{"gene":"TRPV5","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2005,"finding":"Klotho, acting as a beta-glucuronidase, hydrolyzes extracellular N-linked oligosaccharides on TRPV5, entrapping the channel in the plasma membrane and sustaining calcium channel activity at the cell surface.","method":"Biochemical enzymatic assay, Ca2+ influx measurements, cell surface expression studies in HEK293 cells","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct enzymatic activity demonstrated in vitro with functional readout; replicated by multiple subsequent studies confirming the same mechanism","pmids":["16239475"],"is_preprint":false},{"year":2008,"finding":"Klotho removes terminal alpha2,6-linked sialic acids from TRPV5 N-glycan chains, exposing galactose-N-acetylglucosamine that binds galectin-1, which forms a lattice retaining TRPV5 at the plasma membrane. Knockdown of ST6Gal-1 prevents this regulation.","method":"RNAi knockdown of ST6Gal-1, glycan analysis, Ca2+ influx measurements, co-expression in cell lines lacking endogenous ST6Gal-1 with rescue by recombinant ST6Gal-1","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal methods (RNAi, cell-line complementation, glycan biochemistry), single lab but rigorous mechanistic dissection","pmids":["18606998"],"is_preprint":false},{"year":2003,"finding":"TRPV5-knockout mice exhibit severely diminished active Ca2+ reabsorption in the early distal convoluted tubule (confirmed by in vivo micropuncture), severe hypercalciuria despite elevated vitamin D, compensatory intestinal Ca2+ hyperabsorption, and reduced trabecular and cortical bone thickness, establishing TRPV5 as the essential luminal entry gate for active Ca2+ reabsorption in the kidney.","method":"Germline knockout mouse model, in vivo micropuncture, urinary and serum Ca2+ measurements, bone histomorphometry","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — knockout with multiple orthogonal phenotypic readouts including direct in vivo micropuncture; widely replicated","pmids":["14679186"],"is_preprint":false},{"year":2003,"finding":"TRPV5 and TRPV6 assemble as homotetramers (400 kDa complexes) and can form heterotetramers; different heterotetrameric compositions produce Ca2+ channels with distinct Ca2+-dependent inactivation, Ba2+ selectivity, and pharmacological properties.","method":"Sucrose gradient sedimentation, immunoprecipitation, electrophysiology of concatemeric channels with pore mutants in HEK293 cells","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstitution with concatemeric constructs plus electrophysiology and biochemical sedimentation; multiple orthogonal methods","pmids":["12574114"],"is_preprint":false},{"year":2003,"finding":"S100A10 directly binds the conserved C-terminal VATTV sequence of TRPV5 (critical residue T599) and forms a heterotetrameric complex with annexin 2; this S100A10-annexin 2 complex is required to route TRPV5 to the plasma membrane. Annexin 2 siRNA knockdown abolishes TRPV5-mediated currents.","method":"Yeast two-hybrid, GST pulldown, co-immunoprecipitation, site-directed mutagenesis, siRNA knockdown, electrophysiology","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — yeast two-hybrid confirmed by GST pulldown and co-IP; functional mutagenesis and siRNA rescue with electrophysiological readout; multiple orthogonal methods","pmids":["12660155"],"is_preprint":false},{"year":2014,"finding":"FGF23 promotes renal Ca2+ reabsorption and apical membrane abundance of TRPV5 in distal tubules through binding the FGF receptor-αKlotho complex and activating an ERK1/2-SGK1-WNK4 signaling cascade, not through αKlotho's glycosidase activity.","method":"Fgf23 and αKlotho knockout mice, immunolocalization, signaling pathway inhibition, renal Ca2+ balance measurements","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockouts of FGF23 and αKlotho with pathway analysis; multiple orthogonal approaches; replicated in knockout models","pmids":["24434184"],"is_preprint":false},{"year":2018,"finding":"Cryo-EM structures of TRPV5 with PI(4,5)P2 reveal a binding site between the N-linker, S4-S5 linker and S6 helix; PI(4,5)P2 binding induces conformational rearrangements in the lower gate that open the channel. Cryo-EM with calmodulin shows two TRPV5 C-terminal peptides anchoring one CaM molecule, with Lys116 on CaM C-lobe forming a cation-π interaction with Trp583 at the intracellular gate to mediate Ca2+-dependent inhibition.","method":"Cryo-electron microscopy, molecular dynamics simulations","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct cryo-EM structures with multiple modulators, plus MD simulations; atomic resolution mechanistic insight","pmids":["30305626"],"is_preprint":false},{"year":2018,"finding":"Cryo-EM structure of full-length rabbit TRPV5 in complex with econazole reveals the inhibitor occupies a hydrophobic pocket analogous to the phosphatidylinositide/vanilloid-binding site in TRPV1; the econazole-bound structure adopts a closed conformation with a distinct lower gate that occludes Ca2+ permeation.","method":"Cryo-electron microscopy, molecular dynamics simulations","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution cryo-EM structure with functional corroboration from MD simulations; landmark structural study","pmids":["29323279"],"is_preprint":false},{"year":2009,"finding":"PTH activates TRPV5 via the cAMP-PKA pathway by phosphorylating threonine-709 on TRPV5, increasing channel open probability without altering surface expression. Alanine substitution of T709 abolishes both in vitro phosphorylation and PTH-mediated TRPV5 stimulation.","method":"FRET-based cAMP and Ca2+ dynamics, cell-surface biotinylation, PKA catalytic subunit application, site-directed mutagenesis, patch-clamp electrophysiology","journal":"Journal of the American Society of Nephrology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro phosphorylation with mutagenesis, electrophysiology, and FRET; multiple orthogonal methods in single study","pmids":["19423690"],"is_preprint":false},{"year":2005,"finding":"PIP2 activates TRPV5 by a mechanism independent of Mg2+ binding to the selectivity filter and reduces TRPV5 sensitivity to Mg2+-induced slow conformational channel closure. Mutation of aspartate-542 (critical Mg2+-binding site in selectivity filter) abolishes Mg2+-induced slow inhibition; PLC activation sensitizes TRPV5 to this Mg2+-induced slow inhibition by hydrolyzing PIP2.","method":"Whole-cell patch-clamp, site-directed mutagenesis, intracellular perfusion with PIP2 or receptor-mediated PLC activation","journal":"The Journal of general physiology","confidence":"High","confidence_rationale":"Tier 1 / Strong — electrophysiology combined with mutagenesis of selectivity filter and biochemical modulation; two orthogonal approaches","pmids":["16230466"],"is_preprint":false},{"year":2005,"finding":"TRPV5 is localized to the ruffled border membrane of osteoclasts; TRPV5 knockout osteoclasts show increased number and size but nearly absent bone resorption in pit assays, establishing TRPV5-mediated Ca2+ transport as essential for osteoclastic bone resorption.","method":"Immunostaining, TRPV5 knockout mouse analysis, in vitro bone marrow osteoclast cultures, resorption pit assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with multiple phenotypic readouts (immunolocalization, pit assay, in vivo bone markers); well-controlled","pmids":["16291808"],"is_preprint":false},{"year":2006,"finding":"Calbindin-D28K translocates to the plasma membrane at low intracellular Ca2+ and directly associates with TRPV5, where it buffers Ca2+ entering through the channel in close proximity to the pore, preventing Ca2+-dependent channel inactivation and facilitating high transcellular Ca2+ transport rates.","method":"Protein-binding analysis, subcellular fractionation, evanescent-field (TIRF) microscopy, 45Ca2+ uptake, electrophysiology, transcellular Ca2+ transport assays in primary tubule cells infected with lentivirus","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct binding demonstrated with functional electrophysiology in primary cells; multiple orthogonal methods","pmids":["16763551"],"is_preprint":false},{"year":2008,"finding":"TRPV5 undergoes constitutive caveolae-mediated (caveolin-1-dependent, clathrin-independent) endocytosis. PKC activators increase TRPV5 cell-surface abundance by inhibiting this endocytosis via phosphorylation of Ser-299 and Ser-654 on TRPV5. PTH also increases TRPV5 surface abundance through this PKC-caveolin pathway.","method":"Dominant-negative dynamin, siRNA knockdown of caveolin-1 and clathrin, caveolin-1 knockout cells with rescue, site-directed mutagenesis, whole-cell patch-clamp, cell-surface biotinylation","journal":"American journal of physiology. Renal physiology","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis + KO cell rescue + siRNA + dominant-negative approaches; multiple orthogonal methods","pmids":["18305097"],"is_preprint":false},{"year":2006,"finding":"Tissue kallikrein (TK) stimulates Ca2+ reabsorption via bradykinin receptor type 2 activation of the PLC/DAG/PKC pathway, which phosphorylates TRPV5 at Ser-299 and Ser-654, increasing TRPV5 plasma membrane abundance by delaying its retrieval.","method":"Primary renal epithelial cell cultures, PKC pharmacology, BK receptor antagonism, cell-surface labeling, mutagenesis of PKC sites (S299A, S654A)","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — pharmacological pathway dissection combined with site-directed mutagenesis of PKC phosphorylation sites; multiple methods","pmids":["17006539"],"is_preprint":false},{"year":2004,"finding":"The intracellular N-tail (residues 64–77) and C-tail (residues 596–601) of TRPV5 mediate homotetrameric assembly via N-N, C-C, and N-C interactions; deletion of either tail alone causes dominant-negative suppression by preventing plasma membrane trafficking of the channel complex.","method":"GST pulldown, co-immunoprecipitation, site-directed mutagenesis, patch-clamp, 45Ca2+ uptake in oocytes","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct biochemical binding assays with mutagenesis and functional electrophysiology; multiple orthogonal methods","pmids":["15489237"],"is_preprint":false},{"year":2003,"finding":"Extracellular acidification inhibits TRPV5 with pKa ~6.55; glutamate-522 in the extracellular loop between TM5 and TM6 is the extracellular pH sensor. E522Q mutation decreases pH sensitivity and abolishes proton-mediated reduction of open probability without altering single-channel conductance block component.","method":"Whole-cell and single-channel patch-clamp, site-directed mutagenesis, methanethiosulfonate (MTS) cysteine accessibility","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis combined with single-channel analysis and cysteine accessibility method; multiple orthogonal approaches","pmids":["14525991"],"is_preprint":false},{"year":2005,"finding":"Intracellular acidification causes clockwise rotation of the TRPV5 pore helix (detected by substituted cysteine accessibility method), which facilitates closure of the selectivity filter gate by extracellular protons; internal and external pH sensors cross-regulate each other via this pore helix conformational change.","method":"Substituted cysteine accessibility method (SCAM), whole-cell patch-clamp, site-directed mutagenesis","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — SCAM with mutagenesis and electrophysiology; rigorous structural-functional analysis","pmids":["16121193"],"is_preprint":false},{"year":2011,"finding":"Calmodulin binds Ca2+-dependently to a C-terminal fragment (residues 696–729) of TRPV5 in a 1 CaM:2 TRPV5 C-terminus stoichiometry; TRPV5 mutations W702A and R706E abolish CaM binding and strongly reduce Ca2+-dependent channel inactivation. PTH-induced PKA phosphorylation of T709 diminishes CaM binding to TRPV5, thereby enhancing channel open probability.","method":"NMR spectroscopy, site-directed mutagenesis, patch-clamp electrophysiology","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution NMR structural data combined with mutagenesis and functional electrophysiology; multiple orthogonal methods in single study","pmids":["21576356"],"is_preprint":false},{"year":2007,"finding":"TRPV5 is constitutively internalized via dynamin- and clathrin-dependent endocytosis; internalized channels traffic to Rab11a-positive perinuclear recycling endosomes and can return to the cell surface. The recycling kinetics are decreased by intracellular Ca2+ chelation (BAPTA-AM), indicating Ca2+-controlled recycling.","method":"Dynamin dominant-negative, clathrin siRNA, Rab11a co-localization, brefeldin A block, fluorescence microscopy, electrophysiology","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — dominant-negative and siRNA approaches with multiple trafficking markers; well-controlled mechanistic study","pmids":["18077461"],"is_preprint":false},{"year":2009,"finding":"Activation of the Ca2+-sensing receptor (CaR) co-localized with TRPV5 at the luminal membrane of distal tubule stimulates TRPV5-mediated Ca2+ influx via PMA-insensitive PKC isoforms acting on Ser-299 and Ser-654 of TRPV5; dominant-negative CaR(R185Q) and S299A/S654A TRPV5 mutations abolish this effect.","method":"Patch-clamp electrophysiology, Fura-2 Ca2+ imaging, site-directed mutagenesis, dominant-negative CaR construct","journal":"Cell calcium","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — electrophysiology with mutagenesis, single lab, two orthogonal methods","pmids":["19157541"],"is_preprint":false},{"year":2004,"finding":"80K-H directly interacts with TRPV5, co-localizes in kidney, and acts as a Ca2+ sensor that modulates TRPV5 activity via its EF-hand structures, glutamic stretch, and HDEL sequence; inactivation of the EF-hands reduces TRPV5-mediated Ca2+ current and increases TRPV5 sensitivity to intracellular Ca2+, accelerating feedback inhibition.","method":"cDNA microarray identification, co-immunoprecipitation, 45Ca2+ binding assays, site-directed mutagenesis of EF-hands, patch-clamp electrophysiology","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP with mutagenesis and electrophysiology; single lab with two orthogonal approaches","pmids":["15100231"],"is_preprint":false},{"year":2006,"finding":"Extracellular alkalinization rapidly recruits a pool of TRPV5-containing vesicles to the cell surface via a 'kiss and linger' vesicular mechanism without full membrane fusion, increasing functional TRPV5 channel activity; extracellular acidification reverses this, retrieving vesicles from the surface.","method":"Total internal reflection fluorescence (TIRF) microscopy, cell surface protein labeling, electrophysiology, 45Ca2+ uptake, functional channel recovery after chemobleaching","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — TIRF with multiple complementary methods in single lab","pmids":["17178838"],"is_preprint":false},{"year":2008,"finding":"Klotho (beta-glucuronidase) selectively activates TRPV5 and TRPV6 but not TRPV4 or TRPM6 among renal ion channels, indicating channel-type specificity of the glycan-hydrolysis mechanism.","method":"Ca2+ influx measurements in HEK293 cells expressing different renal channels, endoglycosidase-F deglycosylation control","journal":"Nephrology, dialysis, transplantation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic screen with appropriate negative controls; single lab, single method type","pmids":["18495742"],"is_preprint":false},{"year":2014,"finding":"NDPK-B (histidine kinase) activates TRPV5 channel activity and Ca2+ flux through phosphorylation of histidine-711 in the TRPV5 C-terminal tail; the histidine phosphatase PHPT1 reverses this activation. NDPK-B knockdown decreases TRPV5 activity, and NDPK-B knockout mice show increased urinary Ca2+ excretion on high-Ca2+ diet.","method":"Inside-out patch-clamp, NDPK-B shRNA knockdown, NDPK-B knockout mice urinary Ca2+ measurements, site-directed mutagenesis of H711","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro patch phosphorylation with phosphatase reversal, mutagenesis of target residue, and in vivo knockout phenotype; multiple orthogonal methods","pmids":["24523290"],"is_preprint":false},{"year":2013,"finding":"Uromodulin (UMOD) upregulates TRPV5 cell-surface abundance by acting extracellularly to impair caveolin-1-mediated endocytosis; UMOD has no effect in caveolin-1-null cells or with N-glycan-deficient TRPV5; disease mutant UMOD with reduced secretion fails to increase TRPV5 activity. Immunofluorescence shows reduced TRPV5 expression in Umod-/- mouse kidneys.","method":"Whole-cell patch-clamp, cell-surface biotinylation, siRNA knockdown, caveolin-1 knockout cells with rescue, immunofluorescence in Umod-/- mice","journal":"Kidney international","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO cells, rescue, siRNA, and in vivo confirmation; multiple orthogonal methods","pmids":["23466996"],"is_preprint":false},{"year":2005,"finding":"FKBP52 physically interacts specifically with TRPV5 and co-localizes in the distal nephron; it inhibits TRPV5-mediated Ca2+ influx through its peptidyl-prolyl cis-trans isomerase (PPIase) domain, as PPIase domain mutation abolishes the inhibitory effect.","method":"Co-immunoprecipitation, 45Ca2+ uptake, patch-clamp electrophysiology, siRNA knockdown, FK-506 pharmacology, PPIase domain mutagenesis","journal":"American journal of physiology. Renal physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP with functional mutagenesis and electrophysiology; single lab, two orthogonal methods","pmids":["16352746"],"is_preprint":false},{"year":2006,"finding":"BSPRY interacts with TRPV5 (confirmed by GST pulldown and co-IP), co-localizes in mouse kidney, and reduces TRPV5-mediated Ca2+ influx without altering channel surface abundance, indicating direct modulation of channel activity.","method":"GST pulldown, co-immunoprecipitation, 45Ca2+ uptake in MDCK-TRPV5 cells, immunostaining","journal":"Journal of the American Society of Nephrology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal binding assays with functional Ca2+ uptake readout; single lab, two orthogonal methods","pmids":["16380433"],"is_preprint":false},{"year":2008,"finding":"WNK4 decreases TRPV5 cell-surface abundance by stimulating caveolae-mediated endocytosis via a region outside its kinase domain; this WNK4-dependent tonic inhibition lowers basal TRPV5 levels and thereby amplifies the dynamic range of PKC- and PTH-mediated stimulation of TRPV5.","method":"Deletion analysis of WNK4 domains, patch-clamp electrophysiology, cell-surface biotinylation, PKC activator co-stimulation experiments","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain deletion analysis with electrophysiology; single lab, two orthogonal approaches","pmids":["20061383"],"is_preprint":false},{"year":2008,"finding":"WNK3 positively regulates TRPV5 plasma membrane expression via a kinase-dependent mechanism (kinase-inactive D294A mutation abolishes effect); WNK3 promotes complex glycosylation and exocytosis of TRPV5 via microtubule-dependent secretory pathway, as colchicine blocks this effect.","method":"Xenopus oocyte 45Ca2+ uptake, voltage-clamp electrophysiology, kinase-inactive mutagenesis, colchicine treatment, glycosylation analysis","journal":"American journal of physiology. Renal physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — kinase-dead mutagenesis with functional Ca2+ uptake and electrophysiology; single lab, multiple methods","pmids":["18768590"],"is_preprint":false},{"year":2004,"finding":"SGK1 and SGK3 (but not SGK2) together with scaffold protein NHERF2 stimulate TRPV5-mediated Ca2+ entry in Xenopus oocytes; the second PDZ domain of NHERF2 is required for stabilization/targeting of TRPV5 to the plasma membrane, and TRPV5 C-tail interacts with NHERF2 in a Ca2+-independent manner.","method":"Xenopus oocyte 45Ca2+ uptake, voltage-clamp, NHERF2 PDZ deletion mutants, GST pulldown, overlay assays, chemiluminescence surface quantification","journal":"Cellular physiology and biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding assays with functional electrophysiology and domain deletion analysis; single lab","pmids":["15319523","15665527"],"is_preprint":false},{"year":2006,"finding":"NHERF4 (PDZK2) interacts with the C-terminus of TRPV5 through its fourth PDZ domain (PDZ1 also contributes); confirmed by yeast two-hybrid, GST pulldown with in vitro translated NHERF4 and oocyte lysates, and co-immunoprecipitation in HEK293 cells.","method":"Yeast two-hybrid, GST pulldown, co-immunoprecipitation, PDZ domain deletion analysis","journal":"Pflugers Archiv","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — binding interaction confirmed by multiple methods but no functional consequence demonstrated for this specific interaction","pmids":["16565876"],"is_preprint":false},{"year":2002,"finding":"The C-terminal tail of TRPV5 (ECaC1) mediates Ca2+-dependent channel inactivation; deletion of the last 30 amino acids (G701X) and truncations around residues 649-653 significantly reduce or abolish Ca2+-dependent inactivation, identifying two critical C-terminal domains.","method":"Carboxyl-terminal truncation mutagenesis, patch-clamp electrophysiology in HEK293 cells","journal":"Pflugers Archiv","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro mutagenesis with electrophysiology; single lab, single method type","pmids":["12634930"],"is_preprint":false},{"year":2019,"finding":"High-resolution cryo-EM structures of full-length TRPV5 in lipid nanodiscs, a TRPV5 W583A mutant, and TRPV5 in complex with CaM reveal a flexible stoichiometry of CaM binding and the mechanism of calcium-dependent regulation; W583 at the intracellular gate is critical for CaM-mediated inactivation.","method":"Cryo-electron microscopy, lipid nanodisc reconstitution, mutagenesis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple cryo-EM structures with mutagenesis validation; independent replication of CaM-TRPV5 structural interaction","pmids":["30975749"],"is_preprint":false},{"year":2022,"finding":"Cryo-EM shows that low pH inhibits TRPV5 by precluding PI(4,5)P2 binding/activation; intermediate conformations at low pH reveal transition from open to closed state. PI(4,5)P2 is the primary modulator of channel gating; PKA controls TRPV5 by preventing CaM binding and channel inactivation rather than directly opening the gate.","method":"Cryo-electron microscopy, structural analysis of multiple pH and modulator conditions","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple cryo-EM structures capturing different conformational states with two distinct modulators; rigorous structural mechanistic study","pmids":["35476976"],"is_preprint":false},{"year":2017,"finding":"Tryptophan-583 at the terminus of the intracellular pore forms a gate for Ca2+ permeation through TRPV5; W583 mutants show profoundly enhanced Ca2+ influx and increased cell death; a glycine residue above W583 may act as a flexible hinge to rearrange the tryptophan gate.","method":"Site-directed mutagenesis, patch-clamp electrophysiology, biochemical analysis, homology modeling","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — mutagenesis with electrophysiology; single lab, homology model (not direct structure)","pmids":["28374795"],"is_preprint":false},{"year":2013,"finding":"Inflammatory cytokines (TNF-α, IFN-γ, IL-1β) induce TRPV5 interaction with E3 ubiquitin ligase UBR4, leading to ubiquitin-dependent TRPV5 degradation; UBR4 siRNA prevents cytokine-induced TRPV5 degradation. Klotho protects TRPV5 from hypersialylation and cytokine-induced endocytosis and degradation in colitis.","method":"Co-immunoprecipitation (TRPV5-UBR4), siRNA knockdown of UBR4, adenoviral TRPV5 transduction, transgenic Klotho overexpression mice with colitis model","journal":"Gastroenterology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP with siRNA and in vivo genetic rescue; single lab, two orthogonal methods","pmids":["23747339"],"is_preprint":false},{"year":2014,"finding":"Klotho up-regulates TRPV5 from both inside (as membrane-bound Klotho, mKL, via enhanced forward trafficking blocked by brefeldin A) and outside (as secreted Klotho, sKL, via inhibition of dynamin-dependent endocytosis); both effects require putative sialidase activity of Klotho, and mKL acts via intracellular N-glycosylation-dependent mechanisms.","method":"Whole-cell patch-clamp, brefeldin A blockade, dominant-negative dynamin II, sialidase inhibitor, sialidase activity site mutations, cell-surface biotinylation","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological and genetic dissection of trafficking pathways; single lab, multiple complementary approaches","pmids":["25378396"],"is_preprint":false},{"year":2013,"finding":"Klotho increases TRPV5 plasma membrane stability via the TRPV5 N-glycan through a mechanism distinct from sialidase: sialidase activates TRPV5 by inhibiting lipid raft-mediated internalization independently of the N-glycan (N358Q mutant equally activated). Galectin-3 (not galectin-1) is expressed in the distal convoluted tubule, and galectin-3 treatment increases TRPV5-mediated Ca2+ uptake.","method":"Biochemical glycan analysis (isoelectric focusing), TIRF microscopy, Ca2+ uptake assays, N-glycosylation mutant analysis, galectin treatment","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple methods dissecting klotho vs. sialidase mechanisms; single lab","pmids":["23970553"],"is_preprint":false},{"year":2016,"finding":"Mucin-1 (MUC1) increases TRPV5 activity by impairing dynamin-2- and caveolin-1-mediated endocytosis; MUC1 physically interacts with TRPV5 (co-immunoprecipitation), and the MUC1 effect requires N-glycan on TRPV5 and galectin-3 (not galectin-1) binding to VNTR repeats of MUC1, forming a lattice.","method":"Patch-clamp electrophysiology, co-immunoprecipitation, dominant-negative dynamin-2, siRNA knockdown of galectins, caveolin-1 analysis, N-glycosylation mutant TRPV5","journal":"Journal of the American Society of Nephrology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus mutagenesis and siRNA with electrophysiological readout; single lab, multiple methods","pmids":["27036738"],"is_preprint":false},{"year":2013,"finding":"TRPML3 associates with TRPV5 to form a novel heteromeric ion channel with distinct pharmacological and biophysical properties compared to TRPML3 or TRPV5 homomers; the heteromeric channel occurs in potentially distinct stoichiometric configurations.","method":"Co-immunoprecipitation, single-channel patch-clamp analysis, pharmacological characterization","journal":"PLoS one","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single co-IP with limited functional characterization; single lab, single paper","pmids":["23469151"],"is_preprint":false},{"year":2019,"finding":"Structure-based virtual screening at the econazole binding site identified novel TRPV5 inhibitors; cryo-EM structures of TRPV5 with these inhibitors revealed novel binding sites distinct from the econazole site, suggesting multiple inhibitory mechanisms.","method":"Structure-based virtual screening, cryo-electron microscopy, electrophysiology screening","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — cryo-EM structures with functional electrophysiology; single lab but rigorous structural approach","pmids":["31647410"],"is_preprint":false},{"year":2003,"finding":"Cysteine scanning mutagenesis (SCAM) of the ECaC-TRPV5 outer pore defined three structural domains: a coiled structure (Glu515–Tyr526) connected to a pore helix (~15 amino acids, 527–539) followed by the selectivity filter around Asp542 and a coiled structure before S6. The pore helix is alpha-helical and optimized for cation entry; Asp542 is at the high-Ca2+ affinity site.","method":"Substituted cysteine accessibility method (SCAM) with MTSET/MTSES reagents, whole-cell patch-clamp","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — systematic SCAM analysis of 44 positions; single lab, rigorous structural-functional method","pmids":["14630907"],"is_preprint":false},{"year":2011,"finding":"A loss-of-function mutation in the single zebrafish TRPV5/6 orthologue (trpv5/6) causes lethal severe bone ossification defects with 68% reduced calcium content; ambient high calcium (25 mM) partially rescues the phenotype. The mutant channel expressed in HEK293 cells lacks Ca2+-selective inward transport activity, directly linking epithelial TRPV5/6-mediated Ca2+ uptake to bone formation.","method":"Forward genetic screen in zebrafish, HEK293 cell functional expression, calcium content measurements, genetic rescue with ambient Ca2+","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — forward genetics combined with functional channel reconstitution and in vivo rescue; multiple orthogonal methods","pmids":["21670068"],"is_preprint":false},{"year":2017,"finding":"L530R variation in TRPV5 (associated with recurrent kidney stones) abolishes Ca2+ uptake activity in Xenopus oocytes, drastically reduces complex glycosylation, and molecular dynamics simulations show disruption of hydrophobic interaction between L530 and L502, damaging TM5 secondary structure and shifting the Ca2+-selective filter residue D542.","method":"Xenopus oocyte 45Ca2+ uptake, western blot glycosylation analysis, molecular dynamics simulations, TRPV6 crystal structure-based homology modeling","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — functional assay with glycosylation analysis and structural modeling; single lab, simulation not direct structure","pmids":["28847730"],"is_preprint":false}],"current_model":"TRPV5 is a highly Ca2+-selective tetrameric TRP channel localized to the apical membrane of renal distal tubule epithelial cells and osteoclast ruffled borders, where it functions as the rate-limiting entry gate for active transcellular Ca2+ reabsorption; its activity and surface abundance are controlled by a complex network including Klotho-mediated sialidase deglycosylation that traps the channel via galectin-galactosyl-GlcNAc lattices, FGF23-FGFR-αKlotho-ERK1/2-SGK1-WNK4 signaling, PTH-cAMP-PKA phosphorylation of T709 (which also disrupts inhibitory calmodulin binding), PKC phosphorylation of S299/S654 that blocks caveolin-1-dependent endocytosis, histidine-711 phosphorylation by NDPK-B, direct binding to calbindin-D28K that buffers peri-channel Ca2+ to prevent inactivation, and intracellular pH and PI(4,5)P2 that respectively close and open the lower gate through pore-helix conformational changes, with the gating residue W583 acting as the intracellular gate controlled by Ca2+-bound calmodulin."},"narrative":{"mechanistic_narrative":"TRPV5 is a highly Ca2+-selective, homotetrameric TRP channel that serves as the rate-limiting apical entry gate for active transcellular Ca2+ reabsorption, established genetically by knockout mice that show severe renal Ca2+ wasting, compensatory intestinal hyperabsorption, and bone thinning [PMID:14679186], and by a zebrafish loss-of-function ortholog causing lethal ossification defects [PMID:21670068]. Beyond the kidney, TRPV5 resides in the osteoclast ruffled border where its Ca2+ transport is essential for bone resorption [PMID:16291808]. The channel assembles via reciprocal N- and C-tail interactions into 400 kDa tetramers that can co-assemble with TRPV6 to generate channels of distinct permeation properties [PMID:12574114, PMID:15489237], with the outer pore organized around a pore helix and an Asp542 selectivity-filter high-affinity Ca2+ site [PMID:14630907] and an intracellular W583 gate [PMID:28374795]. Channel gating is governed structurally by PI(4,5)P2, which binds at the N-linker/S4-S5/S6 interface to open the lower gate, and by Ca2+-loaded calmodulin, whose C-lobe Lys116 engages Trp583 to drive Ca2+-dependent inactivation [PMID:30305626, PMID:30975749]; protons sensed extracellularly at Glu522 and intracellularly via pore-helix rotation close the channel, and low pH inhibits by precluding PI(4,5)P2 binding [PMID:14525991, PMID:16121193, PMID:35476976]. Surface abundance and activity are set by a regulatory network: Klotho hydrolyzes TRPV5 N-glycans to entrap the channel through galectin lattices [PMID:16239475, PMID:18606998], FGF23 acts through the FGFR-αKlotho complex via an ERK1/2-SGK1-WNK4 cascade [PMID:24434184], PTH-PKA phosphorylates Thr709 to relieve inhibitory calmodulin binding [PMID:19423690, PMID:21576356], PKC phosphorylation of Ser299/Ser654 blocks caveolin-1-dependent endocytosis [PMID:18305097, PMID:17006539], and NDPK-B phosphorylates His711 to activate the channel [PMID:24523290]. Trafficking and stability are further tuned by the S100A10-annexin-2 complex required for plasma membrane delivery [PMID:12660155], constitutive clathrin- and caveolae-mediated endocytosis with Rab11a-dependent recycling [PMID:18077461], and calbindin-D28K, which buffers peri-pore Ca2+ to prevent inactivation and sustain high transport rates [PMID:16763551]. A recurrent kidney-stone-associated L530R variant abolishes Ca2+ transport by disrupting TM5 packing near the selectivity filter [PMID:28847730].","teleology":[{"year":2002,"claim":"Established that the TRPV5 C-terminal tail encodes the determinants of Ca2+-dependent inactivation, localizing feedback regulation to a defined cytoplasmic region.","evidence":"C-terminal truncation mutagenesis with patch-clamp in HEK293 cells","pmids":["12634930"],"confidence":"Medium","gaps":["Did not identify the molecular partner mediating inactivation","Mapped regions but not specific residues or interacting Ca2+ sensor"]},{"year":2003,"claim":"Defined the channel as a tetramer and resolved outer-pore architecture, answering how TRPV5 achieves Ca2+ selectivity and how subunit composition diversifies channel behavior.","evidence":"Sucrose gradient/IP and concatemeric electrophysiology for tetramer assembly; SCAM pore mapping; knockout micropuncture for in vivo function","pmids":["12574114","14630907","14679186"],"confidence":"High","gaps":["Atomic structure not yet available","Heterotetramer stoichiometry in native tissue unresolved"]},{"year":2003,"claim":"Identified S100A10-annexin-2 as a trafficking chaperone required to route TRPV5 to the plasma membrane via its C-terminal VATTV motif, clarifying how the channel reaches its functional site.","evidence":"Yeast two-hybrid, GST pulldown, co-IP, mutagenesis (T599), siRNA, electrophysiology","pmids":["12660155"],"confidence":"High","gaps":["Mechanism of vesicular routing downstream of S100A10 not detailed","Did not address regulated vs constitutive delivery"]},{"year":2003,"claim":"Localized extracellular pH sensing to Glu522, defining how luminal acidification gates the channel.","evidence":"Single-channel patch-clamp, mutagenesis, MTS cysteine accessibility","pmids":["14525991"],"confidence":"High","gaps":["Did not connect extracellular sensor to intracellular conformational changes (resolved later)"]},{"year":2004,"claim":"Showed reciprocal N- and C-tail interactions drive tetrameric assembly and surface trafficking, explaining the dominant-negative effect of tail deletions.","evidence":"GST pulldown, co-IP, mutagenesis, oocyte 45Ca2+ uptake and patch-clamp","pmids":["15489237"],"confidence":"High","gaps":["Structural basis of tail-tail contacts not resolved","Did not address regulation of assembly"]},{"year":2004,"claim":"Identified accessory Ca2+ sensors and kinase/scaffold inputs (80K-H, SGK1/3-NHERF2) modulating activity and surface targeting, broadening the regulatory network.","evidence":"Co-IP, 45Ca2+ binding/uptake, EF-hand and PDZ deletion mutagenesis, oocyte electrophysiology","pmids":["15100231","15319523","15665527"],"confidence":"Medium","gaps":["Single-lab interactions without in vivo confirmation","Physiological hierarchy among regulators unclear"]},{"year":2005,"claim":"Revealed Klotho as a glycosidase that traps TRPV5 at the surface by hydrolyzing N-glycans, providing a hormone-independent mechanism to sustain Ca2+ entry.","evidence":"Enzymatic assay, Ca2+ influx, surface expression in HEK293; osteoclast KO; PIP2 electrophysiology","pmids":["16239475","16291808","16230466"],"confidence":"High","gaps":["Initial enzymatic identity (glucuronidase vs sialidase) later refined","Did not resolve glycan-lattice partner at this stage"]},{"year":2006,"claim":"Established calbindin-D28K as a peri-channel Ca2+ buffer preventing inactivation, and identified additional modulators (BSPRY, FKBP52) plus a pH-driven vesicular surface-recruitment mechanism.","evidence":"TIRF microscopy, binding/fractionation, 45Ca2+ uptake, electrophysiology, PPIase mutagenesis","pmids":["16763551","16380433","16352746","17178838"],"confidence":"Medium","gaps":["Relative contribution of buffering vs structural inactivation control unquantified","Modulator interactions mostly single-lab"]},{"year":2008,"claim":"Distinguished endocytic routes and their hormonal control, showing PKC phosphorylation of Ser299/Ser654 inhibits caveolin-1-mediated retrieval while WNK4 promotes it, tuning surface abundance.","evidence":"Dominant-negative dynamin, caveolin-1/clathrin siRNA and KO rescue, mutagenesis, biotinylation, electrophysiology","pmids":["18305097","17006539","20061383","18077461"],"confidence":"High","gaps":["In vivo significance of caveolar vs clathrin routes not fully separated","WNK4 mechanism outside kinase domain undefined"]},{"year":2009,"claim":"Defined PTH dual action: PKA phosphorylation of Thr709 raising open probability and CaR-coupled PKC stimulation, linking systemic calcium-regulating signals to channel gating and trafficking.","evidence":"FRET cAMP/Ca2+, biotinylation, PKA application, mutagenesis, patch-clamp, CaR dominant-negative","pmids":["19423690","19157541"],"confidence":"High","gaps":["T709 phosphorylation mechanism on CaM binding not yet structurally defined (resolved 2011)","CaR coupling single-lab"]},{"year":2011,"claim":"Resolved the calmodulin-TRPV5 interaction and stoichiometry and showed PKA-Thr709 phosphorylation enhances activity by displacing inhibitory CaM, unifying gating and hormonal control.","evidence":"NMR spectroscopy, mutagenesis (W702A, R706E), patch-clamp; zebrafish ossification genetics","pmids":["21576356","21670068"],"confidence":"High","gaps":["Full-channel structural context of CaM binding not yet available (resolved by cryo-EM)"]},{"year":2013,"claim":"Expanded surface regulation to disease-relevant extracellular factors (UMOD, MUC1) and degradation (UBR4), and refined the Klotho glycan/galectin mechanism toward galectin-3.","evidence":"Co-IP, siRNA, caveolin-1 KO rescue, N-glycan mutants, in vivo Umod-/- and Klotho colitis models, glycan analysis","pmids":["23466996","27036738","23747339","23970553"],"confidence":"Medium","gaps":["Galectin-1 vs galectin-3 identity in DCT remained debated across studies","UBR4 ubiquitination sites unmapped"]},{"year":2014,"claim":"Separated Klotho's hormonal (FGF23-FGFR-αKlotho-ERK1/2-SGK1-WNK4) and enzymatic trafficking roles, and identified NDPK-B/PHPT1 histidine phosphorylation of His711 as a distinct activation switch.","evidence":"FGF23/αKlotho KO mice, pathway inhibition; inside-out patch, NDPK-B shRNA/KO mice, H711 mutagenesis; trafficking dissection","pmids":["24434184","24523290","25378396"],"confidence":"High","gaps":["Integration of multiple Klotho mechanisms in vivo incomplete","Histidine-phosphorylation dynamics in tissue unquantified"]},{"year":2018,"claim":"Provided atomic structures showing how PI(4,5)P2 opens the lower gate, how calmodulin's Lys116 engages Trp583 for Ca2+-dependent inhibition, and how inhibitors occlude the pore, giving a structural basis for gating.","evidence":"Cryo-EM of full-length TRPV5 with PI(4,5)P2, CaM, and econazole; MD simulations; W583 mutagenesis","pmids":["30305626","29323279","28374795"],"confidence":"High","gaps":["Dynamic transitions between states inferred not directly captured at first","Native-membrane lipid composition effects unaddressed"]},{"year":2019,"claim":"Captured CaM-binding stoichiometric flexibility in nanodiscs and used structure-guided screening to find new inhibitor sites, advancing both mechanism and pharmacology.","evidence":"Cryo-EM in lipid nanodiscs, W583A mutant, virtual screening with electrophysiology","pmids":["30975749","31647410"],"confidence":"High","gaps":["Therapeutic selectivity over TRPV6 not established","Functional consequence of variable CaM stoichiometry in vivo unknown"]},{"year":2022,"claim":"Unified gating modulators structurally, showing low pH inhibits by precluding PI(4,5)P2 activation and that PKA acts by preventing CaM-mediated inactivation rather than directly opening the gate.","evidence":"Cryo-EM across multiple pH and modulator conditions capturing intermediate states","pmids":["35476976"],"confidence":"High","gaps":["Kinetics of state transitions in living cells not resolved","Coupling between His711/Thr709 phosphorylation and structural states untested"]},{"year":null,"claim":"How the many converging regulatory inputs (glycan trapping, hormonal kinases, lipid and pH gating, calmodulin, accessory chaperones) are integrated and prioritized within a single native distal-tubule cell to set Ca2+ reabsorption set-points remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No quantitative model integrating trafficking and gating regulators in vivo","Tissue-specific stoichiometry of TRPV5 heteromers (TRPV6, TRPML3) unknown","Human disease genetics beyond a single variant largely uncharacterized in this corpus"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[2,3,9,10,42]},{"term_id":"GO:0140299","term_label":"molecular sensor activity","supporting_discovery_ids":[15,33]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[11,17,20]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,2,4,10,12]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[18,21]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[18]}],"pathway":[{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[2,3,9,10]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[5,8,12,19]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[12,18,21,24]}],"complexes":["TRPV5 homotetramer","TRPV5/TRPV6 heterotetramer","S100A10-annexin 2 complex","TRPV5-calmodulin complex"],"partners":["KLOTHO","S100A10","CALBINDIN-D28K","CALMODULIN","NHERF2","MUC1","UBR4","FKBP52"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9NQA5","full_name":"Transient receptor potential cation channel subfamily V member 5","aliases":["Calcium transport protein 2","CaT2","Epithelial calcium channel 1","ECaC","ECaC1","Osm-9-like TRP channel 3","OTRPC3"],"length_aa":729,"mass_kda":82.6,"function":"Constitutively active calcium selective cation channel thought to be involved in Ca(2+) reabsorption in kidney and intestine (PubMed:11549322, PubMed:18768590). Required for normal Ca(2+) reabsorption in the kidney distal convoluted tubules (By similarity). The channel is activated by low internal calcium level and the current exhibits an inward rectification (PubMed:11549322, PubMed:18768590). A Ca(2+)-dependent feedback regulation includes fast channel inactivation and slow current decay (By similarity). Heteromeric assembly with TRPV6 seems to modify channel properties. TRPV5-TRPV6 heteromultimeric concatemers exhibit voltage-dependent gating (By similarity)","subcellular_location":"Apical cell membrane","url":"https://www.uniprot.org/uniprotkb/Q9NQA5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TRPV5","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/TRPV5","total_profiled":1310},"omim":[{"mim_id":"619683","title":"B-BOX- AND SPRY DOMAIN-CONTAINING PROTEIN; BSPRY","url":"https://www.omim.org/entry/619683"},{"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"},{"mim_id":"604824","title":"KLOTHO; KL","url":"https://www.omim.org/entry/604824"},{"mim_id":"600968","title":"SOLUTE CARRIER FAMILY 12 (SODIUM/CHLORIDE TRANSPORTER), MEMBER 3; SLC12A3","url":"https://www.omim.org/entry/600968"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"brain","ntpm":1.6},{"tissue":"kidney","ntpm":4.0}],"url":"https://www.proteinatlas.org/search/TRPV5"},"hgnc":{"alias_symbol":["CaT2"],"prev_symbol":["ECAC1"]},"alphafold":{"accession":"Q9NQA5","domains":[{"cath_id":"1.25.40.20","chopping":"48-259","consensus_level":"medium","plddt":92.8126,"start":48,"end":259},{"cath_id":"-","chopping":"314-358_367-469","consensus_level":"high","plddt":91.1326,"start":314,"end":469},{"cath_id":"1.10.287","chopping":"481-608","consensus_level":"medium","plddt":89.7791,"start":481,"end":608}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NQA5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NQA5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NQA5-F1-predicted_aligned_error_v6.png","plddt_mean":82.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TRPV5","jax_strain_url":"https://www.jax.org/strain/search?query=TRPV5"},"sequence":{"accession":"Q9NQA5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9NQA5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9NQA5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NQA5"}},"corpus_meta":[{"pmid":"16239475","id":"PMC_16239475","title":"The 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Cell physiology","url":"https://pubmed.ncbi.nlm.nih.gov/16371438","citation_count":24,"is_preprint":false},{"pmid":"12019295","id":"PMC_12019295","title":"Expression of the novel epithelial Ca2+ channel ECaC1 in rat pancreatic islets.","date":"2002","source":"The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society","url":"https://pubmed.ncbi.nlm.nih.gov/12019295","citation_count":23,"is_preprint":false},{"pmid":"17397797","id":"PMC_17397797","title":"Rapamycin stimulates arginine influx through CAT2 transporters in human endothelial cells.","date":"2007","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/17397797","citation_count":23,"is_preprint":false},{"pmid":"17217059","id":"PMC_17217059","title":"TRPV5, the gateway to Ca2+ homeostasis.","date":"2007","source":"Handbook of experimental pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/17217059","citation_count":22,"is_preprint":false},{"pmid":"12446172","id":"PMC_12446172","title":"Cat2 L-arginine transporter-deficient fibroblasts can sustain nitric oxide production.","date":"2002","source":"Nitric oxide : biology and chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12446172","citation_count":22,"is_preprint":false},{"pmid":"23469151","id":"PMC_23469151","title":"A novel ion channel formed by interaction of TRPML3 with TRPV5.","date":"2013","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/23469151","citation_count":21,"is_preprint":false},{"pmid":"28847730","id":"PMC_28847730","title":"The L530R variation associated with recurrent kidney stones impairs the structure and function of TRPV5.","date":"2017","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/28847730","citation_count":21,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":53218,"output_tokens":10873,"usd":0.161375,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":21560,"output_tokens":5517,"usd":0.122862,"stage2_stop_reason":"end_turn"},"total_usd":0.284237,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2005,\n      \"finding\": \"Klotho, acting as a beta-glucuronidase, hydrolyzes extracellular N-linked oligosaccharides on TRPV5, entrapping the channel in the plasma membrane and sustaining calcium channel activity at the cell surface.\",\n      \"method\": \"Biochemical enzymatic assay, Ca2+ influx measurements, cell surface expression studies in HEK293 cells\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct enzymatic activity demonstrated in vitro with functional readout; replicated by multiple subsequent studies confirming the same mechanism\",\n      \"pmids\": [\"16239475\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Klotho removes terminal alpha2,6-linked sialic acids from TRPV5 N-glycan chains, exposing galactose-N-acetylglucosamine that binds galectin-1, which forms a lattice retaining TRPV5 at the plasma membrane. Knockdown of ST6Gal-1 prevents this regulation.\",\n      \"method\": \"RNAi knockdown of ST6Gal-1, glycan analysis, Ca2+ influx measurements, co-expression in cell lines lacking endogenous ST6Gal-1 with rescue by recombinant ST6Gal-1\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal methods (RNAi, cell-line complementation, glycan biochemistry), single lab but rigorous mechanistic dissection\",\n      \"pmids\": [\"18606998\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"TRPV5-knockout mice exhibit severely diminished active Ca2+ reabsorption in the early distal convoluted tubule (confirmed by in vivo micropuncture), severe hypercalciuria despite elevated vitamin D, compensatory intestinal Ca2+ hyperabsorption, and reduced trabecular and cortical bone thickness, establishing TRPV5 as the essential luminal entry gate for active Ca2+ reabsorption in the kidney.\",\n      \"method\": \"Germline knockout mouse model, in vivo micropuncture, urinary and serum Ca2+ measurements, bone histomorphometry\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — knockout with multiple orthogonal phenotypic readouts including direct in vivo micropuncture; widely replicated\",\n      \"pmids\": [\"14679186\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"TRPV5 and TRPV6 assemble as homotetramers (400 kDa complexes) and can form heterotetramers; different heterotetrameric compositions produce Ca2+ channels with distinct Ca2+-dependent inactivation, Ba2+ selectivity, and pharmacological properties.\",\n      \"method\": \"Sucrose gradient sedimentation, immunoprecipitation, electrophysiology of concatemeric channels with pore mutants in HEK293 cells\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstitution with concatemeric constructs plus electrophysiology and biochemical sedimentation; multiple orthogonal methods\",\n      \"pmids\": [\"12574114\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"S100A10 directly binds the conserved C-terminal VATTV sequence of TRPV5 (critical residue T599) and forms a heterotetrameric complex with annexin 2; this S100A10-annexin 2 complex is required to route TRPV5 to the plasma membrane. Annexin 2 siRNA knockdown abolishes TRPV5-mediated currents.\",\n      \"method\": \"Yeast two-hybrid, GST pulldown, co-immunoprecipitation, site-directed mutagenesis, siRNA knockdown, electrophysiology\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — yeast two-hybrid confirmed by GST pulldown and co-IP; functional mutagenesis and siRNA rescue with electrophysiological readout; multiple orthogonal methods\",\n      \"pmids\": [\"12660155\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"FGF23 promotes renal Ca2+ reabsorption and apical membrane abundance of TRPV5 in distal tubules through binding the FGF receptor-αKlotho complex and activating an ERK1/2-SGK1-WNK4 signaling cascade, not through αKlotho's glycosidase activity.\",\n      \"method\": \"Fgf23 and αKlotho knockout mice, immunolocalization, signaling pathway inhibition, renal Ca2+ balance measurements\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockouts of FGF23 and αKlotho with pathway analysis; multiple orthogonal approaches; replicated in knockout models\",\n      \"pmids\": [\"24434184\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Cryo-EM structures of TRPV5 with PI(4,5)P2 reveal a binding site between the N-linker, S4-S5 linker and S6 helix; PI(4,5)P2 binding induces conformational rearrangements in the lower gate that open the channel. Cryo-EM with calmodulin shows two TRPV5 C-terminal peptides anchoring one CaM molecule, with Lys116 on CaM C-lobe forming a cation-π interaction with Trp583 at the intracellular gate to mediate Ca2+-dependent inhibition.\",\n      \"method\": \"Cryo-electron microscopy, molecular dynamics simulations\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct cryo-EM structures with multiple modulators, plus MD simulations; atomic resolution mechanistic insight\",\n      \"pmids\": [\"30305626\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Cryo-EM structure of full-length rabbit TRPV5 in complex with econazole reveals the inhibitor occupies a hydrophobic pocket analogous to the phosphatidylinositide/vanilloid-binding site in TRPV1; the econazole-bound structure adopts a closed conformation with a distinct lower gate that occludes Ca2+ permeation.\",\n      \"method\": \"Cryo-electron microscopy, molecular dynamics simulations\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution cryo-EM structure with functional corroboration from MD simulations; landmark structural study\",\n      \"pmids\": [\"29323279\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"PTH activates TRPV5 via the cAMP-PKA pathway by phosphorylating threonine-709 on TRPV5, increasing channel open probability without altering surface expression. Alanine substitution of T709 abolishes both in vitro phosphorylation and PTH-mediated TRPV5 stimulation.\",\n      \"method\": \"FRET-based cAMP and Ca2+ dynamics, cell-surface biotinylation, PKA catalytic subunit application, site-directed mutagenesis, patch-clamp electrophysiology\",\n      \"journal\": \"Journal of the American Society of Nephrology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro phosphorylation with mutagenesis, electrophysiology, and FRET; multiple orthogonal methods in single study\",\n      \"pmids\": [\"19423690\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"PIP2 activates TRPV5 by a mechanism independent of Mg2+ binding to the selectivity filter and reduces TRPV5 sensitivity to Mg2+-induced slow conformational channel closure. Mutation of aspartate-542 (critical Mg2+-binding site in selectivity filter) abolishes Mg2+-induced slow inhibition; PLC activation sensitizes TRPV5 to this Mg2+-induced slow inhibition by hydrolyzing PIP2.\",\n      \"method\": \"Whole-cell patch-clamp, site-directed mutagenesis, intracellular perfusion with PIP2 or receptor-mediated PLC activation\",\n      \"journal\": \"The Journal of general physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — electrophysiology combined with mutagenesis of selectivity filter and biochemical modulation; two orthogonal approaches\",\n      \"pmids\": [\"16230466\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"TRPV5 is localized to the ruffled border membrane of osteoclasts; TRPV5 knockout osteoclasts show increased number and size but nearly absent bone resorption in pit assays, establishing TRPV5-mediated Ca2+ transport as essential for osteoclastic bone resorption.\",\n      \"method\": \"Immunostaining, TRPV5 knockout mouse analysis, in vitro bone marrow osteoclast cultures, resorption pit assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with multiple phenotypic readouts (immunolocalization, pit assay, in vivo bone markers); well-controlled\",\n      \"pmids\": [\"16291808\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Calbindin-D28K translocates to the plasma membrane at low intracellular Ca2+ and directly associates with TRPV5, where it buffers Ca2+ entering through the channel in close proximity to the pore, preventing Ca2+-dependent channel inactivation and facilitating high transcellular Ca2+ transport rates.\",\n      \"method\": \"Protein-binding analysis, subcellular fractionation, evanescent-field (TIRF) microscopy, 45Ca2+ uptake, electrophysiology, transcellular Ca2+ transport assays in primary tubule cells infected with lentivirus\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct binding demonstrated with functional electrophysiology in primary cells; multiple orthogonal methods\",\n      \"pmids\": [\"16763551\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"TRPV5 undergoes constitutive caveolae-mediated (caveolin-1-dependent, clathrin-independent) endocytosis. PKC activators increase TRPV5 cell-surface abundance by inhibiting this endocytosis via phosphorylation of Ser-299 and Ser-654 on TRPV5. PTH also increases TRPV5 surface abundance through this PKC-caveolin pathway.\",\n      \"method\": \"Dominant-negative dynamin, siRNA knockdown of caveolin-1 and clathrin, caveolin-1 knockout cells with rescue, site-directed mutagenesis, whole-cell patch-clamp, cell-surface biotinylation\",\n      \"journal\": \"American journal of physiology. Renal physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis + KO cell rescue + siRNA + dominant-negative approaches; multiple orthogonal methods\",\n      \"pmids\": [\"18305097\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Tissue kallikrein (TK) stimulates Ca2+ reabsorption via bradykinin receptor type 2 activation of the PLC/DAG/PKC pathway, which phosphorylates TRPV5 at Ser-299 and Ser-654, increasing TRPV5 plasma membrane abundance by delaying its retrieval.\",\n      \"method\": \"Primary renal epithelial cell cultures, PKC pharmacology, BK receptor antagonism, cell-surface labeling, mutagenesis of PKC sites (S299A, S654A)\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — pharmacological pathway dissection combined with site-directed mutagenesis of PKC phosphorylation sites; multiple methods\",\n      \"pmids\": [\"17006539\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"The intracellular N-tail (residues 64–77) and C-tail (residues 596–601) of TRPV5 mediate homotetrameric assembly via N-N, C-C, and N-C interactions; deletion of either tail alone causes dominant-negative suppression by preventing plasma membrane trafficking of the channel complex.\",\n      \"method\": \"GST pulldown, co-immunoprecipitation, site-directed mutagenesis, patch-clamp, 45Ca2+ uptake in oocytes\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct biochemical binding assays with mutagenesis and functional electrophysiology; multiple orthogonal methods\",\n      \"pmids\": [\"15489237\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Extracellular acidification inhibits TRPV5 with pKa ~6.55; glutamate-522 in the extracellular loop between TM5 and TM6 is the extracellular pH sensor. E522Q mutation decreases pH sensitivity and abolishes proton-mediated reduction of open probability without altering single-channel conductance block component.\",\n      \"method\": \"Whole-cell and single-channel patch-clamp, site-directed mutagenesis, methanethiosulfonate (MTS) cysteine accessibility\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis combined with single-channel analysis and cysteine accessibility method; multiple orthogonal approaches\",\n      \"pmids\": [\"14525991\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Intracellular acidification causes clockwise rotation of the TRPV5 pore helix (detected by substituted cysteine accessibility method), which facilitates closure of the selectivity filter gate by extracellular protons; internal and external pH sensors cross-regulate each other via this pore helix conformational change.\",\n      \"method\": \"Substituted cysteine accessibility method (SCAM), whole-cell patch-clamp, site-directed mutagenesis\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — SCAM with mutagenesis and electrophysiology; rigorous structural-functional analysis\",\n      \"pmids\": [\"16121193\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Calmodulin binds Ca2+-dependently to a C-terminal fragment (residues 696–729) of TRPV5 in a 1 CaM:2 TRPV5 C-terminus stoichiometry; TRPV5 mutations W702A and R706E abolish CaM binding and strongly reduce Ca2+-dependent channel inactivation. PTH-induced PKA phosphorylation of T709 diminishes CaM binding to TRPV5, thereby enhancing channel open probability.\",\n      \"method\": \"NMR spectroscopy, site-directed mutagenesis, patch-clamp electrophysiology\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution NMR structural data combined with mutagenesis and functional electrophysiology; multiple orthogonal methods in single study\",\n      \"pmids\": [\"21576356\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"TRPV5 is constitutively internalized via dynamin- and clathrin-dependent endocytosis; internalized channels traffic to Rab11a-positive perinuclear recycling endosomes and can return to the cell surface. The recycling kinetics are decreased by intracellular Ca2+ chelation (BAPTA-AM), indicating Ca2+-controlled recycling.\",\n      \"method\": \"Dynamin dominant-negative, clathrin siRNA, Rab11a co-localization, brefeldin A block, fluorescence microscopy, electrophysiology\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — dominant-negative and siRNA approaches with multiple trafficking markers; well-controlled mechanistic study\",\n      \"pmids\": [\"18077461\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Activation of the Ca2+-sensing receptor (CaR) co-localized with TRPV5 at the luminal membrane of distal tubule stimulates TRPV5-mediated Ca2+ influx via PMA-insensitive PKC isoforms acting on Ser-299 and Ser-654 of TRPV5; dominant-negative CaR(R185Q) and S299A/S654A TRPV5 mutations abolish this effect.\",\n      \"method\": \"Patch-clamp electrophysiology, Fura-2 Ca2+ imaging, site-directed mutagenesis, dominant-negative CaR construct\",\n      \"journal\": \"Cell calcium\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — electrophysiology with mutagenesis, single lab, two orthogonal methods\",\n      \"pmids\": [\"19157541\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"80K-H directly interacts with TRPV5, co-localizes in kidney, and acts as a Ca2+ sensor that modulates TRPV5 activity via its EF-hand structures, glutamic stretch, and HDEL sequence; inactivation of the EF-hands reduces TRPV5-mediated Ca2+ current and increases TRPV5 sensitivity to intracellular Ca2+, accelerating feedback inhibition.\",\n      \"method\": \"cDNA microarray identification, co-immunoprecipitation, 45Ca2+ binding assays, site-directed mutagenesis of EF-hands, patch-clamp electrophysiology\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP with mutagenesis and electrophysiology; single lab with two orthogonal approaches\",\n      \"pmids\": [\"15100231\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Extracellular alkalinization rapidly recruits a pool of TRPV5-containing vesicles to the cell surface via a 'kiss and linger' vesicular mechanism without full membrane fusion, increasing functional TRPV5 channel activity; extracellular acidification reverses this, retrieving vesicles from the surface.\",\n      \"method\": \"Total internal reflection fluorescence (TIRF) microscopy, cell surface protein labeling, electrophysiology, 45Ca2+ uptake, functional channel recovery after chemobleaching\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — TIRF with multiple complementary methods in single lab\",\n      \"pmids\": [\"17178838\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Klotho (beta-glucuronidase) selectively activates TRPV5 and TRPV6 but not TRPV4 or TRPM6 among renal ion channels, indicating channel-type specificity of the glycan-hydrolysis mechanism.\",\n      \"method\": \"Ca2+ influx measurements in HEK293 cells expressing different renal channels, endoglycosidase-F deglycosylation control\",\n      \"journal\": \"Nephrology, dialysis, transplantation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic screen with appropriate negative controls; single lab, single method type\",\n      \"pmids\": [\"18495742\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"NDPK-B (histidine kinase) activates TRPV5 channel activity and Ca2+ flux through phosphorylation of histidine-711 in the TRPV5 C-terminal tail; the histidine phosphatase PHPT1 reverses this activation. NDPK-B knockdown decreases TRPV5 activity, and NDPK-B knockout mice show increased urinary Ca2+ excretion on high-Ca2+ diet.\",\n      \"method\": \"Inside-out patch-clamp, NDPK-B shRNA knockdown, NDPK-B knockout mice urinary Ca2+ measurements, site-directed mutagenesis of H711\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro patch phosphorylation with phosphatase reversal, mutagenesis of target residue, and in vivo knockout phenotype; multiple orthogonal methods\",\n      \"pmids\": [\"24523290\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Uromodulin (UMOD) upregulates TRPV5 cell-surface abundance by acting extracellularly to impair caveolin-1-mediated endocytosis; UMOD has no effect in caveolin-1-null cells or with N-glycan-deficient TRPV5; disease mutant UMOD with reduced secretion fails to increase TRPV5 activity. Immunofluorescence shows reduced TRPV5 expression in Umod-/- mouse kidneys.\",\n      \"method\": \"Whole-cell patch-clamp, cell-surface biotinylation, siRNA knockdown, caveolin-1 knockout cells with rescue, immunofluorescence in Umod-/- mice\",\n      \"journal\": \"Kidney international\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO cells, rescue, siRNA, and in vivo confirmation; multiple orthogonal methods\",\n      \"pmids\": [\"23466996\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"FKBP52 physically interacts specifically with TRPV5 and co-localizes in the distal nephron; it inhibits TRPV5-mediated Ca2+ influx through its peptidyl-prolyl cis-trans isomerase (PPIase) domain, as PPIase domain mutation abolishes the inhibitory effect.\",\n      \"method\": \"Co-immunoprecipitation, 45Ca2+ uptake, patch-clamp electrophysiology, siRNA knockdown, FK-506 pharmacology, PPIase domain mutagenesis\",\n      \"journal\": \"American journal of physiology. Renal physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP with functional mutagenesis and electrophysiology; single lab, two orthogonal methods\",\n      \"pmids\": [\"16352746\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"BSPRY interacts with TRPV5 (confirmed by GST pulldown and co-IP), co-localizes in mouse kidney, and reduces TRPV5-mediated Ca2+ influx without altering channel surface abundance, indicating direct modulation of channel activity.\",\n      \"method\": \"GST pulldown, co-immunoprecipitation, 45Ca2+ uptake in MDCK-TRPV5 cells, immunostaining\",\n      \"journal\": \"Journal of the American Society of Nephrology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal binding assays with functional Ca2+ uptake readout; single lab, two orthogonal methods\",\n      \"pmids\": [\"16380433\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"WNK4 decreases TRPV5 cell-surface abundance by stimulating caveolae-mediated endocytosis via a region outside its kinase domain; this WNK4-dependent tonic inhibition lowers basal TRPV5 levels and thereby amplifies the dynamic range of PKC- and PTH-mediated stimulation of TRPV5.\",\n      \"method\": \"Deletion analysis of WNK4 domains, patch-clamp electrophysiology, cell-surface biotinylation, PKC activator co-stimulation experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain deletion analysis with electrophysiology; single lab, two orthogonal approaches\",\n      \"pmids\": [\"20061383\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"WNK3 positively regulates TRPV5 plasma membrane expression via a kinase-dependent mechanism (kinase-inactive D294A mutation abolishes effect); WNK3 promotes complex glycosylation and exocytosis of TRPV5 via microtubule-dependent secretory pathway, as colchicine blocks this effect.\",\n      \"method\": \"Xenopus oocyte 45Ca2+ uptake, voltage-clamp electrophysiology, kinase-inactive mutagenesis, colchicine treatment, glycosylation analysis\",\n      \"journal\": \"American journal of physiology. Renal physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — kinase-dead mutagenesis with functional Ca2+ uptake and electrophysiology; single lab, multiple methods\",\n      \"pmids\": [\"18768590\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"SGK1 and SGK3 (but not SGK2) together with scaffold protein NHERF2 stimulate TRPV5-mediated Ca2+ entry in Xenopus oocytes; the second PDZ domain of NHERF2 is required for stabilization/targeting of TRPV5 to the plasma membrane, and TRPV5 C-tail interacts with NHERF2 in a Ca2+-independent manner.\",\n      \"method\": \"Xenopus oocyte 45Ca2+ uptake, voltage-clamp, NHERF2 PDZ deletion mutants, GST pulldown, overlay assays, chemiluminescence surface quantification\",\n      \"journal\": \"Cellular physiology and biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding assays with functional electrophysiology and domain deletion analysis; single lab\",\n      \"pmids\": [\"15319523\", \"15665527\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"NHERF4 (PDZK2) interacts with the C-terminus of TRPV5 through its fourth PDZ domain (PDZ1 also contributes); confirmed by yeast two-hybrid, GST pulldown with in vitro translated NHERF4 and oocyte lysates, and co-immunoprecipitation in HEK293 cells.\",\n      \"method\": \"Yeast two-hybrid, GST pulldown, co-immunoprecipitation, PDZ domain deletion analysis\",\n      \"journal\": \"Pflugers Archiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — binding interaction confirmed by multiple methods but no functional consequence demonstrated for this specific interaction\",\n      \"pmids\": [\"16565876\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"The C-terminal tail of TRPV5 (ECaC1) mediates Ca2+-dependent channel inactivation; deletion of the last 30 amino acids (G701X) and truncations around residues 649-653 significantly reduce or abolish Ca2+-dependent inactivation, identifying two critical C-terminal domains.\",\n      \"method\": \"Carboxyl-terminal truncation mutagenesis, patch-clamp electrophysiology in HEK293 cells\",\n      \"journal\": \"Pflugers Archiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro mutagenesis with electrophysiology; single lab, single method type\",\n      \"pmids\": [\"12634930\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"High-resolution cryo-EM structures of full-length TRPV5 in lipid nanodiscs, a TRPV5 W583A mutant, and TRPV5 in complex with CaM reveal a flexible stoichiometry of CaM binding and the mechanism of calcium-dependent regulation; W583 at the intracellular gate is critical for CaM-mediated inactivation.\",\n      \"method\": \"Cryo-electron microscopy, lipid nanodisc reconstitution, mutagenesis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple cryo-EM structures with mutagenesis validation; independent replication of CaM-TRPV5 structural interaction\",\n      \"pmids\": [\"30975749\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Cryo-EM shows that low pH inhibits TRPV5 by precluding PI(4,5)P2 binding/activation; intermediate conformations at low pH reveal transition from open to closed state. PI(4,5)P2 is the primary modulator of channel gating; PKA controls TRPV5 by preventing CaM binding and channel inactivation rather than directly opening the gate.\",\n      \"method\": \"Cryo-electron microscopy, structural analysis of multiple pH and modulator conditions\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple cryo-EM structures capturing different conformational states with two distinct modulators; rigorous structural mechanistic study\",\n      \"pmids\": [\"35476976\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Tryptophan-583 at the terminus of the intracellular pore forms a gate for Ca2+ permeation through TRPV5; W583 mutants show profoundly enhanced Ca2+ influx and increased cell death; a glycine residue above W583 may act as a flexible hinge to rearrange the tryptophan gate.\",\n      \"method\": \"Site-directed mutagenesis, patch-clamp electrophysiology, biochemical analysis, homology modeling\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis with electrophysiology; single lab, homology model (not direct structure)\",\n      \"pmids\": [\"28374795\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Inflammatory cytokines (TNF-α, IFN-γ, IL-1β) induce TRPV5 interaction with E3 ubiquitin ligase UBR4, leading to ubiquitin-dependent TRPV5 degradation; UBR4 siRNA prevents cytokine-induced TRPV5 degradation. Klotho protects TRPV5 from hypersialylation and cytokine-induced endocytosis and degradation in colitis.\",\n      \"method\": \"Co-immunoprecipitation (TRPV5-UBR4), siRNA knockdown of UBR4, adenoviral TRPV5 transduction, transgenic Klotho overexpression mice with colitis model\",\n      \"journal\": \"Gastroenterology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP with siRNA and in vivo genetic rescue; single lab, two orthogonal methods\",\n      \"pmids\": [\"23747339\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Klotho up-regulates TRPV5 from both inside (as membrane-bound Klotho, mKL, via enhanced forward trafficking blocked by brefeldin A) and outside (as secreted Klotho, sKL, via inhibition of dynamin-dependent endocytosis); both effects require putative sialidase activity of Klotho, and mKL acts via intracellular N-glycosylation-dependent mechanisms.\",\n      \"method\": \"Whole-cell patch-clamp, brefeldin A blockade, dominant-negative dynamin II, sialidase inhibitor, sialidase activity site mutations, cell-surface biotinylation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological and genetic dissection of trafficking pathways; single lab, multiple complementary approaches\",\n      \"pmids\": [\"25378396\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Klotho increases TRPV5 plasma membrane stability via the TRPV5 N-glycan through a mechanism distinct from sialidase: sialidase activates TRPV5 by inhibiting lipid raft-mediated internalization independently of the N-glycan (N358Q mutant equally activated). Galectin-3 (not galectin-1) is expressed in the distal convoluted tubule, and galectin-3 treatment increases TRPV5-mediated Ca2+ uptake.\",\n      \"method\": \"Biochemical glycan analysis (isoelectric focusing), TIRF microscopy, Ca2+ uptake assays, N-glycosylation mutant analysis, galectin treatment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple methods dissecting klotho vs. sialidase mechanisms; single lab\",\n      \"pmids\": [\"23970553\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Mucin-1 (MUC1) increases TRPV5 activity by impairing dynamin-2- and caveolin-1-mediated endocytosis; MUC1 physically interacts with TRPV5 (co-immunoprecipitation), and the MUC1 effect requires N-glycan on TRPV5 and galectin-3 (not galectin-1) binding to VNTR repeats of MUC1, forming a lattice.\",\n      \"method\": \"Patch-clamp electrophysiology, co-immunoprecipitation, dominant-negative dynamin-2, siRNA knockdown of galectins, caveolin-1 analysis, N-glycosylation mutant TRPV5\",\n      \"journal\": \"Journal of the American Society of Nephrology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus mutagenesis and siRNA with electrophysiological readout; single lab, multiple methods\",\n      \"pmids\": [\"27036738\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"TRPML3 associates with TRPV5 to form a novel heteromeric ion channel with distinct pharmacological and biophysical properties compared to TRPML3 or TRPV5 homomers; the heteromeric channel occurs in potentially distinct stoichiometric configurations.\",\n      \"method\": \"Co-immunoprecipitation, single-channel patch-clamp analysis, pharmacological characterization\",\n      \"journal\": \"PLoS one\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single co-IP with limited functional characterization; single lab, single paper\",\n      \"pmids\": [\"23469151\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Structure-based virtual screening at the econazole binding site identified novel TRPV5 inhibitors; cryo-EM structures of TRPV5 with these inhibitors revealed novel binding sites distinct from the econazole site, suggesting multiple inhibitory mechanisms.\",\n      \"method\": \"Structure-based virtual screening, cryo-electron microscopy, electrophysiology screening\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structures with functional electrophysiology; single lab but rigorous structural approach\",\n      \"pmids\": [\"31647410\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Cysteine scanning mutagenesis (SCAM) of the ECaC-TRPV5 outer pore defined three structural domains: a coiled structure (Glu515–Tyr526) connected to a pore helix (~15 amino acids, 527–539) followed by the selectivity filter around Asp542 and a coiled structure before S6. The pore helix is alpha-helical and optimized for cation entry; Asp542 is at the high-Ca2+ affinity site.\",\n      \"method\": \"Substituted cysteine accessibility method (SCAM) with MTSET/MTSES reagents, whole-cell patch-clamp\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — systematic SCAM analysis of 44 positions; single lab, rigorous structural-functional method\",\n      \"pmids\": [\"14630907\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"A loss-of-function mutation in the single zebrafish TRPV5/6 orthologue (trpv5/6) causes lethal severe bone ossification defects with 68% reduced calcium content; ambient high calcium (25 mM) partially rescues the phenotype. The mutant channel expressed in HEK293 cells lacks Ca2+-selective inward transport activity, directly linking epithelial TRPV5/6-mediated Ca2+ uptake to bone formation.\",\n      \"method\": \"Forward genetic screen in zebrafish, HEK293 cell functional expression, calcium content measurements, genetic rescue with ambient Ca2+\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — forward genetics combined with functional channel reconstitution and in vivo rescue; multiple orthogonal methods\",\n      \"pmids\": [\"21670068\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"L530R variation in TRPV5 (associated with recurrent kidney stones) abolishes Ca2+ uptake activity in Xenopus oocytes, drastically reduces complex glycosylation, and molecular dynamics simulations show disruption of hydrophobic interaction between L530 and L502, damaging TM5 secondary structure and shifting the Ca2+-selective filter residue D542.\",\n      \"method\": \"Xenopus oocyte 45Ca2+ uptake, western blot glycosylation analysis, molecular dynamics simulations, TRPV6 crystal structure-based homology modeling\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — functional assay with glycosylation analysis and structural modeling; single lab, simulation not direct structure\",\n      \"pmids\": [\"28847730\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TRPV5 is a highly Ca2+-selective tetrameric TRP channel localized to the apical membrane of renal distal tubule epithelial cells and osteoclast ruffled borders, where it functions as the rate-limiting entry gate for active transcellular Ca2+ reabsorption; its activity and surface abundance are controlled by a complex network including Klotho-mediated sialidase deglycosylation that traps the channel via galectin-galactosyl-GlcNAc lattices, FGF23-FGFR-αKlotho-ERK1/2-SGK1-WNK4 signaling, PTH-cAMP-PKA phosphorylation of T709 (which also disrupts inhibitory calmodulin binding), PKC phosphorylation of S299/S654 that blocks caveolin-1-dependent endocytosis, histidine-711 phosphorylation by NDPK-B, direct binding to calbindin-D28K that buffers peri-channel Ca2+ to prevent inactivation, and intracellular pH and PI(4,5)P2 that respectively close and open the lower gate through pore-helix conformational changes, with the gating residue W583 acting as the intracellular gate controlled by Ca2+-bound calmodulin.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TRPV5 is a highly Ca2+-selective, homotetrameric TRP channel that serves as the rate-limiting apical entry gate for active transcellular Ca2+ reabsorption, established genetically by knockout mice that show severe renal Ca2+ wasting, compensatory intestinal hyperabsorption, and bone thinning [#2], and by a zebrafish loss-of-function ortholog causing lethal ossification defects [#42]. Beyond the kidney, TRPV5 resides in the osteoclast ruffled border where its Ca2+ transport is essential for bone resorption [#10]. The channel assembles via reciprocal N- and C-tail interactions into 400 kDa tetramers that can co-assemble with TRPV6 to generate channels of distinct permeation properties [#3, #14], with the outer pore organized around a pore helix and an Asp542 selectivity-filter high-affinity Ca2+ site [#41] and an intracellular W583 gate [#34]. Channel gating is governed structurally by PI(4,5)P2, which binds at the N-linker/S4-S5/S6 interface to open the lower gate, and by Ca2+-loaded calmodulin, whose C-lobe Lys116 engages Trp583 to drive Ca2+-dependent inactivation [#6, #32]; protons sensed extracellularly at Glu522 and intracellularly via pore-helix rotation close the channel, and low pH inhibits by precluding PI(4,5)P2 binding [#15, #16, #33]. Surface abundance and activity are set by a regulatory network: Klotho hydrolyzes TRPV5 N-glycans to entrap the channel through galectin lattices [#0, #1], FGF23 acts through the FGFR-\\u03b1Klotho complex via an ERK1/2-SGK1-WNK4 cascade [#5], PTH-PKA phosphorylates Thr709 to relieve inhibitory calmodulin binding [#8, #17], PKC phosphorylation of Ser299/Ser654 blocks caveolin-1-dependent endocytosis [#12, #13], and NDPK-B phosphorylates His711 to activate the channel [#23]. Trafficking and stability are further tuned by the S100A10-annexin-2 complex required for plasma membrane delivery [#4], constitutive clathrin- and caveolae-mediated endocytosis with Rab11a-dependent recycling [#18], and calbindin-D28K, which buffers peri-pore Ca2+ to prevent inactivation and sustain high transport rates [#11]. A recurrent kidney-stone-associated L530R variant abolishes Ca2+ transport by disrupting TM5 packing near the selectivity filter [#43].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Established that the TRPV5 C-terminal tail encodes the determinants of Ca2+-dependent inactivation, localizing feedback regulation to a defined cytoplasmic region.\",\n      \"evidence\": \"C-terminal truncation mutagenesis with patch-clamp in HEK293 cells\",\n      \"pmids\": [\"12634930\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not identify the molecular partner mediating inactivation\", \"Mapped regions but not specific residues or interacting Ca2+ sensor\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Defined the channel as a tetramer and resolved outer-pore architecture, answering how TRPV5 achieves Ca2+ selectivity and how subunit composition diversifies channel behavior.\",\n      \"evidence\": \"Sucrose gradient/IP and concatemeric electrophysiology for tetramer assembly; SCAM pore mapping; knockout micropuncture for in vivo function\",\n      \"pmids\": [\"12574114\", \"14630907\", \"14679186\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic structure not yet available\", \"Heterotetramer stoichiometry in native tissue unresolved\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Identified S100A10-annexin-2 as a trafficking chaperone required to route TRPV5 to the plasma membrane via its C-terminal VATTV motif, clarifying how the channel reaches its functional site.\",\n      \"evidence\": \"Yeast two-hybrid, GST pulldown, co-IP, mutagenesis (T599), siRNA, electrophysiology\",\n      \"pmids\": [\"12660155\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of vesicular routing downstream of S100A10 not detailed\", \"Did not address regulated vs constitutive delivery\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Localized extracellular pH sensing to Glu522, defining how luminal acidification gates the channel.\",\n      \"evidence\": \"Single-channel patch-clamp, mutagenesis, MTS cysteine accessibility\",\n      \"pmids\": [\"14525991\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not connect extracellular sensor to intracellular conformational changes (resolved later)\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Showed reciprocal N- and C-tail interactions drive tetrameric assembly and surface trafficking, explaining the dominant-negative effect of tail deletions.\",\n      \"evidence\": \"GST pulldown, co-IP, mutagenesis, oocyte 45Ca2+ uptake and patch-clamp\",\n      \"pmids\": [\"15489237\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of tail-tail contacts not resolved\", \"Did not address regulation of assembly\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identified accessory Ca2+ sensors and kinase/scaffold inputs (80K-H, SGK1/3-NHERF2) modulating activity and surface targeting, broadening the regulatory network.\",\n      \"evidence\": \"Co-IP, 45Ca2+ binding/uptake, EF-hand and PDZ deletion mutagenesis, oocyte electrophysiology\",\n      \"pmids\": [\"15100231\", \"15319523\", \"15665527\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab interactions without in vivo confirmation\", \"Physiological hierarchy among regulators unclear\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Revealed Klotho as a glycosidase that traps TRPV5 at the surface by hydrolyzing N-glycans, providing a hormone-independent mechanism to sustain Ca2+ entry.\",\n      \"evidence\": \"Enzymatic assay, Ca2+ influx, surface expression in HEK293; osteoclast KO; PIP2 electrophysiology\",\n      \"pmids\": [\"16239475\", \"16291808\", \"16230466\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Initial enzymatic identity (glucuronidase vs sialidase) later refined\", \"Did not resolve glycan-lattice partner at this stage\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Established calbindin-D28K as a peri-channel Ca2+ buffer preventing inactivation, and identified additional modulators (BSPRY, FKBP52) plus a pH-driven vesicular surface-recruitment mechanism.\",\n      \"evidence\": \"TIRF microscopy, binding/fractionation, 45Ca2+ uptake, electrophysiology, PPIase mutagenesis\",\n      \"pmids\": [\"16763551\", \"16380433\", \"16352746\", \"17178838\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relative contribution of buffering vs structural inactivation control unquantified\", \"Modulator interactions mostly single-lab\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Distinguished endocytic routes and their hormonal control, showing PKC phosphorylation of Ser299/Ser654 inhibits caveolin-1-mediated retrieval while WNK4 promotes it, tuning surface abundance.\",\n      \"evidence\": \"Dominant-negative dynamin, caveolin-1/clathrin siRNA and KO rescue, mutagenesis, biotinylation, electrophysiology\",\n      \"pmids\": [\"18305097\", \"17006539\", \"20061383\", \"18077461\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo significance of caveolar vs clathrin routes not fully separated\", \"WNK4 mechanism outside kinase domain undefined\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Defined PTH dual action: PKA phosphorylation of Thr709 raising open probability and CaR-coupled PKC stimulation, linking systemic calcium-regulating signals to channel gating and trafficking.\",\n      \"evidence\": \"FRET cAMP/Ca2+, biotinylation, PKA application, mutagenesis, patch-clamp, CaR dominant-negative\",\n      \"pmids\": [\"19423690\", \"19157541\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"T709 phosphorylation mechanism on CaM binding not yet structurally defined (resolved 2011)\", \"CaR coupling single-lab\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Resolved the calmodulin-TRPV5 interaction and stoichiometry and showed PKA-Thr709 phosphorylation enhances activity by displacing inhibitory CaM, unifying gating and hormonal control.\",\n      \"evidence\": \"NMR spectroscopy, mutagenesis (W702A, R706E), patch-clamp; zebrafish ossification genetics\",\n      \"pmids\": [\"21576356\", \"21670068\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-channel structural context of CaM binding not yet available (resolved by cryo-EM)\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Expanded surface regulation to disease-relevant extracellular factors (UMOD, MUC1) and degradation (UBR4), and refined the Klotho glycan/galectin mechanism toward galectin-3.\",\n      \"evidence\": \"Co-IP, siRNA, caveolin-1 KO rescue, N-glycan mutants, in vivo Umod-/- and Klotho colitis models, glycan analysis\",\n      \"pmids\": [\"23466996\", \"27036738\", \"23747339\", \"23970553\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Galectin-1 vs galectin-3 identity in DCT remained debated across studies\", \"UBR4 ubiquitination sites unmapped\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Separated Klotho's hormonal (FGF23-FGFR-\\u03b1Klotho-ERK1/2-SGK1-WNK4) and enzymatic trafficking roles, and identified NDPK-B/PHPT1 histidine phosphorylation of His711 as a distinct activation switch.\",\n      \"evidence\": \"FGF23/\\u03b1Klotho KO mice, pathway inhibition; inside-out patch, NDPK-B shRNA/KO mice, H711 mutagenesis; trafficking dissection\",\n      \"pmids\": [\"24434184\", \"24523290\", \"25378396\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Integration of multiple Klotho mechanisms in vivo incomplete\", \"Histidine-phosphorylation dynamics in tissue unquantified\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Provided atomic structures showing how PI(4,5)P2 opens the lower gate, how calmodulin's Lys116 engages Trp583 for Ca2+-dependent inhibition, and how inhibitors occlude the pore, giving a structural basis for gating.\",\n      \"evidence\": \"Cryo-EM of full-length TRPV5 with PI(4,5)P2, CaM, and econazole; MD simulations; W583 mutagenesis\",\n      \"pmids\": [\"30305626\", \"29323279\", \"28374795\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Dynamic transitions between states inferred not directly captured at first\", \"Native-membrane lipid composition effects unaddressed\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Captured CaM-binding stoichiometric flexibility in nanodiscs and used structure-guided screening to find new inhibitor sites, advancing both mechanism and pharmacology.\",\n      \"evidence\": \"Cryo-EM in lipid nanodiscs, W583A mutant, virtual screening with electrophysiology\",\n      \"pmids\": [\"30975749\", \"31647410\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Therapeutic selectivity over TRPV6 not established\", \"Functional consequence of variable CaM stoichiometry in vivo unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Unified gating modulators structurally, showing low pH inhibits by precluding PI(4,5)P2 activation and that PKA acts by preventing CaM-mediated inactivation rather than directly opening the gate.\",\n      \"evidence\": \"Cryo-EM across multiple pH and modulator conditions capturing intermediate states\",\n      \"pmids\": [\"35476976\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinetics of state transitions in living cells not resolved\", \"Coupling between His711/Thr709 phosphorylation and structural states untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the many converging regulatory inputs (glycan trapping, hormonal kinases, lipid and pH gating, calmodulin, accessory chaperones) are integrated and prioritized within a single native distal-tubule cell to set Ca2+ reabsorption set-points remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No quantitative model integrating trafficking and gating regulators in vivo\", \"Tissue-specific stoichiometry of TRPV5 heteromers (TRPV6, TRPML3) unknown\", \"Human disease genetics beyond a single variant largely uncharacterized in this corpus\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [2, 3, 9, 10, 42]},\n      {\"term_id\": \"GO:0005262\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"GO:0140299\", \"supporting_discovery_ids\": [15, 33]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [11, 17, 20]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 2, 4, 10, 12]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [18, 21]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [18]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [2, 3, 9, 10]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [5, 8, 12, 19]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [12, 18, 21, 24]}\n    ],\n    \"complexes\": [\n      \"TRPV5 homotetramer\",\n      \"TRPV5/TRPV6 heterotetramer\",\n      \"S100A10-annexin 2 complex\",\n      \"TRPV5-calmodulin complex\"\n    ],\n    \"partners\": [\n      \"Klotho\",\n      \"S100A10\",\n      \"calbindin-D28K\",\n      \"calmodulin\",\n      \"NHERF2\",\n      \"MUC1\",\n      \"UBR4\",\n      \"FKBP52\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}