEK293 cells\",\n      \"pmids\": [\"12660155\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Step in the trafficking itinerary controlled by the complex not resolved\", \"Regulation of complex assembly addressed only later\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"The basis of Ca2+-dependent feedback regulation was addressed by showing calmodulin binds TRPV6 in a Ca2+-dependent manner at defined N- and C-terminal motifs and positively modulates current.\",\n      \"evidence\": \"GST pull-down, co-IP, whole-cell patch clamp, and chimeric channel analysis in HEK293 cells\",\n      \"pmids\": [\"15123711\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural coupling of CaM binding to gating not defined\", \"Relationship to PIP2-dependent inactivation not integrated\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Whether TRPV6 is constitutively open was tested; under physiological Ca2+ buffering it is not, and divalent block plus the pore aspartate D542 govern permeation behavior.\",\n      \"evidence\": \"Whole-cell/perforated patch clamp, Fura-2, and pore/truncation mutagenesis in HEK293 cells\",\n      \"pmids\": [\"15184369\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular trigger for physiological opening in epithelia not defined here\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Mechanisms of surface regulation and direct gating control were expanded by identifying GDP-Rab11a as a trafficking partner and RGS2 as a direct, trafficking-independent gating inhibitor, with NHERF4 as an additional C-terminal PDZ interactor.\",\n      \"evidence\": \"Co-IP, confocal colocalization, Ca2+ uptake/surface biotinylation, yeast two-hybrid, and GST pull-down in epithelial cells and oocytes\",\n      \"pmids\": [\"16354700\", \"16895908\", \"16565876\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of NHERF4 binding not established\", \"How RGS2 alters gating structurally unknown\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Multiple inputs converging on TRPV6 were defined: cAMP/PKA/calcineurin control of the annexin 2–S100A10 association, CypB isomerase-dependent activation, klotho/β-glucuronidase deglycosylation activation, and Nipsnap1 as a direct gating inhibitor.\",\n      \"evidence\": \"Co-IP, 45Ca uptake, oocyte Ca2+ assays, enzymatic deglycosylation, GST pull-down, and biotinylation across HEK293, Caco-2, oocyte and placental systems\",\n      \"pmids\": [\"18187190\", \"18445599\", \"18495742\", \"18392847\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Several findings rest on single-lab functional assays\", \"Direct enzymatic action of klotho on the channel inferred from specificity not structurally shown\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"The physiological necessity of TRPV6 was established in vivo, showing it mediates maternal-fetal placental Ca2+ transport critical for fetal bone mineralization.\",\n      \"evidence\": \"Trpv6 knockout mice with 45Ca transport assays, immunohistochemistry, and qRT-PCR\",\n      \"pmids\": [\"18348695\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of other Ca2+ pathways in placenta not quantified\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"The lipid and metabolic regulation of TRPV6 was clarified by showing Ca2+ influx activates PLC to deplete PIP2, driving Ca2+-induced inactivation.\",\n      \"evidence\": \"Whole-cell and excised patch clamp with rapamycin-inducible phosphatase and Ca2+ imaging\",\n      \"pmids\": [\"18390907\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the PLC isoform in native epithelia not defined\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Kinase and nucleotide inputs were defined: WNK3 and SGK1/Akt increase activity and surface abundance, while ATP binding to the ARD/C-terminus prevents rundown and PKCβII phosphorylation opposes it.\",\n      \"evidence\": \"Oocyte co-expression, Ca2+/current assays, surface biotinylation, ATP-binding studies, and mutagenesis\",\n      \"pmids\": [\"18768590\", \"19805577\", \"20041238\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"SGK1/PIKfyve effect relies on indirect Cl- current readout (Low confidence)\", \"Direct phosphosites on TRPV6 versus partners not fully resolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"The structural basis of the regulatory cytoplasmic domain was probed by solving the ankyrin repeat domain, showing it does not bind ATP or CaM unlike TRPV1, implying a distinct regulatory role.\",\n      \"evidence\": \"X-ray crystallography at 1.7 Å with ATP- and CaM-agarose pull-downs and SEC\",\n      \"pmids\": [\"18232717\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional role of the variable third-finger loop and its phosphosites not tested in vivo\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Negative protein-stability control was defined by showing Nedd4-2 ubiquitinates TRPV6 and reduces its abundance, predominantly via the proteasome.\",\n      \"evidence\": \"Oocyte co-expression, Ca2+ influx, ubiquitination assays, proteasome/lysosome inhibitors, and HECT/WW domain mutagenesis\",\n      \"pmids\": [\"20843805\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological context regulating Nedd4-2 action on TRPV6 not established\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"PIP2 was demonstrated to be a direct activator using purified, reconstituted channel, decoupling lipid activation from cellular machinery and explaining ATP-dependent recovery via PIP2 resynthesis.\",\n      \"evidence\": \"Planar lipid bilayer reconstitution of purified TRPV6 plus excised inside-out patch clamp and PI4K inhibition\",\n      \"pmids\": [\"21810903\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Lipid-binding site not resolved structurally until later mutational work\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"In vivo significance for fertility was established by showing a pore-dead D541A channel impairs epididymal Ca2+ absorption and male fertility, localizing TRPV6 to apical epididymal epithelium.\",\n      \"evidence\": \"D541A knock-in mice with luminal Ca2+ measurements, absorption assays, immunolocalization, and sperm assays\",\n      \"pmids\": [\"21540454\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream coupling to sperm physiology mechanistic detail limited\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"A receptor-driven mode of TRPV6 activation was identified in prostate cells, with PSGR activating TRPV6 through Src kinase independent of G-protein signaling.\",\n      \"evidence\": \"Electrophysiology, Ca2+ imaging, TRPV6 siRNA, co-IP, and Src inhibition\",\n      \"pmids\": [\"21349844\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct Src target residues on TRPV6 not mapped\", \"Single-lab cancer-cell context\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Genetic null mice confirmed that epididymal and fertility phenotypes arise from loss of channel function rather than channel-independent effects of the pore mutant, and established intestinal Ca2+ absorption requires functional channels; an extended non-AUG-initiated N-terminus enhances trafficking; Numb1 was identified as a direct inhibitor.\",\n      \"evidence\": \"Trpv6 knockout and D541A knock-in mice, 45Ca absorption assays, mass spectrometry of placental protein, and co-IP/FRET/mutagenesis\",\n      \"pmids\": [\"22427671\", \"22878123\", \"23612980\", \"23140583\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tissue distribution and regulation of the extended isoform not fully mapped\", \"Numb1 inhibition mechanism on gating not structurally resolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Atomic structure of TRPV6 revealed the tetrameric assembly, the aspartate ring selectivity filter, and a multi-site Ca2+ permeation pathway, providing the framework for all mechanistic interpretation.\",\n      \"evidence\": \"X-ray crystallography of rat TRPV6 at 3.25 Å\",\n      \"pmids\": [\"27296226\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Single conformational state; gating transition not yet captured at this stage\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"The native epithelial output of TRPV6 Ca2+ entry was linked to a calcium-activated chloride conductance (TMEM16A) coupling in epididymal principal cells, connecting Ca2+ entry to downstream transport.\",\n      \"evidence\": \"Single-cell patch clamp, pharmacology, in vivo luminal perfusion, and immunofluorescence colocalization\",\n      \"pmids\": [\"27481714\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct physical coupling between TRPV6 and TMEM16A not demonstrated\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Open and closed cryo-EM structures defined the gating mechanism as an α-to-π S6 transition at an alanine hinge with iris-like opening, and confirmed PIP2 increases open probability.\",\n      \"evidence\": \"Cryo-EM of human TRPV6 in two states plus electrophysiology\",\n      \"pmids\": [\"29258289\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"PIP2 binding pose not resolved in these maps\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"The pharmacological inhibitor 2-APB was localized to a defined S1–S4 cytoplasmic pocket, showing closure occurs by modulating protein–lipid interactions.\",\n      \"evidence\": \"Crystal/cryo-EM of human and rat TRPV6 with 2-APB plus Y467A mutagenesis and functional assays\",\n      \"pmids\": [\"29941865\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Selectivity of this pocket relative to other TRPV channels not addressed\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Two regulatory questions were answered: basolateral CaSR signaling restrains intestinal TRPV6 Ca2+ absorption via PLC, and TRPV6-mediated constitutive Ca2+ influx enforces epithelial quiescence by activating PP2A to suppress IGF-Akt/Erk signaling.\",\n      \"evidence\": \"Ussing chambers with D541A mice and oocyte co-expression; zebrafish genetic deletion with channel-dead rescue, PP2A assays, and human colon cancer cells\",\n      \"pmids\": [\"31013259\", \"31526479\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct CaSR-PLC-TRPV6 molecular coupling not resolved\", \"Quiescence pathway demonstrated in single-lab models\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"The autoinhibitory architecture of resting TRPV6 was defined as two intramolecular TRP-helix interactions relieved by PIP2 binding at S5/C-terminal cationic residues, unifying lipid activation with gating control.\",\n      \"evidence\": \"Site-directed mutagenesis, blocking peptides, patch clamp, and Ca2+ imaging\",\n      \"pmids\": [\"32829285\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Interactions inferred functionally without a corresponding apo/PIP2-bound structure in this study\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"A skeletal role beyond Ca2+ absorption was proposed, with TRPV6 suppressing osteoclastogenesis through IGF-PI3K-AKT signaling, paralleling its quiescence-promoting role.\",\n      \"evidence\": \"Trpv6 knockout mice, micro-CT, TRAP/pit assays, lentiviral manipulation, and pathway inhibitor rescue in osteoclasts\",\n      \"pmids\": [\"33159483\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cell-autonomous versus systemic Ca2+ contributions not fully separated\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"A pathological gating modality was identified: ethanol and acetaldehyde directly activate TRPV6 via a histidine-containing site, driving Ca2+ influx that disrupts intestinal tight junctions.\",\n      \"evidence\": \"Patch clamp, Ca2+ imaging, Trpv6-/- mice and organoids, Caco-2 monolayers, photoaffinity labeling, and mutagenesis\",\n      \"pmids\": [\"35705057\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural definition of the alcohol-binding pocket not resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the diverse regulatory inputs (CaM, PIP2 autoinhibition, kinases, trafficking partners, ubiquitination) are integrated dynamically within a single epithelial cell to set TRPV6 set-point in vivo remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified quantitative model linking lipid, Ca2+, and post-translational regulation\", \"Structural states bound to physiological protein regulators (CaM, RGS2, S100A10) not solved\", \"Native channel composition and regulation across tissues incompletely mapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [0, 3, 22, 31]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [10, 11, 33]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [4, 22, 30]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1, 7, 18, 34]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [18, 20, 21, 30]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [10, 33, 35]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [1, 7, 25]}\n    ],\n    \"complexes\": [\n      \"TRPV6 homotetramer\",\n      \"TRPV5/TRPV6 heterotetramer\",\n      \"S100A10-annexin 2 complex\"\n    ],\n    \"partners\": [\n      \"TRPV5\",\n      \"S100A10\",\n      \"ANXA2\",\n      \"CALM1\",\n      \"RAB11A\",\n      \"RGS2\",\n      \"NEDD4L\",\n      \"NUMB\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}