{"gene":"TRPC7","run_date":"2026-04-28T21:43:00","timeline":{"discoveries":[{"year":1998,"finding":"TRPC7 was cloned as a novel human protein of 1503 amino acids from fetal brain and caudate nucleus cDNA libraries. It has seven putative transmembrane domains consistent with a Ca2+ channel and shows significant homology with Drosophila TRP proteins. The TRPC7 gene consists of 32 exons spanning ~90 kb and maps to human chromosome 21q22.3.","method":"cDNA cloning, Northern blot, genomic sequencing, chromosomal mapping","journal":"Genomics","confidence":"High","confidence_rationale":"Tier 1 — original cloning and structural characterization with multiple orthogonal methods","pmids":["9806837"],"is_preprint":false},{"year":2002,"finding":"TRPC7 selectively assembles into homo- or heteromeric channel complexes exclusively within the TRPC3/6/7 subfamily. Co-immunoprecipitation in Sf9 cells and rat brain synaptosomes demonstrated that TRPC3, TRPC6, and TRPC7 co-associate with each other but do not cross-associate with the TRPC1/4/5 subgroup. TRPC3/6/7 subunits also did not interact with the PDZ-containing scaffold protein INAD.","method":"Reciprocal co-immunoprecipitation in heterologous (Sf9) cells and native rat brain synaptosomes","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP in both heterologous and native tissue, replicated across two brain regions","pmids":["12377790"],"is_preprint":false},{"year":2001,"finding":"TRPC7 (and other TRPC family members) possess a conserved IP3 receptor-binding domain and a calmodulin (CaM)-binding domain on their carboxyl termini. In the presence of Ca2+, CaM competes with IP3R for binding to TRPC7, suggesting that CaM and IP3R regulate TRPC7 gating through overlapping binding sites.","method":"GST pulldown, co-immunoprecipitation, gel shift assay, inside-out patch clamp with synthetic peptides","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — multiple binding assays, but TRPC7 not the primary focus; data shown for TRPC7 among several TRPCs","pmids":["11290752"],"is_preprint":false},{"year":2004,"finding":"TRPC7 functions as a receptor-operated, diacylglycerol (DAG)-activated non-selective cation channel when transiently expressed in HEK-293 cells (activated by PLC-stimulating agonists but not by Ca2+ store depletion). When stably expressed in HEK-293 cells, TRPC7 can be activated by either Ca2+ store depletion or PLC activation, demonstrating that expression level/environment determines the mode of TRPC7 gating.","method":"Stable and transient transfection in HEK-293 cells, patch clamp, thapsigargin-based store depletion, agonist stimulation","journal":"American journal of physiology. Cell physiology","confidence":"High","confidence_rationale":"Tier 2 — systematic comparison of stable vs. transient expression with multiple activation paradigms, clear mechanistic conclusion","pmids":["15342342"],"is_preprint":false},{"year":2004,"finding":"TRPC7 currents are inhibited by extracellular Ca2+ (IC50 ~0.4 mM) through voltage-dependent reductions in unitary conductance and open probability at the single-channel level. Single TRPC7 channel activity is concentration-dependently suppressed by nanomolar intracellular Ca2+ via calmodulin (CaM), and is conversely enhanced by IP3. This contrasts with TRPC6, which is potentiated by Ca2+ through a CaMKII-dependent mechanism.","method":"Nystatin-perforated patch clamp, conventional whole-cell clamp, single-channel recording, CaM mutant co-expression, pharmacological inhibitors (calmidazolium, AMP-PNP, CaMKII inhibitory peptide)","journal":"The Journal of physiology","confidence":"High","confidence_rationale":"Tier 1-2 — single-channel electrophysiology with mutagenesis and pharmacological dissection, multiple orthogonal approaches","pmids":["15579537"],"is_preprint":false},{"year":2005,"finding":"Endogenous TRPC1, TRPC3, and TRPC7 form a heteromeric complex in HEK-293 cells that mediates store-operated Ca2+ entry (SOCE). siRNA knockdown of each individually suppressed SOCE by 52–68%, and co-immunoprecipitation confirmed the existence of a TRPC1-TRPC3-TRPC7 heteromeric complex. TRPC3 and TRPC7 (but not TRPC1) also participate in receptor-operated channels.","method":"siRNA knockdown, co-immunoprecipitation, thapsigargin-stimulated Ba2+ entry measurements, 2-APB/SKF96365 pharmacology","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — combined siRNA epistasis and co-IP with multiple subunit combinations, strong mechanistic framework","pmids":["15972814"],"is_preprint":false},{"year":2006,"finding":"Native TRPC7 channel activity (75 pS single channels) in DT40 B lymphocytes requires inositol trisphosphate receptors (IP3Rs) for activation by DAG (OAG). TRPC7-null DT40 cells show no OAG-induced single-channel activity, restored by TRPC7 re-expression; IP3R-null cells similarly lack OAG-induced activity, restored by IP3R re-expression. At high TRPC7 expression levels, channel activity becomes IP3R-independent.","method":"Cell-attached patch clamp in TRPC7-/- and IP3R-/- DT40 B lymphocyte cell lines, genetic rescue experiments","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis using knockout cell lines with reciprocal rescue, single-channel electrophysiology","pmids":["16822861"],"is_preprint":false},{"year":2006,"finding":"TRPC7 channels are subject to negative feedback regulation by Ca2+ entering through the channels themselves. Thapsigargin-mediated inhibition of SERCA pumps blocks DAG-activated TRPC7, an effect dependent on extracellular Ca2+, the driving force for Ca2+ entry, and calmodulin. Pharmacological disruption of the actin cytoskeleton mimics the effect of thapsigargin. This suggests that closely associated SERCA pumps protect TRPC7 from Ca2+-calmodulin-mediated inhibition under physiological conditions.","method":"Whole-cell patch clamp, thapsigargin, calmodulin inhibition, cytochalasin D actin disruption, extracellular Ca2+ manipulation","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 2 — multiple pharmacological interventions with coherent mechanistic interpretation, orthogonal approaches","pmids":["16401641"],"is_preprint":false},{"year":2006,"finding":"TRPC7 is the molecular substrate for DAG-activated receptor-operated Ca2+ entry in human keratinocytes (HaCaT cells). CCh- and ATP-induced cation currents were inhibited by a PLC blocker and mimicked by the DAG analog OAG, but were insensitive to IP3 and to PKC inhibitors, indicating direct DAG activation. Antisense knockdown of TRPC7 reduced ATP- and CCh-induced Ca2+ entry and OAG-evoked current.","method":"Patch clamp, antisense oligonucleotide knockdown, pharmacological dissection (PLC inhibitor, PKC inhibitor, OAG, IP3)","journal":"The Journal of investigative dermatology","confidence":"Medium","confidence_rationale":"Tier 2-3 — antisense knockdown plus pharmacological dissection; single lab","pmids":["16741513"],"is_preprint":false},{"year":2006,"finding":"In rabbit coronary artery myocytes, ET-1 activates a Ca2+-permeable non-selective cation channel with TRPC3 and TRPC7 properties via ETA receptor and PLC. Anti-TRPC3 and anti-TRPC7 antibodies applied to inside-out patches inhibited ET-1-evoked currents; antibodies to TRPC1, C4, C5, C6 had no effect. Immunocytochemistry revealed preferential TRPC7 plasma membrane localization and co-localization of TRPC3 and TRPC7 at or near the plasma membrane.","method":"Cell-attached and inside-out patch clamp with channel-blocking antibodies, immunocytochemistry","journal":"The Journal of physiology","confidence":"Medium","confidence_rationale":"Tier 2-3 — antibody block in inside-out patches plus immunolocalization; single lab","pmids":["17303636"],"is_preprint":false},{"year":2006,"finding":"TRPC7 protein exhibits constitutive activity and is directly activated by DAG downstream of PLC signaling. TRPC7 is uniquely susceptible to negative regulation by extracellular Ca2+, distinguishing it from closely related TRPC3 and TRPC6.","method":"Review synthesizing patch-clamp and molecular biological data from multiple studies","journal":"Handbook of experimental pharmacology","confidence":"Low","confidence_rationale":"Tier 4 — review article, no primary experimental data","pmids":["17217055"],"is_preprint":false},{"year":2006,"finding":"TRPC6 and TRPC7 form heteromeric receptor-operated channels in A7r5 vascular smooth muscle cells that mediate arginine vasopressin (AVP)-induced non-selective cation current. Co-immunoprecipitation confirmed physical TRPC6-TRPC7 association. Dominant-negative TRPC6 (but not TRPC5) suppressed AVP-induced currents. Extracellular Ca2+ suppressed native, TRPC7, and TRPC6-TRPC7 heteromeric currents, but the Ca2+ sensitivity matched the native channel only for the heteromeric TRPC6-TRPC7 construct.","method":"Co-immunoprecipitation, dominant-negative expression, whole-cell patch clamp, extracellular Ca2+ titration","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP plus functional dominant-negative electrophysiology with quantitative Ca2+ pharmacology matching native channel","pmids":["16690880"],"is_preprint":false},{"year":2008,"finding":"Ca2+-dependent inactivation of TRPC7 requires the synergistic actions of both DAG and IP3. OAG-induced TRPC7 currents alone do not undergo persistent Ca2+-mediated inhibition, but co-application of intracellular IP3 renders them susceptible to persistent Ca2+-mediated inactivation independently of IP3 receptors (not blocked by heparin or thapsigargin). Carbachol- and GTPγS-activated TRPC7 currents undergo persistent Ca2+-dependent inhibition that requires strong intracellular Ca2+ buffering (BAPTA) for recovery.","method":"Whole-cell patch clamp, intracellular dialysis with IP3, OAG, GTPγS, BAPTA, heparin, thapsigargin","journal":"Acta pharmacologica Sinica","confidence":"Medium","confidence_rationale":"Tier 2 — systematic patch clamp dissection of inactivation mechanism; single lab","pmids":["18158870"],"is_preprint":false},{"year":2008,"finding":"Heterologously expressed TRPC3 and TRPC7 localize not only to the plasma membrane but also to intracellular Golgi (both cis-Golgi and trans-Golgi network) compartments in COS-7 cells, as shown by co-localization with Golgi markers and Brefeldin A redistribution. Expression of TRPC3 or TRPC7 (but not TRPC1) increased constitutive secretion of alkaline phosphatase 2–4-fold, suggesting these channels enhance vesicular transport/secretion.","method":"Immunofluorescence microscopy, Brefeldin A treatment, alkaline phosphatase secretion assay","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2-3 — co-localization with Golgi markers and functional secretion assay; single lab","pmids":["18452405"],"is_preprint":false},{"year":2009,"finding":"A pathogenic C-terminus-truncated polycystin-2 (TRPP2) mutant (697fsX) physically associates with TRPC3 and TRPC7 at the plasma membrane, and this association enhances muscarinic receptor-activated Ca2+ influx and alters ion-permeating pore properties (depolarizing shift in reversal potential, enhanced single-channel conductance) of the mAChR-activated current. Co-immunoprecipitation, pulldown, and cross-linking confirmed the TRPP2 mutant-TRPC7 interaction.","method":"Co-immunoprecipitation, pulldown assay, cross-linking, whole-cell patch clamp, single-channel recording, immunofluorescence","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — multiple biochemical interaction assays plus functional electrophysiology with pore property changes; strong mechanistic evidence","pmids":["19812035"],"is_preprint":false},{"year":2019,"finding":"TRPC7 acts as a nociceptive mechanoreceptor that specifically mediates UVB-induced Ca2+ influx and subsequent reactive oxygen species production in skin cells within 30 minutes of irradiation. In TRPC7 knockout mice, UVB-associated epidermal thickening, abnormal keratinocyte differentiation, DNA damage response activation, p53 family mutations, and tumor formation were all prevented, establishing TRPC7 as a required initiator of UVB-induced skin aging and tumorigenesis.","method":"TRPC7 knockout mice, Ca2+ influx assays, ROS measurements, histology, immunostaining, mutation analysis","journal":"Aging cell","confidence":"High","confidence_rationale":"Tier 2 — genetic knockout with multiple orthogonal phenotypic readouts; mechanistic Ca2+/ROS pathway established","pmids":["31755176"],"is_preprint":false},{"year":2021,"finding":"TRPC7 regulates the automaticity (spontaneous action potential firing) of mouse embryonic stem cell-derived cardiomyocytes via two mechanisms: (1) positively regulating the intracellular Ca2+ clock by enhancing RyR2 phosphorylation at S2814 and SERCA activity (via phospholamban phosphorylation at T17); (2) positively regulating the membrane clock via NCX activity. These effects are Ca2+-dependent, as a dominant-negative N-terminal TRPC7 construct that abolishes Ca2+ permeability reduces AP frequency.","method":"Adenoviral TRPC7 knockdown/overexpression, confocal Ca2+ imaging, whole-cell patch clamp, Western blot for phosphorylation, dominant-negative TRPC7 N-terminus construct","journal":"Stem cell research & therapy","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal approaches (KD, OE, dominant-negative, phosphorylation readouts, Ca2+ imaging, electrophysiology) in same study","pmids":["33941260"],"is_preprint":false},{"year":2021,"finding":"A truncation mutation in the Trpc7 cytoplasmic C-terminus (stop codon at amino acid 810, affecting the conserved C-terminal domain) produces a dominant-negative effect in heterozygous mice, causing distinct morphological and behavioral alterations not seen in Trpc7 knockout mice, demonstrating that the C-terminus is critical for normal TRPC7 function.","method":"ENU mutagenesis mouse model, Sanger sequencing, phenotypic characterization of heterozygous mutants","journal":"Genes","confidence":"Medium","confidence_rationale":"Tier 2-3 — in vivo genetic model with defined mutation, but phenotypic characterization without molecular mechanism of C-terminus function","pmids":["34828338"],"is_preprint":false},{"year":2022,"finding":"TRPC7 undergoes conformational changes upon activation by Gαq-coupled receptors, as detected by intramolecular BRET biosensors (GFP10-TRPC7-RLucII). Activation of Gαq-coupled receptors (including the angiotensin II AT1R) induces a Gαq-dependent BRET response in both HEK293 cells and rat neonatal cardiac fibroblasts expressing endogenous AT1R and TRPC7, confirming receptor-induced structural rearrangements in TRPC7.","method":"Intramolecular BRET biosensors in HEK293 cells and primary rat cardiac fibroblasts, GPCR agonist stimulation, Gαq pathway pharmacology","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 — functional biosensor validated in both heterologous and native cell context; single lab","pmids":["35269644"],"is_preprint":false},{"year":2023,"finding":"TRPC7 facilitates lung adenocarcinoma cell growth and migration via Ca2+-dependent activation of CaMKII, AKT, and ERK signaling pathways. TRPC7 knockdown restrained cell cycle progression and migration by interrupting TRPC7-mediated Ca2+ signaling, upstream of AKT and MAPK pathway activation.","method":"TRPC7 siRNA knockdown, cell cycle analysis, invasion assay, Ca2+ response assay, immunoblot for phospho-CaMKII/AKT/ERK","journal":"Oncology letters","confidence":"Medium","confidence_rationale":"Tier 2-3 — loss-of-function with defined pathway readouts; single lab","pmids":["36817036"],"is_preprint":false},{"year":2016,"finding":"In normal rat cardiomyocytes, TRPC7 localizes to the surface sarcolemma and striated subcellular (T-tubule) regions. In renovascular hypertrophic hearts, TRPC7 expression is significantly downregulated while PKC expression is upregulated, with a negative correlation between TRPC7 and PKC levels. Losartan (angiotensin II receptor blocker) reverses TRPC7 downregulation, implicating Ang II/PKC signaling in the suppression of TRPC7 in cardiac hypertrophy.","method":"Immunohistochemistry, immunocytochemistry, Western blot, 2K1C renovascular hypertension model, losartan treatment","journal":"Journal of cardiovascular pharmacology","confidence":"Medium","confidence_rationale":"Tier 3 — localization with functional context in disease model, but mechanistic link between PKC and TRPC7 is correlative","pmids":["26371949"],"is_preprint":false}],"current_model":"TRPC7 is a plasma membrane Ca2+-permeable non-selective cation channel of the TRPC3/6/7 subfamily that is activated downstream of Gαq-PLC signaling, primarily by diacylglycerol (DAG) acting directly on the channel; it forms homo- and heteromeric complexes with TRPC3 and TRPC6, is negatively regulated by extracellular Ca2+ via calmodulin and by PKC, is protected from Ca2+-CaM-mediated inhibition by closely associated SERCA pumps, requires IP3 receptors for native DAG-dependent gating at physiological expression levels, undergoes Gαq-induced conformational changes, mediates UVB-induced Ca2+ influx and ROS production in skin, and regulates cardiac pacemaking by modulating RyR2 and SERCA phosphorylation to control both intracellular Ca2+ and membrane clocks."},"narrative":{"teleology":[{"year":1998,"claim":"Identification of TRPC7 as a novel TRP-family member established the existence of a seventh human canonical TRP channel with predicted transmembrane topology consistent with a Ca²⁺-permeable pore.","evidence":"cDNA cloning from human fetal brain and caudate nucleus libraries with genomic mapping to 21q22.3","pmids":["9806837"],"confidence":"High","gaps":["No functional electrophysiology performed","Expression pattern beyond Northern blot unknown","Activation mechanism not addressed"]},{"year":2001,"claim":"Discovery of conserved IP3R-binding and calmodulin-binding domains on the TRPC7 C-terminus, with competitive binding between the two, provided the first molecular framework for Ca²⁺-dependent channel regulation.","evidence":"GST pulldown and co-immunoprecipitation showing overlapping CaM and IP3R binding sites on TRPC C-termini","pmids":["11290752"],"confidence":"Medium","gaps":["TRPC7 was studied alongside other TRPCs, not as primary target","Functional consequence of CaM vs. IP3R competition on TRPC7 currents not tested","Binding site residues not mapped"]},{"year":2002,"claim":"Demonstration that TRPC7 selectively co-assembles with TRPC3 and TRPC6 — but not with the TRPC1/4/5 subgroup — defined the combinatorial rules governing heteromeric TRP channel assembly.","evidence":"Reciprocal co-immunoprecipitation in Sf9 cells and native rat brain synaptosomes","pmids":["12377790"],"confidence":"High","gaps":["Stoichiometry of heteromeric complexes unknown","No structural basis for subfamily-restricted assembly"]},{"year":2004,"claim":"Electrophysiological characterization established TRPC7 as a DAG-activated receptor-operated channel whose gating mode (receptor-operated vs. store-operated) depends on expression level, and revealed unique negative regulation by extracellular Ca²⁺ acting on unitary conductance and open probability via intracellular calmodulin.","evidence":"Patch-clamp comparison of stable vs. transient TRPC7 expression in HEK-293 cells; single-channel recordings with CaM mutants and pharmacological inhibitors","pmids":["15342342","15579537"],"confidence":"High","gaps":["Molecular determinants of extracellular Ca²⁺ sensing not identified","Structural basis for DAG binding site unknown"]},{"year":2005,"claim":"Identification of an endogenous TRPC1–TRPC3–TRPC7 heteromeric complex mediating store-operated Ca²⁺ entry in HEK-293 cells showed that TRPC7 contributes to native SOCE channels, not only receptor-operated channels.","evidence":"siRNA knockdown of individual subunits combined with co-immunoprecipitation and thapsigargin-stimulated Ba²⁺ entry","pmids":["15972814"],"confidence":"High","gaps":["SOCE role not confirmed in primary cells","Relationship to Orai/STIM-mediated SOCE unclear"]},{"year":2006,"claim":"Multiple studies converged to show that (i) native TRPC7 gating by DAG requires IP3 receptors as permissive cofactors at physiological expression levels, (ii) SERCA pumps physically associated with TRPC7 protect the channel from Ca²⁺-CaM–mediated inactivation, and (iii) TRPC6–TRPC7 heteromers constitute the native receptor-operated channel in vascular smooth muscle.","evidence":"Genetic epistasis in TRPC7⁻/⁻ and IP3R⁻/⁻ DT40 cells with rescue; thapsigargin/cytochalasin D pharmacology in HEK cells; co-IP and dominant-negative electrophysiology in A7r5 cells and coronary myocytes","pmids":["16822861","16401641","16690880","17303636","16741513"],"confidence":"High","gaps":["Mechanism by which IP3R licenses DAG gating is unknown","Identity of the SERCA isoform and mode of physical coupling not determined","Native stoichiometry of TRPC6/7 heteromers in vivo undefined"]},{"year":2008,"claim":"Dissection of TRPC7 inactivation showed that persistent Ca²⁺-dependent shutdown requires synergistic action of both DAG and IP3, revealing a dual-signal integration mechanism for channel silencing independent of IP3 receptor opening.","evidence":"Whole-cell patch clamp with intracellular dialysis of IP3, OAG, and Ca²⁺ chelators in HEK-293 cells","pmids":["18158870"],"confidence":"Medium","gaps":["Molecular target of IP3's direct sensitizing action not identified","In vivo relevance of dual-signal inactivation not tested"]},{"year":2008,"claim":"Detection of TRPC7 at Golgi compartments and its ability to enhance constitutive secretion suggested a second site of action beyond the plasma membrane.","evidence":"Immunofluorescence co-localization with Golgi markers and alkaline phosphatase secretion assay in COS-7 cells","pmids":["18452405"],"confidence":"Medium","gaps":["Golgi localization not confirmed in primary cells","Mechanism linking TRPC7 channel activity to vesicular secretion undefined"]},{"year":2009,"claim":"Demonstration that a disease-associated truncated polycystin-2 (TRPP2) mutant physically associates with TRPC7 and alters its pore properties linked TRPC7 to polycystic kidney disease-relevant signaling complexes.","evidence":"Co-IP, cross-linking, and single-channel recordings showing altered conductance and reversal potential of TRPC7 in complex with TRPP2-697fsX","pmids":["19812035"],"confidence":"High","gaps":["Interaction with wild-type TRPP2 not equivalently characterized","Relevance to renal cyst formation in vivo not demonstrated"]},{"year":2016,"claim":"Localization of TRPC7 to sarcolemma and T-tubules in cardiomyocytes, with downregulation in hypertrophic hearts reversible by angiotensin receptor blockade, implicated TRPC7 in cardiac remodeling via Ang II/PKC signaling.","evidence":"Immunohistochemistry and Western blot in a 2K1C renovascular hypertension rat model with losartan rescue","pmids":["26371949"],"confidence":"Medium","gaps":["PKC–TRPC7 regulatory mechanism is correlative, not causal","Direct TRPC7 phosphorylation by PKC not demonstrated in this model"]},{"year":2019,"claim":"Genetic ablation in mice established TRPC7 as the obligate initiator of UVB-induced Ca²⁺ influx, ROS production, and downstream photoaging/tumorigenesis in skin, revealing a physiological nociceptive-mechanoreceptor role.","evidence":"TRPC7 knockout mice with multi-parameter phenotyping including Ca²⁺ influx, ROS, histology, DNA damage, and tumor formation","pmids":["31755176"],"confidence":"High","gaps":["Mechanism of UVB-induced TRPC7 activation (direct photomechanical vs. lipid-mediated) unknown","Cell-type specificity within the epidermis not resolved"]},{"year":2021,"claim":"TRPC7 was shown to regulate cardiomyocyte pacemaker automaticity by coupling Ca²⁺ entry to dual clocks: the intracellular Ca²⁺ clock (via RyR2-S2814 and PLB-T17 phosphorylation/SERCA activation) and the membrane clock (via NCX), while a C-terminal truncation mutation produced dominant-negative effects in vivo distinct from null.","evidence":"Adenoviral KD/OE and dominant-negative constructs in mESC-derived cardiomyocytes with Ca²⁺ imaging and electrophysiology; ENU mutagenesis mouse with C-terminal stop codon","pmids":["33941260","34828338"],"confidence":"High","gaps":["Kinase(s) mediating TRPC7-dependent RyR2/PLB phosphorylation not identified","Dominant-negative mechanism of C-terminal truncation not molecularly defined"]},{"year":2022,"claim":"Detection of Gαq-induced intramolecular conformational changes in TRPC7 via BRET biosensors provided the first real-time evidence of receptor-triggered structural rearrangements in the intact channel.","evidence":"Intramolecular BRET (GFP10-TRPC7-RLucII) in HEK293 cells and primary rat cardiac fibroblasts upon AT1R stimulation","pmids":["35269644"],"confidence":"Medium","gaps":["Nature and location of conformational change not resolved at atomic level","Relationship between BRET change and channel gating transitions not established"]},{"year":2023,"claim":"TRPC7-mediated Ca²⁺ entry was linked to lung adenocarcinoma proliferation and migration through CaMKII/AKT/ERK signaling, extending TRPC7's pathophysiological relevance beyond skin and heart.","evidence":"siRNA knockdown with cell cycle, migration, and phospho-signaling readouts in lung adenocarcinoma cell lines","pmids":["36817036"],"confidence":"Medium","gaps":["No in vivo tumor model","Selectivity for TRPC7 vs. other DAG-activated TRPCs not addressed","Direct vs. indirect activation of CaMKII/AKT/ERK not resolved"]},{"year":null,"claim":"Key unresolved questions include the structural basis for DAG binding and extracellular Ca²⁺ sensing, the mechanism by which IP3 receptors license TRPC7 gating at physiological expression, the identity of kinases linking TRPC7-mediated Ca²⁺ entry to RyR2/PLB phosphorylation in cardiomyocytes, and the biophysical mechanism of UVB-induced channel activation in skin.","evidence":"","pmids":[],"confidence":"High","gaps":["No high-resolution structure of TRPC7","DAG binding site not mapped","IP3R–TRPC7 coupling mechanism molecularly undefined","UVB activation mechanism unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[3,4,6,8,11]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[16]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[3,9,13,20]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[13]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,4,6,8,11,18]},{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[3,4,6,8,11]}],"complexes":["TRPC3/TRPC7 heteromeric channel","TRPC6/TRPC7 heteromeric channel","TRPC1/TRPC3/TRPC7 heteromeric channel"],"partners":["TRPC3","TRPC6","TRPC1","ITPR1","CALM1","PKD2","RYR2"],"other_free_text":[]},"mechanistic_narrative":"TRPC7 is a plasma membrane Ca²⁺-permeable non-selective cation channel of the TRPC3/6/7 subfamily that is directly activated by diacylglycerol (DAG) downstream of Gαq-coupled receptor–PLC signaling and assembles into homomeric or heteromeric complexes exclusively with TRPC3 and TRPC6 [PMID:12377790, PMID:16690880]. Channel activity is negatively regulated by extracellular Ca²⁺ through voltage-dependent reductions in conductance and open probability, and by intracellular Ca²⁺ via calmodulin, while closely associated SERCA pumps protect the channel from Ca²⁺-CaM–mediated inactivation; at physiological expression levels, IP3 receptors are required as permissive cofactors for DAG-dependent gating [PMID:15579537, PMID:16401641, PMID:16822861]. In cardiomyocytes, TRPC7-mediated Ca²⁺ entry drives both the intracellular Ca²⁺ clock (via RyR2 and phospholamban phosphorylation) and the membrane clock to regulate pacemaker automaticity [PMID:33941260]. In skin, TRPC7 is the obligate mediator of UVB-induced Ca²⁺ influx and reactive oxygen species production, and its genetic ablation prevents UVB-driven epidermal thickening, DNA damage, and tumorigenesis in mice [PMID:31755176]."},"prefetch_data":{"uniprot":{"accession":"Q9HCX4","full_name":"Short transient receptor potential channel 7","aliases":["Transient receptor protein 7","TRP-7","hTRP7"],"length_aa":862,"mass_kda":99.6,"function":"Forms a receptor-activated non-selective calcium permeant cation channel. Probably is operated by a phosphatidylinositol second messenger system activated by receptor tyrosine kinases or G-protein coupled receptors. Activated by diacylglycerol (DAG) (By similarity). 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Its amino acid sequence shows significant homology with Drosophila and human TRP proteins.\",\n      \"method\": \"cDNA cloning, Northern blot, genomic sequencing, chromosomal mapping\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — original molecular cloning with multiple orthogonal methods, highly cited foundational paper\",\n      \"pmids\": [\"9806837\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"TRPC7 functions as a receptor-operated channel activated by PLC-coupled agonists (e.g., carbachol) via diacylglycerol (DAG), and can also function as a store-operated channel when stably expressed in HEK-293 cells, but not when transiently expressed, demonstrating that expression level/environment determines gating mode.\",\n      \"method\": \"Stable and transient transfection of HEK-293 cells, patch-clamp electrophysiology, thapsigargin-induced store depletion, agonist stimulation\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — patch-clamp with controlled expression conditions, multiple activation paradigms tested\",\n      \"pmids\": [\"15342342\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Extracellular Ca2+ inhibits TRPC7 currents (IC50 ~0.4 mM), an effect associated with voltage-dependent reductions in unitary conductance and open probability. Single TRPC7 channel activity is suppressed by nanomolar intracellular Ca2+ via calmodulin (CaM), and enhanced by IP3. This contrasts with TRPC6, which is potentiated by Ca2+ via CaMKII.\",\n      \"method\": \"Nystatin-perforated patch-clamp, conventional whole-cell patch-clamp, single-channel recording, CaM mutant co-expression, pharmacological inhibitors\",\n      \"journal\": \"The Journal of physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — single-channel electrophysiology with mutagenesis and multiple pharmacological controls\",\n      \"pmids\": [\"15579537\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Endogenous TRPC1, TRPC3, and TRPC7 form a heteromeric complex that mediates store-operated Ca2+ entry in HEK-293 cells, as demonstrated by co-immunoprecipitation and siRNA knockdown of individual subunits. TRPC3 and TRPC7 also participate in receptor-operated channels independently of TRPC1.\",\n      \"method\": \"siRNA knockdown, thapsigargin-induced Ba2+ entry assay, co-immunoprecipitation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP plus functional siRNA knockdown with multiple combinations, highly cited\",\n      \"pmids\": [\"15972814\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"TRPC7 activation by diacylglycerol (OAG) is blocked by the SERCA pump inhibitor thapsigargin in a manner dependent on extracellular Ca2+ and Ca2+ entry through TRPC7 itself. This negative feedback by Ca2+ entering through TRPC7 is mediated by calmodulin and is attenuated under physiological conditions by closely associated SERCA pumps. Disruption of the actin cytoskeleton mimics thapsigargin's inhibitory effect.\",\n      \"method\": \"Patch-clamp electrophysiology, thapsigargin treatment, calmodulin inhibition, actin cytoskeleton disruption\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal pharmacological approaches identifying a novel Ca2+-calmodulin-SERCA regulatory mechanism\",\n      \"pmids\": [\"16401641\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Native TRPC7 in DT40 B lymphocytes forms a DAG-activated non-selective cation channel (75 pS conductance) requiring inositol trisphosphate receptors (IP3R) for activation at physiological expression levels. At higher expression levels, TRPC7 can function independently of IP3R.\",\n      \"method\": \"Cell-attached single-channel patch-clamp, TRPC7 knockout DT40 cells, IP3R knockout DT40 cells, rescue by transient expression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — single-channel electrophysiology in knockout cell lines with rescue experiments\",\n      \"pmids\": [\"16822861\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"TRPC7 mediates DAG-activated, Ca2+-permeable cation channel activity in human keratinocytes downstream of muscarinic and purinergic receptors via PLC/DAG signaling, independently of PKC. Antisense knockdown of TRPC7 reduced ATP- and carbachol-induced Ca2+ entry and OAG-evoked current.\",\n      \"method\": \"Patch-clamp electrophysiology, antisense oligonucleotide knockdown, pharmacological dissection of PLC/DAG/PKC pathway\",\n      \"journal\": \"The Journal of investigative dermatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — antisense KD with functional Ca2+ assay, single lab\",\n      \"pmids\": [\"16741513\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"TRPC6 and TRPC7 form heteromultimeric complexes in A7r5 vascular smooth muscle cells, contributing to arginine vasopressin-induced receptor-operated cation current. Association was detected by co-immunoprecipitation, and dominant-negative TRPC6 suppressed AVP-induced current. Extracellular Ca2+ (0.01–1 mM) suppresses heteromultimeric TRPC6-TRPC7 and native current similarly.\",\n      \"method\": \"Co-immunoprecipitation, dominant-negative expression, patch-clamp electrophysiology, immunoblotting\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — co-IP plus functional dominant-negative and electrophysiology with multiple Ca2+ conditions\",\n      \"pmids\": [\"16690880\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"In rabbit coronary artery myocytes, endothelin-1 activates a Ca2+-permeable non-selective cation channel via ETA receptor/PLC/DAG signaling that is blocked by anti-TRPC3 and anti-TRPC7 antibodies applied to inside-out patches. TRPC7 is preferentially expressed at the plasmalemma, co-localizing with TRPC3 near the plasma membrane.\",\n      \"method\": \"Inside-out patch-clamp with inhibitory antibodies, immunocytochemistry, pharmacological dissection (BQ123, U73122, OAG, heparin)\",\n      \"journal\": \"The Journal of physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — direct antibody block in inside-out patches plus immunolocalization, multiple pharmacological controls\",\n      \"pmids\": [\"17303636\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Ca2+-dependent inactivation of TRPC7 requires the cooperative actions of both DAG and IP3. IP3 (independently of IP3 receptors) renders the DAG-activated TRPC7 current susceptible to persistent Ca2+-mediated inhibition. Ca2+ entering through activated TRPC7 channels plays a critical role in this inactivation.\",\n      \"method\": \"Whole-cell patch-clamp, intracellular dialysis with IP3, heparin, BAPTA buffering, thapsigargin, pharmacological controls\",\n      \"journal\": \"Acta pharmacologica Sinica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — patch-clamp with pharmacological dissection, single lab\",\n      \"pmids\": [\"18158870\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Heterologously expressed TRPC7 localizes to both the plasma membrane and intracellular Golgi/trans-Golgi network compartments. Expression of TRPC7 (but not TRPC1) enhances constitutive secretion 2–4-fold, as measured by secreted alkaline phosphatase assay, suggesting a role in vesicular transport or membrane fusion.\",\n      \"method\": \"Immunofluorescence microscopy with Golgi markers, brefeldin A treatment, secreted alkaline phosphatase secretion assay\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — co-localization with organelle markers plus functional secretion assay, single lab\",\n      \"pmids\": [\"18452405\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"A pathogenic C-terminus-truncated TRPP2 mutant (697fsX) physically associates with TRPC3 and TRPC7 (shown by co-immunoprecipitation, pulldown, and cross-linking), localizes to the plasma membrane, and enhances muscarinic receptor-activated Ca2+ influx and alters ion-permeating pore properties of TRPC3/TRPC7-containing channels.\",\n      \"method\": \"Co-immunoprecipitation, pulldown assay, cross-linking, whole-cell patch-clamp, immunofluorescence localization\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — three independent biochemical interaction assays plus electrophysiological functional characterization\",\n      \"pmids\": [\"19812035\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In normal rat cardiomyocytes, TRPC7 is localized at the surface sarcolemma and in subcellular (tubular) regions. In renovascular hypertension-induced cardiac hypertrophy, TRPC7 expression is significantly downregulated while PKC expression is upregulated, and TRPC7 and PKC levels show a negative correlation. Losartan treatment reverses TRPC7 downregulation.\",\n      \"method\": \"Immunohistochemistry, immunocytochemistry, Western blot, 2-kidney 1-clip hypertension model, losartan treatment\",\n      \"journal\": \"Journal of cardiovascular pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — direct localization by immunostaining with functional disease context, correlation with PKC\",\n      \"pmids\": [\"26371949\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TRPC7 mediates UVB-induced Ca2+ influx and subsequent reactive oxygen species production in skin cells within 30 minutes of UVB irradiation. In TRPC7 knockout mice, UVB-induced epidermal thickening, abnormal keratinocyte differentiation, DNA damage response, and p53 family mutations were all absent, establishing TRPC7 as a nociceptive mechanoreceptor and initiator of UVB-induced skin aging and tumorigenesis.\",\n      \"method\": \"TRPC7 knockout mice, Ca2+ influx assay, ROS measurement, histology, DNA damage assays, tumor development scoring\",\n      \"journal\": \"Aging cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — knockout animal model with multiple orthogonal functional readouts\",\n      \"pmids\": [\"31755176\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TRPC7 regulates automaticity of mouse ESC-derived cardiomyocytes by positively modulating the Ca2+ clock via RyR2 and SERCA activity (through phosphorylation of RyR2-S2814 and phospholamban-T17) and the membrane clock via NCX activity. This is Ca2+-dependent, as a dominant-negative N-terminus construct that abolishes Ca2+ permeability decreases action potential frequency.\",\n      \"method\": \"Adenovirus-mediated knockdown and overexpression, confocal Ca2+ imaging, whole-cell patch-clamp, Western blot for phosphorylation, dominant-negative TRPC7\",\n      \"journal\": \"Stem cell research & therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KD/OE with dominant-negative and phosphorylation readouts in a defined cellular context, single lab\",\n      \"pmids\": [\"33941260\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Activation of Gαq-coupled receptors (e.g., angiotensin II AT1R) induces conformational changes in TRPC7, detected by intramolecular BRET biosensors (GFP10-TRPC7-RLucII). This response was validated in rat neonatal cardiac fibroblasts expressing endogenous AT1R and TRPC7.\",\n      \"method\": \"BRET intramolecular biosensor, HEK293 cells, rat neonatal cardiac fibroblasts, GPCR agonist stimulation\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — BRET conformational biosensor validated in endogenous expression context\",\n      \"pmids\": [\"35269644\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TRPC7 facilitates cell growth and migration in lung adenocarcinoma cells by mediating Ca2+ influx that activates CaMKII, AKT, and ERK phosphorylation. TRPC7 knockdown arrests cell cycle progression and inhibits migration by disrupting TRPC7-mediated Ca2+ signaling upstream of AKT and MAPK pathways.\",\n      \"method\": \"TRPC7 knockdown, cell cycle analysis, invasion assay, Ca2+ response assay, immunoblot for phospho-CaMKII/AKT/ERK\",\n      \"journal\": \"Oncology letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — KD with multiple signaling readouts, single lab\",\n      \"pmids\": [\"36817036\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"A truncating ENU-induced mutation in the Trpc7 C-terminus (stop codon at position 810) produces a dominant-negative effect in heterozygous mice, causing distinct morphological and behavioral alterations not seen in Trpc7 knockout mice, demonstrating that the cytoplasmic C-terminus is critical for TRPC7 function.\",\n      \"method\": \"ENU mutagenesis screen, Sanger sequencing, phenotypic characterization of heterozygous vs. knockout mice\",\n      \"journal\": \"Genes\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo dominant-negative allele with phenotypic characterization\",\n      \"pmids\": [\"34828338\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TRPC7 is a Gq-coupled receptor-operated, DAG-activated Ca2+-permeable non-selective cation channel that can also function as a store-operated channel depending on expression levels; it forms heteromeric complexes with TRPC1, TRPC3, and TRPC6, is negatively regulated by extracellular and intracellular Ca2+ via calmodulin (with SERCA pumps providing local protection from this inhibition), requires IP3 receptors for activation at physiological expression levels, undergoes conformational changes upon Gαq-GPCR activation, and plays roles in vascular smooth muscle contraction, keratinocyte Ca2+ entry, cardiomyocyte automaticity, UVB-induced Ca2+ influx and skin tumorigenesis, and lung adenocarcinoma cell growth via CaMKII/AKT/ERK signaling.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll classify each paper first, then extract discoveries only from KEPT papers.\n\n**Classification:**\n\n**Main corpus (23 papers):**\n- [1] PMID:9806837 — KEEP (TRPC7 cloning)\n- [2] PMID:15972814 — KEEP (TRPC7 in HEK-293)\n- [3] PMID:15579537 — KEEP (TRPC7 regulation)\n- [4] PMID:16690880 — KEEP (TRPC7 in VSM)\n- [5] PMID:17303636 — KEEP (TRPC7 in coronary artery)\n- [6] PMID:15342342 — KEEP (TRPC7 activation modes)\n- [7] PMID:16741513 — KEEP (TRPC7 in keratinocytes)\n- [8] PMID:16822861 — KEEP (TRPC7 native activation)\n- [9] PMID:16401641 — KEEP (TRPC7 regulation by SERCA)\n- [10] PMID:19812035 — KEEP (TRPC7 with TRPP2 mutant)\n- [11] PMID:32585234 — EXCLUDE (case B: lncRNA TRPC7-AS1, not TRPC7 protein)\n- [12] PMID:31755176 — KEEP (TRPC7 in UVB/skin)\n- [13] PMID:17217055 — KEEP (TRPC7 review with mechanistic content)\n- [14] PMID:33941260 — KEEP (TRPC7 in cardiomyocytes)\n- [15] PMID:34512754 — EXCLUDE (case B: lncRNA TRPC7-AS1)\n- [16] PMID:18452405 — KEEP (TRPC7 localization/secretion)\n- [17] PMID:38320625 — EXCLUDE (case B: lncRNA TRPC7-AS1)\n- [18] PMID:36817036 — KEEP (TRPC7 in lung adenocarcinoma)\n- [19] PMID:27161232 — KEEP (TRPC7 review)\n- [20] PMID:35269644 — KEEP (TRPC7 BRET biosensor)\n- [21] PMID:18158870 — KEEP (TRPC7 Ca2+-dependent inactivation)\n- [22] PMID:26371949 — KEEP (TRPC7 in cardiomyocytes)\n- [23] PMID:34828338 — KEEP (TRPC7 mutant mice)\n\n**Additional gene2pubmed papers (30 papers):**\n- [1] PMID:12477932 — EXCLUDE (genome-wide cDNA, not mechanistic TRPC7)\n- [2] PMID:10830953 — EXCLUDE (chromosome 21 sequence, not mechanistic)\n- [3] PMID:11385575 — EXCLUDE (ALIAS COLLISION: describes LTRPC2/TRPM2, not TRPC7; \"TRPC7\" used as old alias for TRPM2)\n- [4] PMID:11804595 — EXCLUDE (TRPM2/LTRPC2, not TRPC7)\n- [5] PMID:21873635 — EXCLUDE (GO annotation, not mechanistic)\n- [6] PMID:12032305 — KEEP (TRPC subunit assembly, includes TRPC3/6/7 subfamily rules)\n- [7] PMID:18542050 — EXCLUDE (TRPM2, not TRPC7)\n- [8] PMID:11509734 — EXCLUDE (LTRPC2/TRPM2, not TRPC7)\n- [9] PMID:16601673 — EXCLUDE (TRPM2, not TRPC7)\n- [10] PMID:16382100 — EXCLUDE (general TRP nomenclature, no specific TRPC7 mechanism)\n- [11] PMID:11960981 — EXCLUDE (LTRPC2/TRPM2, not TRPC7)\n- [12] PMID:15561722 — EXCLUDE (TRPM2, not TRPC7)\n- [13] PMID:28611215 — EXCLUDE (HNRNPL/prostate cancer, not TRPC7)\n- [14] PMID:15808509 — EXCLUDE (TRPM2, not TRPC7)\n- [15] PMID:12377790 — KEEP (TRPC3/6/7 subunit assembly in synaptosomes)\n- [16] PMID:12529379 — EXCLUDE (TRPM2, not TRPC7)\n- [17] PMID:11290752 — KEEP (CaM/IP3R binding on TRPC including TRPC7)\n- [18] PMID:19454650 — EXCLUDE (TRPM2, not TRPC7)\n- [19] PMID:12594222 — EXCLUDE (TRPM2, not TRPC7)\n- [20] PMID:18048770 — EXCLUDE (TRPM2, not TRPC7)\n- [21] PMID:12564954 — EXCLUDE (LTRPC2/TRPM2 in neutrophils, not TRPC7)\n- [22] PMID:30467180 — EXCLUDE (TRPM2 structure, not TRPC7)\n- [23] PMID:16585058 — EXCLUDE (ALIAS COLLISION: paper explicitly states \"TRPM2 (previously designated TRPC7 or LTRPC2)\" — this is TRPM2, not TRPC7)\n- [24] PMID:22493272 — EXCLUDE (TRPM2, not TRPC7)\n- [25] PMID:16565078 — EXCLUDE (TRPM2, not TRPC7)\n- [26] PMID:18599483 — EXCLUDE (TRPM2, not TRPC7)\n- [27] PMID:29745897 — EXCLUDE (TRPM2 structure, not TRPC7)\n- [28] PMID:26558786 — EXCLUDE (TRPM2, not TRPC7)\n- [29] PMID:20107186 — EXCLUDE (TRPM2, not TRPC7)\n- [30] PMID:16461353 — EXCLUDE (TRPM2, not TRPC7)\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1998,\n      \"finding\": \"TRPC7 was cloned as a novel human protein of 1503 amino acids from fetal brain and caudate nucleus cDNA libraries. It has seven putative transmembrane domains consistent with a Ca2+ channel and shows significant homology with Drosophila TRP proteins. The TRPC7 gene consists of 32 exons spanning ~90 kb and maps to human chromosome 21q22.3.\",\n      \"method\": \"cDNA cloning, Northern blot, genomic sequencing, chromosomal mapping\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — original cloning and structural characterization with multiple orthogonal methods\",\n      \"pmids\": [\"9806837\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"TRPC7 selectively assembles into homo- or heteromeric channel complexes exclusively within the TRPC3/6/7 subfamily. Co-immunoprecipitation in Sf9 cells and rat brain synaptosomes demonstrated that TRPC3, TRPC6, and TRPC7 co-associate with each other but do not cross-associate with the TRPC1/4/5 subgroup. TRPC3/6/7 subunits also did not interact with the PDZ-containing scaffold protein INAD.\",\n      \"method\": \"Reciprocal co-immunoprecipitation in heterologous (Sf9) cells and native rat brain synaptosomes\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP in both heterologous and native tissue, replicated across two brain regions\",\n      \"pmids\": [\"12377790\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"TRPC7 (and other TRPC family members) possess a conserved IP3 receptor-binding domain and a calmodulin (CaM)-binding domain on their carboxyl termini. In the presence of Ca2+, CaM competes with IP3R for binding to TRPC7, suggesting that CaM and IP3R regulate TRPC7 gating through overlapping binding sites.\",\n      \"method\": \"GST pulldown, co-immunoprecipitation, gel shift assay, inside-out patch clamp with synthetic peptides\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple binding assays, but TRPC7 not the primary focus; data shown for TRPC7 among several TRPCs\",\n      \"pmids\": [\"11290752\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"TRPC7 functions as a receptor-operated, diacylglycerol (DAG)-activated non-selective cation channel when transiently expressed in HEK-293 cells (activated by PLC-stimulating agonists but not by Ca2+ store depletion). When stably expressed in HEK-293 cells, TRPC7 can be activated by either Ca2+ store depletion or PLC activation, demonstrating that expression level/environment determines the mode of TRPC7 gating.\",\n      \"method\": \"Stable and transient transfection in HEK-293 cells, patch clamp, thapsigargin-based store depletion, agonist stimulation\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — systematic comparison of stable vs. transient expression with multiple activation paradigms, clear mechanistic conclusion\",\n      \"pmids\": [\"15342342\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"TRPC7 currents are inhibited by extracellular Ca2+ (IC50 ~0.4 mM) through voltage-dependent reductions in unitary conductance and open probability at the single-channel level. Single TRPC7 channel activity is concentration-dependently suppressed by nanomolar intracellular Ca2+ via calmodulin (CaM), and is conversely enhanced by IP3. This contrasts with TRPC6, which is potentiated by Ca2+ through a CaMKII-dependent mechanism.\",\n      \"method\": \"Nystatin-perforated patch clamp, conventional whole-cell clamp, single-channel recording, CaM mutant co-expression, pharmacological inhibitors (calmidazolium, AMP-PNP, CaMKII inhibitory peptide)\",\n      \"journal\": \"The Journal of physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — single-channel electrophysiology with mutagenesis and pharmacological dissection, multiple orthogonal approaches\",\n      \"pmids\": [\"15579537\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Endogenous TRPC1, TRPC3, and TRPC7 form a heteromeric complex in HEK-293 cells that mediates store-operated Ca2+ entry (SOCE). siRNA knockdown of each individually suppressed SOCE by 52–68%, and co-immunoprecipitation confirmed the existence of a TRPC1-TRPC3-TRPC7 heteromeric complex. TRPC3 and TRPC7 (but not TRPC1) also participate in receptor-operated channels.\",\n      \"method\": \"siRNA knockdown, co-immunoprecipitation, thapsigargin-stimulated Ba2+ entry measurements, 2-APB/SKF96365 pharmacology\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — combined siRNA epistasis and co-IP with multiple subunit combinations, strong mechanistic framework\",\n      \"pmids\": [\"15972814\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Native TRPC7 channel activity (75 pS single channels) in DT40 B lymphocytes requires inositol trisphosphate receptors (IP3Rs) for activation by DAG (OAG). TRPC7-null DT40 cells show no OAG-induced single-channel activity, restored by TRPC7 re-expression; IP3R-null cells similarly lack OAG-induced activity, restored by IP3R re-expression. At high TRPC7 expression levels, channel activity becomes IP3R-independent.\",\n      \"method\": \"Cell-attached patch clamp in TRPC7-/- and IP3R-/- DT40 B lymphocyte cell lines, genetic rescue experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis using knockout cell lines with reciprocal rescue, single-channel electrophysiology\",\n      \"pmids\": [\"16822861\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"TRPC7 channels are subject to negative feedback regulation by Ca2+ entering through the channels themselves. Thapsigargin-mediated inhibition of SERCA pumps blocks DAG-activated TRPC7, an effect dependent on extracellular Ca2+, the driving force for Ca2+ entry, and calmodulin. Pharmacological disruption of the actin cytoskeleton mimics the effect of thapsigargin. This suggests that closely associated SERCA pumps protect TRPC7 from Ca2+-calmodulin-mediated inhibition under physiological conditions.\",\n      \"method\": \"Whole-cell patch clamp, thapsigargin, calmodulin inhibition, cytochalasin D actin disruption, extracellular Ca2+ manipulation\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple pharmacological interventions with coherent mechanistic interpretation, orthogonal approaches\",\n      \"pmids\": [\"16401641\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"TRPC7 is the molecular substrate for DAG-activated receptor-operated Ca2+ entry in human keratinocytes (HaCaT cells). CCh- and ATP-induced cation currents were inhibited by a PLC blocker and mimicked by the DAG analog OAG, but were insensitive to IP3 and to PKC inhibitors, indicating direct DAG activation. Antisense knockdown of TRPC7 reduced ATP- and CCh-induced Ca2+ entry and OAG-evoked current.\",\n      \"method\": \"Patch clamp, antisense oligonucleotide knockdown, pharmacological dissection (PLC inhibitor, PKC inhibitor, OAG, IP3)\",\n      \"journal\": \"The Journal of investigative dermatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — antisense knockdown plus pharmacological dissection; single lab\",\n      \"pmids\": [\"16741513\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"In rabbit coronary artery myocytes, ET-1 activates a Ca2+-permeable non-selective cation channel with TRPC3 and TRPC7 properties via ETA receptor and PLC. Anti-TRPC3 and anti-TRPC7 antibodies applied to inside-out patches inhibited ET-1-evoked currents; antibodies to TRPC1, C4, C5, C6 had no effect. Immunocytochemistry revealed preferential TRPC7 plasma membrane localization and co-localization of TRPC3 and TRPC7 at or near the plasma membrane.\",\n      \"method\": \"Cell-attached and inside-out patch clamp with channel-blocking antibodies, immunocytochemistry\",\n      \"journal\": \"The Journal of physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — antibody block in inside-out patches plus immunolocalization; single lab\",\n      \"pmids\": [\"17303636\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"TRPC7 protein exhibits constitutive activity and is directly activated by DAG downstream of PLC signaling. TRPC7 is uniquely susceptible to negative regulation by extracellular Ca2+, distinguishing it from closely related TRPC3 and TRPC6.\",\n      \"method\": \"Review synthesizing patch-clamp and molecular biological data from multiple studies\",\n      \"journal\": \"Handbook of experimental pharmacology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 — review article, no primary experimental data\",\n      \"pmids\": [\"17217055\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"TRPC6 and TRPC7 form heteromeric receptor-operated channels in A7r5 vascular smooth muscle cells that mediate arginine vasopressin (AVP)-induced non-selective cation current. Co-immunoprecipitation confirmed physical TRPC6-TRPC7 association. Dominant-negative TRPC6 (but not TRPC5) suppressed AVP-induced currents. Extracellular Ca2+ suppressed native, TRPC7, and TRPC6-TRPC7 heteromeric currents, but the Ca2+ sensitivity matched the native channel only for the heteromeric TRPC6-TRPC7 construct.\",\n      \"method\": \"Co-immunoprecipitation, dominant-negative expression, whole-cell patch clamp, extracellular Ca2+ titration\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP plus functional dominant-negative electrophysiology with quantitative Ca2+ pharmacology matching native channel\",\n      \"pmids\": [\"16690880\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Ca2+-dependent inactivation of TRPC7 requires the synergistic actions of both DAG and IP3. OAG-induced TRPC7 currents alone do not undergo persistent Ca2+-mediated inhibition, but co-application of intracellular IP3 renders them susceptible to persistent Ca2+-mediated inactivation independently of IP3 receptors (not blocked by heparin or thapsigargin). Carbachol- and GTPγS-activated TRPC7 currents undergo persistent Ca2+-dependent inhibition that requires strong intracellular Ca2+ buffering (BAPTA) for recovery.\",\n      \"method\": \"Whole-cell patch clamp, intracellular dialysis with IP3, OAG, GTPγS, BAPTA, heparin, thapsigargin\",\n      \"journal\": \"Acta pharmacologica Sinica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — systematic patch clamp dissection of inactivation mechanism; single lab\",\n      \"pmids\": [\"18158870\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Heterologously expressed TRPC3 and TRPC7 localize not only to the plasma membrane but also to intracellular Golgi (both cis-Golgi and trans-Golgi network) compartments in COS-7 cells, as shown by co-localization with Golgi markers and Brefeldin A redistribution. Expression of TRPC3 or TRPC7 (but not TRPC1) increased constitutive secretion of alkaline phosphatase 2–4-fold, suggesting these channels enhance vesicular transport/secretion.\",\n      \"method\": \"Immunofluorescence microscopy, Brefeldin A treatment, alkaline phosphatase secretion assay\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — co-localization with Golgi markers and functional secretion assay; single lab\",\n      \"pmids\": [\"18452405\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"A pathogenic C-terminus-truncated polycystin-2 (TRPP2) mutant (697fsX) physically associates with TRPC3 and TRPC7 at the plasma membrane, and this association enhances muscarinic receptor-activated Ca2+ influx and alters ion-permeating pore properties (depolarizing shift in reversal potential, enhanced single-channel conductance) of the mAChR-activated current. Co-immunoprecipitation, pulldown, and cross-linking confirmed the TRPP2 mutant-TRPC7 interaction.\",\n      \"method\": \"Co-immunoprecipitation, pulldown assay, cross-linking, whole-cell patch clamp, single-channel recording, immunofluorescence\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple biochemical interaction assays plus functional electrophysiology with pore property changes; strong mechanistic evidence\",\n      \"pmids\": [\"19812035\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TRPC7 acts as a nociceptive mechanoreceptor that specifically mediates UVB-induced Ca2+ influx and subsequent reactive oxygen species production in skin cells within 30 minutes of irradiation. In TRPC7 knockout mice, UVB-associated epidermal thickening, abnormal keratinocyte differentiation, DNA damage response activation, p53 family mutations, and tumor formation were all prevented, establishing TRPC7 as a required initiator of UVB-induced skin aging and tumorigenesis.\",\n      \"method\": \"TRPC7 knockout mice, Ca2+ influx assays, ROS measurements, histology, immunostaining, mutation analysis\",\n      \"journal\": \"Aging cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic knockout with multiple orthogonal phenotypic readouts; mechanistic Ca2+/ROS pathway established\",\n      \"pmids\": [\"31755176\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TRPC7 regulates the automaticity (spontaneous action potential firing) of mouse embryonic stem cell-derived cardiomyocytes via two mechanisms: (1) positively regulating the intracellular Ca2+ clock by enhancing RyR2 phosphorylation at S2814 and SERCA activity (via phospholamban phosphorylation at T17); (2) positively regulating the membrane clock via NCX activity. These effects are Ca2+-dependent, as a dominant-negative N-terminal TRPC7 construct that abolishes Ca2+ permeability reduces AP frequency.\",\n      \"method\": \"Adenoviral TRPC7 knockdown/overexpression, confocal Ca2+ imaging, whole-cell patch clamp, Western blot for phosphorylation, dominant-negative TRPC7 N-terminus construct\",\n      \"journal\": \"Stem cell research & therapy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal approaches (KD, OE, dominant-negative, phosphorylation readouts, Ca2+ imaging, electrophysiology) in same study\",\n      \"pmids\": [\"33941260\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"A truncation mutation in the Trpc7 cytoplasmic C-terminus (stop codon at amino acid 810, affecting the conserved C-terminal domain) produces a dominant-negative effect in heterozygous mice, causing distinct morphological and behavioral alterations not seen in Trpc7 knockout mice, demonstrating that the C-terminus is critical for normal TRPC7 function.\",\n      \"method\": \"ENU mutagenesis mouse model, Sanger sequencing, phenotypic characterization of heterozygous mutants\",\n      \"journal\": \"Genes\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — in vivo genetic model with defined mutation, but phenotypic characterization without molecular mechanism of C-terminus function\",\n      \"pmids\": [\"34828338\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TRPC7 undergoes conformational changes upon activation by Gαq-coupled receptors, as detected by intramolecular BRET biosensors (GFP10-TRPC7-RLucII). Activation of Gαq-coupled receptors (including the angiotensin II AT1R) induces a Gαq-dependent BRET response in both HEK293 cells and rat neonatal cardiac fibroblasts expressing endogenous AT1R and TRPC7, confirming receptor-induced structural rearrangements in TRPC7.\",\n      \"method\": \"Intramolecular BRET biosensors in HEK293 cells and primary rat cardiac fibroblasts, GPCR agonist stimulation, Gαq pathway pharmacology\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional biosensor validated in both heterologous and native cell context; single lab\",\n      \"pmids\": [\"35269644\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TRPC7 facilitates lung adenocarcinoma cell growth and migration via Ca2+-dependent activation of CaMKII, AKT, and ERK signaling pathways. TRPC7 knockdown restrained cell cycle progression and migration by interrupting TRPC7-mediated Ca2+ signaling, upstream of AKT and MAPK pathway activation.\",\n      \"method\": \"TRPC7 siRNA knockdown, cell cycle analysis, invasion assay, Ca2+ response assay, immunoblot for phospho-CaMKII/AKT/ERK\",\n      \"journal\": \"Oncology letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — loss-of-function with defined pathway readouts; single lab\",\n      \"pmids\": [\"36817036\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In normal rat cardiomyocytes, TRPC7 localizes to the surface sarcolemma and striated subcellular (T-tubule) regions. In renovascular hypertrophic hearts, TRPC7 expression is significantly downregulated while PKC expression is upregulated, with a negative correlation between TRPC7 and PKC levels. Losartan (angiotensin II receptor blocker) reverses TRPC7 downregulation, implicating Ang II/PKC signaling in the suppression of TRPC7 in cardiac hypertrophy.\",\n      \"method\": \"Immunohistochemistry, immunocytochemistry, Western blot, 2K1C renovascular hypertension model, losartan treatment\",\n      \"journal\": \"Journal of cardiovascular pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — localization with functional context in disease model, but mechanistic link between PKC and TRPC7 is correlative\",\n      \"pmids\": [\"26371949\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TRPC7 is a plasma membrane Ca2+-permeable non-selective cation channel of the TRPC3/6/7 subfamily that is activated downstream of Gαq-PLC signaling, primarily by diacylglycerol (DAG) acting directly on the channel; it forms homo- and heteromeric complexes with TRPC3 and TRPC6, is negatively regulated by extracellular Ca2+ via calmodulin and by PKC, is protected from Ca2+-CaM-mediated inhibition by closely associated SERCA pumps, requires IP3 receptors for native DAG-dependent gating at physiological expression levels, undergoes Gαq-induced conformational changes, mediates UVB-induced Ca2+ influx and ROS production in skin, and regulates cardiac pacemaking by modulating RyR2 and SERCA phosphorylation to control both intracellular Ca2+ and membrane clocks.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"TRPC7 is a non-selective, Ca²⁺-permeable cation channel activated downstream of Gq-coupled receptor–PLC signaling primarily via diacylglycerol (DAG), functioning as both a receptor-operated and, under certain expression conditions, a store-operated channel [PMID:15342342, PMID:16822861]. The channel is negatively regulated by extracellular and intracellular Ca²⁺ through calmodulin, with closely associated SERCA pumps providing local Ca²⁺ clearance that sustains channel activity; IP3 receptors are required for activation at physiological expression levels and IP3 itself cooperates with DAG to confer Ca²⁺-dependent inactivation [PMID:15579537, PMID:16401641, PMID:18158870]. TRPC7 assembles into heteromeric complexes with TRPC1, TRPC3, and TRPC6 to mediate receptor-operated and store-operated Ca²⁺ entry in diverse cell types including vascular smooth muscle, keratinocytes, and cardiomyocytes [PMID:15972814, PMID:16690880, PMID:17303636]. In vivo, TRPC7 mediates UVB-induced Ca²⁺ influx and subsequent ROS production, DNA damage, and skin tumorigenesis—processes abolished in TRPC7-knockout mice—and promotes lung adenocarcinoma cell growth via CaMKII/AKT/ERK signaling [PMID:31755176, PMID:36817036].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Molecular identification of TRPC7 established it as a new seven-transmembrane-domain TRP family member enriched in brain, mapping to human chromosome 21q22.3 and providing the sequence foundation for all subsequent functional studies.\",\n      \"evidence\": \"cDNA cloning, Northern blot, and chromosomal mapping\",\n      \"pmids\": [\"9806837\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No functional characterization of ion conductance or gating\", \"Expression pattern limited to Northern blot across tissues\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Electrophysiological characterization revealed that TRPC7 is a DAG-activated receptor-operated channel that can also operate as a store-operated channel depending on expression level, resolving the longstanding question of whether individual TRP channels can exhibit dual gating modes.\",\n      \"evidence\": \"Patch-clamp electrophysiology comparing stable vs. transient HEK-293 expression with thapsigargin and agonist stimulation\",\n      \"pmids\": [\"15342342\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis for expression-level-dependent gating switch unknown\", \"Endogenous cell context not tested\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Discovery that extracellular Ca²⁺ inhibits TRPC7 with an IC50 of ~0.4 mM and that intracellular Ca²⁺ inhibits via calmodulin—while IP3 enhances activity—established the core regulatory framework distinguishing TRPC7 from the Ca²⁺-potentiated TRPC6.\",\n      \"evidence\": \"Single-channel recording, calmodulin mutant co-expression, and pharmacological inhibitors\",\n      \"pmids\": [\"15579537\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Calmodulin binding site on TRPC7 not mapped\", \"Structural basis of Ca²⁺-dependent gating changes unresolved\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Demonstration that endogenous TRPC1, TRPC3, and TRPC7 form heteromeric complexes mediating store-operated Ca²⁺ entry showed that TRPC7 does not operate exclusively as a homomeric channel and that subunit composition determines gating properties.\",\n      \"evidence\": \"Co-immunoprecipitation and siRNA knockdown with thapsigargin-induced Ba²⁺ entry in HEK-293 cells\",\n      \"pmids\": [\"15972814\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of heteromeric complexes unknown\", \"No structural data on subunit interfaces\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Multiple studies in 2006 collectively defined TRPC7's physiological roles in vascular smooth muscle and keratinocytes, its heteromeric assembly with TRPC6, and the critical role of SERCA pumps in protecting the channel from Ca²⁺/calmodulin-mediated inactivation during sustained signaling.\",\n      \"evidence\": \"Patch-clamp with SERCA inhibition and actin disruption (FASEB J); antisense knockdown in keratinocytes (J Invest Dermatol); Co-IP and dominant-negative TRPC6 in A7r5 smooth muscle cells (Circ Res); single-channel recording in TRPC7/IP3R-knockout DT40 cells (JBC)\",\n      \"pmids\": [\"16401641\", \"16741513\", \"16690880\", \"16822861\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular determinants of TRPC6–TRPC7 heteromeric assembly not identified\", \"In vivo relevance of SERCA-mediated protection not tested\", \"IP3R's physical versus functional coupling to TRPC7 unresolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Antibody-blocking experiments in inside-out patches from coronary artery myocytes demonstrated that native TRPC3/TRPC7 heteromers mediate endothelin-1-activated cation conductance, extending the heteromeric channel concept to a native vascular preparation.\",\n      \"evidence\": \"Inside-out patch-clamp with inhibitory anti-TRPC3 and anti-TRPC7 antibodies in rabbit coronary artery myocytes\",\n      \"pmids\": [\"17303636\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of TRPC3 vs. TRPC7 subunits to channel properties not separated\", \"No genetic confirmation (knockdown/knockout) in this tissue\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identification that IP3 cooperates with DAG to render TRPC7 susceptible to persistent Ca²⁺-dependent inactivation—independently of IP3 receptors—added a second layer of IP3-mediated regulation beyond IP3R-dependent activation, while Golgi localization and enhancement of constitutive secretion pointed to potential roles in intracellular membrane trafficking.\",\n      \"evidence\": \"Whole-cell patch-clamp with intracellular IP3 dialysis and heparin (Acta Pharmacol Sin); immunofluorescence with Golgi markers and secreted alkaline phosphatase assay (Biochem J)\",\n      \"pmids\": [\"18158870\", \"18452405\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"IP3 binding site on TRPC7 not identified\", \"Secretion enhancement mechanism (vesicle fusion vs. Ca²⁺-dependent) not dissected\", \"Golgi localization not confirmed endogenously\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Pathogenic truncated TRPP2 was shown to physically associate with TRPC3/TRPC7 and alter their pore properties, linking TRPC7-containing channels to polycystic kidney disease-associated Ca²⁺ dysregulation.\",\n      \"evidence\": \"Co-immunoprecipitation, pulldown, cross-linking, and whole-cell patch-clamp\",\n      \"pmids\": [\"19812035\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether wild-type TRPP2 similarly modulates TRPC7 under physiological conditions not tested\", \"Relevance to renal pathology not examined in vivo\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"TRPC7-knockout mice demonstrated that TRPC7 is the primary mediator of UVB-induced Ca²⁺ influx, ROS production, DNA damage, and eventual skin tumorigenesis, providing the first in vivo genetic evidence for a specific TRPC7 physiological function.\",\n      \"evidence\": \"TRPC7 knockout mice with Ca²⁺ influx, ROS, histology, DNA damage, and tumor scoring after UVB exposure\",\n      \"pmids\": [\"31755176\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which UVB activates TRPC7 (direct photosensing vs. PLC-mediated) unknown\", \"Whether other TRPC channels compensate in knockout mice not assessed\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"TRPC7 was shown to modulate cardiomyocyte automaticity through both Ca²⁺ clock (RyR2/SERCA phosphorylation) and membrane clock (NCX) mechanisms, and a dominant-negative ENU-induced C-terminal truncation demonstrated that the cytoplasmic C-terminus is essential for TRPC7 function in vivo.\",\n      \"evidence\": \"Adenoviral KD/OE with Ca²⁺ imaging and patch-clamp in ESC-derived cardiomyocytes (Stem Cell Res Ther); ENU mutagenesis with phenotypic characterization of heterozygous vs. KO mice (Genes)\",\n      \"pmids\": [\"33941260\", \"34828338\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific C-terminal domains mediating dominant-negative effect not mapped\", \"Whether TRPC7 modulates adult cardiomyocyte pacemaking in vivo not tested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"BRET-based intramolecular biosensors detected Gαq-GPCR-induced conformational changes in TRPC7, providing the first biophysical evidence that receptor activation directly alters TRPC7 structure prior to gating.\",\n      \"evidence\": \"BRET intramolecular biosensor in HEK293 cells and rat neonatal cardiac fibroblasts with GPCR agonist stimulation\",\n      \"pmids\": [\"35269644\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Nature of conformational change (pore opening vs. trafficking vs. protein–protein interaction) unresolved\", \"No high-resolution structural data for TRPC7\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"TRPC7-mediated Ca²⁺ influx was linked to lung adenocarcinoma proliferation and migration through CaMKII/AKT/ERK signaling, extending TRPC7's pathophysiological significance to cancer biology.\",\n      \"evidence\": \"TRPC7 knockdown with cell cycle analysis, invasion assay, and immunoblot for phospho-CaMKII/AKT/ERK in lung adenocarcinoma cells\",\n      \"pmids\": [\"36817036\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vivo tumor model\", \"Whether TRPC7 is a driver or passenger in lung adenocarcinoma unclear\", \"Specific agonist activating TRPC7 in tumor cells not identified\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Major unresolved questions include the high-resolution structure of TRPC7, the molecular basis of its dual gating modes, the identity of the calmodulin-binding site, and how UVB irradiation activates the channel.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No cryo-EM or crystal structure available\", \"Calmodulin and IP3 binding sites unmapped\", \"Mechanism of UVB-induced activation unknown\", \"In vivo roles beyond skin and cardiomyocytes poorly defined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [1, 2, 5, 6, 7, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1, 6, 8, 10, 12]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [10]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 2, 5, 6, 7, 8, 15, 16]},\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [1, 2, 5, 6]}\n    ],\n    \"complexes\": [\n      \"TRPC1/TRPC3/TRPC7 heteromer\",\n      \"TRPC6/TRPC7 heteromer\",\n      \"TRPC3/TRPC7 heteromer\"\n    ],\n    \"partners\": [\n      \"TRPC1\",\n      \"TRPC3\",\n      \"TRPC6\",\n      \"CALM1\",\n      \"ITPR1\",\n      \"PKD2\",\n      \"RYR2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"TRPC7 is a plasma membrane Ca²⁺-permeable non-selective cation channel of the TRPC3/6/7 subfamily that is directly activated by diacylglycerol (DAG) downstream of Gαq-coupled receptor–PLC signaling and assembles into homomeric or heteromeric complexes exclusively with TRPC3 and TRPC6 [PMID:12377790, PMID:16690880]. Channel activity is negatively regulated by extracellular Ca²⁺ through voltage-dependent reductions in conductance and open probability, and by intracellular Ca²⁺ via calmodulin, while closely associated SERCA pumps protect the channel from Ca²⁺-CaM–mediated inactivation; at physiological expression levels, IP3 receptors are required as permissive cofactors for DAG-dependent gating [PMID:15579537, PMID:16401641, PMID:16822861]. In cardiomyocytes, TRPC7-mediated Ca²⁺ entry drives both the intracellular Ca²⁺ clock (via RyR2 and phospholamban phosphorylation) and the membrane clock to regulate pacemaker automaticity [PMID:33941260]. In skin, TRPC7 is the obligate mediator of UVB-induced Ca²⁺ influx and reactive oxygen species production, and its genetic ablation prevents UVB-driven epidermal thickening, DNA damage, and tumorigenesis in mice [PMID:31755176].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Identification of TRPC7 as a novel TRP-family member established the existence of a seventh human canonical TRP channel with predicted transmembrane topology consistent with a Ca²⁺-permeable pore.\",\n      \"evidence\": \"cDNA cloning from human fetal brain and caudate nucleus libraries with genomic mapping to 21q22.3\",\n      \"pmids\": [\"9806837\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No functional electrophysiology performed\", \"Expression pattern beyond Northern blot unknown\", \"Activation mechanism not addressed\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Discovery of conserved IP3R-binding and calmodulin-binding domains on the TRPC7 C-terminus, with competitive binding between the two, provided the first molecular framework for Ca²⁺-dependent channel regulation.\",\n      \"evidence\": \"GST pulldown and co-immunoprecipitation showing overlapping CaM and IP3R binding sites on TRPC C-termini\",\n      \"pmids\": [\"11290752\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"TRPC7 was studied alongside other TRPCs, not as primary target\", \"Functional consequence of CaM vs. IP3R competition on TRPC7 currents not tested\", \"Binding site residues not mapped\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Demonstration that TRPC7 selectively co-assembles with TRPC3 and TRPC6 — but not with the TRPC1/4/5 subgroup — defined the combinatorial rules governing heteromeric TRP channel assembly.\",\n      \"evidence\": \"Reciprocal co-immunoprecipitation in Sf9 cells and native rat brain synaptosomes\",\n      \"pmids\": [\"12377790\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of heteromeric complexes unknown\", \"No structural basis for subfamily-restricted assembly\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Electrophysiological characterization established TRPC7 as a DAG-activated receptor-operated channel whose gating mode (receptor-operated vs. store-operated) depends on expression level, and revealed unique negative regulation by extracellular Ca²⁺ acting on unitary conductance and open probability via intracellular calmodulin.\",\n      \"evidence\": \"Patch-clamp comparison of stable vs. transient TRPC7 expression in HEK-293 cells; single-channel recordings with CaM mutants and pharmacological inhibitors\",\n      \"pmids\": [\"15342342\", \"15579537\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular determinants of extracellular Ca²⁺ sensing not identified\", \"Structural basis for DAG binding site unknown\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identification of an endogenous TRPC1–TRPC3–TRPC7 heteromeric complex mediating store-operated Ca²⁺ entry in HEK-293 cells showed that TRPC7 contributes to native SOCE channels, not only receptor-operated channels.\",\n      \"evidence\": \"siRNA knockdown of individual subunits combined with co-immunoprecipitation and thapsigargin-stimulated Ba²⁺ entry\",\n      \"pmids\": [\"15972814\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"SOCE role not confirmed in primary cells\", \"Relationship to Orai/STIM-mediated SOCE unclear\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Multiple studies converged to show that (i) native TRPC7 gating by DAG requires IP3 receptors as permissive cofactors at physiological expression levels, (ii) SERCA pumps physically associated with TRPC7 protect the channel from Ca²⁺-CaM–mediated inactivation, and (iii) TRPC6–TRPC7 heteromers constitute the native receptor-operated channel in vascular smooth muscle.\",\n      \"evidence\": \"Genetic epistasis in TRPC7⁻/⁻ and IP3R⁻/⁻ DT40 cells with rescue; thapsigargin/cytochalasin D pharmacology in HEK cells; co-IP and dominant-negative electrophysiology in A7r5 cells and coronary myocytes\",\n      \"pmids\": [\"16822861\", \"16401641\", \"16690880\", \"17303636\", \"16741513\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which IP3R licenses DAG gating is unknown\", \"Identity of the SERCA isoform and mode of physical coupling not determined\", \"Native stoichiometry of TRPC6/7 heteromers in vivo undefined\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Dissection of TRPC7 inactivation showed that persistent Ca²⁺-dependent shutdown requires synergistic action of both DAG and IP3, revealing a dual-signal integration mechanism for channel silencing independent of IP3 receptor opening.\",\n      \"evidence\": \"Whole-cell patch clamp with intracellular dialysis of IP3, OAG, and Ca²⁺ chelators in HEK-293 cells\",\n      \"pmids\": [\"18158870\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular target of IP3's direct sensitizing action not identified\", \"In vivo relevance of dual-signal inactivation not tested\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Detection of TRPC7 at Golgi compartments and its ability to enhance constitutive secretion suggested a second site of action beyond the plasma membrane.\",\n      \"evidence\": \"Immunofluorescence co-localization with Golgi markers and alkaline phosphatase secretion assay in COS-7 cells\",\n      \"pmids\": [\"18452405\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Golgi localization not confirmed in primary cells\", \"Mechanism linking TRPC7 channel activity to vesicular secretion undefined\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Demonstration that a disease-associated truncated polycystin-2 (TRPP2) mutant physically associates with TRPC7 and alters its pore properties linked TRPC7 to polycystic kidney disease-relevant signaling complexes.\",\n      \"evidence\": \"Co-IP, cross-linking, and single-channel recordings showing altered conductance and reversal potential of TRPC7 in complex with TRPP2-697fsX\",\n      \"pmids\": [\"19812035\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Interaction with wild-type TRPP2 not equivalently characterized\", \"Relevance to renal cyst formation in vivo not demonstrated\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Localization of TRPC7 to sarcolemma and T-tubules in cardiomyocytes, with downregulation in hypertrophic hearts reversible by angiotensin receptor blockade, implicated TRPC7 in cardiac remodeling via Ang II/PKC signaling.\",\n      \"evidence\": \"Immunohistochemistry and Western blot in a 2K1C renovascular hypertension rat model with losartan rescue\",\n      \"pmids\": [\"26371949\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"PKC–TRPC7 regulatory mechanism is correlative, not causal\", \"Direct TRPC7 phosphorylation by PKC not demonstrated in this model\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Genetic ablation in mice established TRPC7 as the obligate initiator of UVB-induced Ca²⁺ influx, ROS production, and downstream photoaging/tumorigenesis in skin, revealing a physiological nociceptive-mechanoreceptor role.\",\n      \"evidence\": \"TRPC7 knockout mice with multi-parameter phenotyping including Ca²⁺ influx, ROS, histology, DNA damage, and tumor formation\",\n      \"pmids\": [\"31755176\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of UVB-induced TRPC7 activation (direct photomechanical vs. lipid-mediated) unknown\", \"Cell-type specificity within the epidermis not resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"TRPC7 was shown to regulate cardiomyocyte pacemaker automaticity by coupling Ca²⁺ entry to dual clocks: the intracellular Ca²⁺ clock (via RyR2-S2814 and PLB-T17 phosphorylation/SERCA activation) and the membrane clock (via NCX), while a C-terminal truncation mutation produced dominant-negative effects in vivo distinct from null.\",\n      \"evidence\": \"Adenoviral KD/OE and dominant-negative constructs in mESC-derived cardiomyocytes with Ca²⁺ imaging and electrophysiology; ENU mutagenesis mouse with C-terminal stop codon\",\n      \"pmids\": [\"33941260\", \"34828338\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase(s) mediating TRPC7-dependent RyR2/PLB phosphorylation not identified\", \"Dominant-negative mechanism of C-terminal truncation not molecularly defined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Detection of Gαq-induced intramolecular conformational changes in TRPC7 via BRET biosensors provided the first real-time evidence of receptor-triggered structural rearrangements in the intact channel.\",\n      \"evidence\": \"Intramolecular BRET (GFP10-TRPC7-RLucII) in HEK293 cells and primary rat cardiac fibroblasts upon AT1R stimulation\",\n      \"pmids\": [\"35269644\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Nature and location of conformational change not resolved at atomic level\", \"Relationship between BRET change and channel gating transitions not established\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"TRPC7-mediated Ca²⁺ entry was linked to lung adenocarcinoma proliferation and migration through CaMKII/AKT/ERK signaling, extending TRPC7's pathophysiological relevance beyond skin and heart.\",\n      \"evidence\": \"siRNA knockdown with cell cycle, migration, and phospho-signaling readouts in lung adenocarcinoma cell lines\",\n      \"pmids\": [\"36817036\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vivo tumor model\", \"Selectivity for TRPC7 vs. other DAG-activated TRPCs not addressed\", \"Direct vs. indirect activation of CaMKII/AKT/ERK not resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis for DAG binding and extracellular Ca²⁺ sensing, the mechanism by which IP3 receptors license TRPC7 gating at physiological expression, the identity of kinases linking TRPC7-mediated Ca²⁺ entry to RyR2/PLB phosphorylation in cardiomyocytes, and the biophysical mechanism of UVB-induced channel activation in skin.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No high-resolution structure of TRPC7\", \"DAG binding site not mapped\", \"IP3R–TRPC7 coupling mechanism molecularly undefined\", \"UVB activation mechanism unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [3, 4, 6, 8, 11]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [16]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [3, 9, 13, 20]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [13]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 4, 6, 8, 11, 18]},\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [3, 4, 6, 8, 11]}\n    ],\n    \"complexes\": [\n      \"TRPC3/TRPC7 heteromeric channel\",\n      \"TRPC6/TRPC7 heteromeric channel\",\n      \"TRPC1/TRPC3/TRPC7 heteromeric channel\"\n    ],\n    \"partners\": [\n      \"TRPC3\",\n      \"TRPC6\",\n      \"TRPC1\",\n      \"ITPR1\",\n      \"CALM1\",\n      \"PKD2\",\n      \"RYR2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}