{"gene":"TRPC4","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2001,"finding":"TRP4-deficient (TRPC4-/-) mice lack a store-operated Ca2+ current in vascular endothelial cells, establishing that TRP4 is an indispensable component of store-operated channels in native endothelial cells and that these channels are required for agonist-induced Ca2+ entry and vasorelaxation.","method":"Knockout mouse model, electrophysiology, Ca2+ imaging, vascular tension measurements","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO mouse with defined cellular and vascular phenotype, replicated across multiple functional readouts","pmids":["11175743"],"is_preprint":false},{"year":2000,"finding":"Murine TRPC4 forms a nonselective cation channel activated by Gq/11-coupled receptors and receptor tyrosine kinases independently of intracellular Ca2+ store depletion; single-channel conductance is 42 pS at -60 mV; store depletion alone fails to activate the channel.","method":"Heterologous expression in HEK293 cells, whole-cell and inside-out patch clamp, Mn2+ quench fluorimetry, GTPγS infusion","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — rigorous electrophysiology with multiple activation paradigms, replicated by independent labs","pmids":["10837492"],"is_preprint":false},{"year":2000,"finding":"Murine TRP4 and phospholipase Cβ1/β2 interact with the first PDZ domain of the scaffolding protein NHERF (EBP50), and TRP4/PLCβ1/NHERF complexes co-immunoprecipitate from HEK293-Trp4 cells and adult mouse brain, linking the channel to the actin cytoskeleton via the ERM-NHERF interaction.","method":"Co-immunoprecipitation from transfected cells and native brain tissue, GST pull-down","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP in heterologous and native systems, multiple binding partners confirmed","pmids":["10980202"],"is_preprint":false},{"year":2000,"finding":"TRP4 protein is abundantly expressed in bovine adrenal cortex cells and contributes essentially to native CRAC-like (store-operated) currents; antisense reduction of TRP4 protein significantly reduces both endogenous CRAC-like currents and native TRP4 protein.","method":"Antisense knockdown, Northern blot, immunoblot, immunohistochemistry, electrophysiology","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — antisense KD with correlated electrophysiological and protein readouts in native cells","pmids":["10816590"],"is_preprint":false},{"year":2002,"finding":"TRPC4-/- lung endothelial cells show drastically reduced agonist (thrombin/PAR-1)-induced Ca2+ influx, lack actin stress fiber formation and cell retraction, and isolated-perfused TRPC4-/- lungs show markedly attenuated increases in microvascular permeability, establishing TRPC4-dependent Ca2+ entry as a key determinant of increased microvascular permeability.","method":"TRPC4 knockout mouse model, Ca2+ imaging, actin staining, isolated-perfused lung microvascular filtration coefficient measurement","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO mouse with multiple orthogonal cellular and organ-level phenotypic readouts","pmids":["12114324"],"is_preprint":false},{"year":2001,"finding":"TRP4 directly binds calmodulin (CaM) in a Ca2+-dependent manner through two C-terminal domains (residues 688-759 and 786-848); half-maximal CaM binding occurs at 16.6 µM (domain 1) and 27.9 µM (domain 2) Ca2+; synthetic peptides from these regions bind dansyl-CaM with Kd 94-189 nM.","method":"CaM-Sepharose affinity chromatography, GST pull-down, synthetic peptide binding assay with dansyl-CaM fluorimetry","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro biochemical binding assays with domain mapping and Kd determination, single lab","pmids":["11311128"],"is_preprint":false},{"year":2006,"finding":"TRPC3 and TRPC4 associate to form a redox-sensitive heteromeric cation channel complex in porcine aortic endothelial cells; co-IP demonstrates physical association; FRET shows close proximity between TRPC4 N-terminus and TRPC3 C-terminus; dominant-negative TRPC4 suppresses TRPC3-related currents and the native redox-sensitive conductance.","method":"Co-immunoprecipitation, FRET, whole-cell electrophysiology, dominant-negative expression","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — four orthogonal lines of evidence including native tissue Co-IP, FRET, and dominant-negative functional suppression","pmids":["16537542"],"is_preprint":false},{"year":2009,"finding":"In intestinal smooth muscle cells, TRPC4 forms a 55 pS cation channel underlying >80% of muscarinic receptor-induced cation current (mICAT); TRPC4-deficient myocytes show greatly reduced carbachol-induced membrane depolarization; TRPC4/C6 double KO slows intestinal transit in vivo, establishing TRPC4 and TRPC6 as the molecular basis of mICAT coupling muscarinic receptors to smooth muscle contraction.","method":"Single and double knockout mice, whole-cell patch clamp, muscle contraction assays, intestinal transit measurement","journal":"Gastroenterology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO mice, single-channel electrophysiology, multiple functional readouts in vitro and in vivo","pmids":["19549525"],"is_preprint":false},{"year":2008,"finding":"TRPC4α (but not TRPC4β) is strongly inhibited by intracellularly applied PIP2 in an isoform-specific manner; PIP2 binds to the C-terminus of TRPC4α but not TRPC4β in vitro; inhibition requires association with actin cytoskeleton via the C-terminal PDZ-binding motif (Thr-Thr-Arg-Leu) that links TRPC4 to F-actin through NHERF and ezrin; PIP2 breakdown is a required step in TRPC4α activation requiring additional Ca2+ and Gi/o proteins.","method":"Whole-cell patch clamp with intracellular PIP2 application, in vitro lipid-binding assay, cytochalasin D treatment, PDZ-motif deletion mutants","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstituted lipid-protein binding assay plus electrophysiology with mutagenesis, single lab with multiple orthogonal approaches","pmids":["18230622"],"is_preprint":false},{"year":2001,"finding":"Human TRPC4α contains a C-terminal autoinhibitory domain: TRPC4β is robustly activated by receptor stimulation across species, whereas TRPC4α shows poor activation; C-terminal truncation of TRPC4α fully restores channel activity; TRPC4α exerts a dominant-negative effect on TRPC4β with cooperativity >2 in heteromultimers; FRET confirms homomultimer and heteromultimer assembly of TRPC4α and TRPC4β.","method":"Heterologous expression, whole-cell patch clamp, FRET in living cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — electrophysiology with deletion mutants, FRET for assembly, multiple isoforms and ratios tested","pmids":["11713258"],"is_preprint":false},{"year":2005,"finding":"Protein 4.1 interacts with TRPC4 and the spectrin-actin membrane skeleton; deletion of the protein 4.1 binding domain on TRPC4 or peptide competition to this domain prevents activation of the endothelial ISOC (store-operated) channel, establishing protein 4.1 interaction with TRPC4 as an essential component of the ISOC gating mechanism.","method":"Co-immunoprecipitation, domain deletion, peptide competition, whole-cell electrophysiology","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — domain deletion and peptide competition combined with electrophysiology, multiple orthogonal approaches","pmids":["16254212"],"is_preprint":false},{"year":2002,"finding":"The PDZ-interacting TRL motif at the TRPC4 C-terminus controls its plasma membrane localization; deletion of TRL causes accumulation in cell outgrowths and reduces plasma membrane expression ~2.4-fold; co-expression with an EBP50 mutant lacking the ERM-binding site retains TRPC4 in a perinuclear (Golgi) compartment.","method":"Immunofluorescence microscopy, cell surface biotinylation, co-expression with EBP50 truncation mutants in HEK293 cells","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Moderate — direct localization experiments with surface biotinylation quantification and deletion mutants, clear functional consequence on membrane targeting","pmids":["12154080"],"is_preprint":false},{"year":2012,"finding":"Gαi subunits, particularly Gαi2, are primary and direct activators of TRPC4, acting through direct interaction with the conserved C-terminal SESTD domain; Gαi2 activation by muscarinic M2 receptors or constitutively active Gαi2 mutants fully activates the channel; two amino acids (K715 and R716) in the TRPC4 C-terminus mediate the interaction with Gαi2.","method":"Co-immunoprecipitation, whole-cell patch clamp, constitutively active Gα mutant expression, site-directed mutagenesis, Ca2+ imaging","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — Co-IP with mutagenesis mapping of interaction residues plus functional electrophysiology, single lab with multiple orthogonal methods","pmids":["22457348"],"is_preprint":false},{"year":2016,"finding":"TRPC4 activation requires coincident stimulation of Gi/o proteins and PLCδ1 (preferentially over PLCβ); PIP2 is required for biphasic TRPC4 activation; reducing PIP2 via phosphatases abolishes biphasic kinetics; dominant-negative PLCδ1 or constitutively active RhoA almost completely eliminates TRPC4 activation; this mechanism differs from the closely related TRPC5.","method":"Whole-cell patch clamp, siRNA knockdown of PLCδ1, dominant-negative constructs, constitutively active RhoA, PIP2 manipulation in HEK293 and A-498 renal carcinoma cells","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple genetic and pharmacological perturbations with electrophysiological readouts in heterologous and native cells","pmids":["26755577"],"is_preprint":false},{"year":2016,"finding":"Dynamic interaction of NHERF1/2 with the C-terminal PDZ-binding motif of TRPC4/5 suppresses DAG sensitivity; PIP2 depletion evokes a C-terminal conformational change leading to NHERF dissociation, which is a prerequisite for DAG-mediated channel activation; PKC inhibition or PDZ-motif mutation confers DAG sensitivity, establishing NHERF as a direct negative regulator of TRPC4/5 DAG sensitivity.","method":"Whole-cell patch clamp, FRET-based conformational analysis, PKC inhibition, site-directed mutagenesis of PDZ-binding motif","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Moderate — FRET conformational readout combined with electrophysiology and mutagenesis, multiple orthogonal approaches in single study","pmids":["27994151"],"is_preprint":false},{"year":2018,"finding":"Cryo-EM structure of mouse TRPC4 at 3.3 Å resolution reveals a unique architecture with a long pore loop stabilized by a disulfide bond, a unique cytosolic N-terminal domain forming extensive aromatic contacts with the TRP and C-terminal domains, and a tetrameric six-transmembrane fold; structural features provide molecular basis for TRPC4 ion selectivity.","method":"Cryo-electron microscopy, 3.3 Å resolution structure determination","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution cryo-EM structure with functional domain interpretation","pmids":["30082700"],"is_preprint":false},{"year":2018,"finding":"Cryo-EM structure of zebrafish TRPC4 at 3.6 Å resolution in unliganded closed state reveals molecular architecture of the cation-conducting pore including selectivity filter and lower gate; cytoplasmic domain contains two key hubs for modulating protein interactions.","method":"Cryo-electron microscopy, 3.6 Å resolution structure determination","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution cryo-EM structure identifying pore elements and cytoplasmic regulatory hubs","pmids":["29717981"],"is_preprint":false},{"year":2020,"finding":"Cryo-EM structures of TRPC4 in complex with calmodulin and three pyridazinone inhibitors reveal that all inhibitors bind to the same cavity in the voltage-sensing-like domain; structural changes propagate from the ligand-binding site to the central ion-conducting pore; CaM binds to the rib helix of TRPC4, ordering a previously disordered region and fixing the channel in a closed conformation — a novel CaM-induced regulatory mechanism.","method":"Cryo-EM structural determination of TRPC4-CaM and TRPC4-inhibitor complexes","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple high-resolution cryo-EM structures with distinct ligands identifying binding site and conformational changes","pmids":["33236980"],"is_preprint":false},{"year":2017,"finding":"TRPC1, TRPC4, and TRPC5 assemble exclusively into heteromultimers with each other (not other TRP family members) in mouse brain, as shown by quantitative mass spectrometry; TRPC1/4/5 triple-KO hippocampal neurons show significantly reduced action potential-triggered EPSCs and impaired hippocampal network cross-frequency coupling and spatial working memory, establishing heteromultimeric TRPC1/4/5 channels as regulators of hippocampal synaptic transmission.","method":"Quantitative high-resolution mass spectrometry for interactome, triple-knockout mouse model, hippocampal slice electrophysiology, in vivo LFP recording, behavioral testing","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — MS-based complex identification combined with KO electrophysiology and behavior, multiple orthogonal methods","pmids":["28790178"],"is_preprint":false},{"year":2015,"finding":"TRPC1/TRPC4 double-KO mice show reduced background Ca2+ entry (BGCE) in cardiomyocytes, lower diastolic and systolic Ca2+ concentrations, and are protected against neurohumoral-induced and pressure overload-induced cardiac hypertrophy and fibrosis; TRPC1 or TRPC4 single-KO mice do not show protection, establishing a cooperative TRPC1/C4 constitutively active BGCE pathway as a driver of pathological cardiac remodeling.","method":"Multiple knockout mouse models, fluorescence Ca2+ imaging of electrically paced cardiomyocytes, Mn2+ quench microfluorimetry, cardiac hypertrophy models (neurohumoral, pressure overload)","journal":"European heart journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — systematic analysis of multiple KO models with mechanistic Ca2+ entry measurements and multiple hypertrophy endpoints","pmids":["26069213"],"is_preprint":false},{"year":2001,"finding":"The alpha1 and beta2 splice variants of human TRPC4 differ in regulation: hTRPC4β forms receptor-operated cation channels when expressed in HEK293 cells, while hTRPC4α is poorly activated by H1 receptor stimulation despite correct plasma membrane targeting; the C-terminal region of hTRPC4α acts as an autoinhibitory domain, as C-terminal truncation fully restores channel activity.","method":"Heterologous expression, whole-cell patch clamp, FRET, GFP-fusion protein trafficking","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — electrophysiology combined with FRET and truncation analysis demonstrating autoinhibitory domain","pmids":["11713258"],"is_preprint":false},{"year":2002,"finding":"TRP4 is localized to caveolae of interstitial cells of Cajal (ICC); Ca2+ oscillations in ICC depend on Ca2+ influx through a non-selective, store-operated, SK&F 96365-sensitive cation channel; this links TRP4 localization in caveolae to pacemaker Ca2+ oscillations in ICC.","method":"Immunofluorescence/caveolae fractionation, Ca2+ imaging with fluo-4, pharmacological inhibition, c-Kit immunoreactivity for ICC identification","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization to caveolae plus correlated functional Ca2+ oscillation data, single lab","pmids":["11897792"],"is_preprint":false},{"year":2001,"finding":"Alpha-hTRP4 (but not beta-hTRP4) C-terminus associates in vitro with the C-terminal domain of InsP3 receptors types 1, 2, and 3, as demonstrated by yeast two-hybrid and GST pull-down; this interaction is regulated by alternative splicing (the 84 aa insert in alpha absent in beta contains the binding region).","method":"Yeast two-hybrid assay, GST pull-down experiments","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two orthogonal in vitro binding methods, single lab","pmids":["11163362"],"is_preprint":false},{"year":2005,"finding":"TRPC4 co-localizes with and co-immunoprecipitates with ZO-1 in human fetal astrocytes via the C-terminal TRL PDZ-binding motif; deletion of TRL retains TRPC4 in a juxtanuclear compartment and reduces plasma membrane expression, demonstrating that TRPC4's PDZ-binding motif controls its surface localization in astrocytes.","method":"Co-immunoprecipitation, confocal microscopy, immunoelectron microscopy, cell surface biotinylation, GST fusion protein binding assay, TRL-motif deletion mutants","journal":"Glia","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple methods confirming ZO-1 interaction and PDZ-motif-dependent membrane targeting, single lab","pmids":["15540229"],"is_preprint":false},{"year":2009,"finding":"TRPC1 and TRPC4 channels are expressed at the sarcolemma of skeletal myotubes and associate with the alpha1-syntrophin–dystrophin complex (DAPC); siRNA silencing of alpha1-syntrophin dysregulates cation influx; deletion of the PDZ-containing domain of alpha1-syntrophin prevents restoration of normal cation entry; TRPC1 and TRPC4 co-immunoprecipitate with alpha1-syntrophin, establishing DAPC-anchored TRPC1/C4 as regulators of cation homeostasis in skeletal muscle.","method":"Co-immunoprecipitation, siRNA knockdown, Ca2+ entry measurements, dominant-negative constructs","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and siRNA with functional Ca2+ readout, single lab","pmids":["19812031"],"is_preprint":false},{"year":2009,"finding":"STIM1 interacts with TRPC4 (but not TRPC1) as demonstrated by co-immunoprecipitation in human mesangial cells; TRPC1/TRPC4 complexes constitute functional SOC subunits; STIM1 knockdown significantly reduces thapsigargin-stimulated membrane currents; simultaneous inhibition of STIM1 and TRPC1 produces no additive effect on SOC over single inhibition, suggesting they act in the same pathway.","method":"Co-immunoprecipitation, siRNA knockdown, patch clamp, Ca2+ imaging","journal":"Experimental biology and medicine (Maywood, N.J.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus functional knockdown, single lab","pmids":["19307462"],"is_preprint":false},{"year":2010,"finding":"SESTD1, a protein containing a SEC14-like lipid-binding domain and spectrin-type cytoskeleton interaction domains, associates with TRPC4 and TRPC5 via the channel's calmodulin- and IP3-receptor-binding (CIRB) domain; SESTD1 binds several phospholipid species in a Ca2+-dependent manner in vitro and is essential for efficient receptor-mediated activation of TRPC5.","method":"Yeast two-hybrid screen, co-immunoprecipitation, in vitro phospholipid binding, functional expression assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid + Co-IP + in vitro lipid binding + functional assay, single lab","pmids":["20164195"],"is_preprint":false},{"year":2013,"finding":"A conserved glycine residue in the cytosolic S4-S5 linker (G503 in TRPC4) is a critical gating element; TRPC4G503S mutant is constitutively active with current-voltage relationships resembling fully activated WT; a second mutation S623A in the predicted S6 helix suppresses constitutive activation, indicating that the S4-S5 linker interacts with S6 during gating.","method":"Site-directed mutagenesis, whole-cell patch clamp, homology modeling","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis of gating residue with gain-of-function electrophysiology and rescue by second-site mutation, single lab","pmids":["23677990"],"is_preprint":false},{"year":2008,"finding":"Gαi protein (specifically Gαi subtype) directly activates TRPC4 channel; among Gα proteins tested, only Gαi activates TRPC4; co-expression with M2 muscarinic receptor induces TRPC4 activation by carbachol that is blocked by pertussis toxin; this selectivity distinguishes TRPC4 from TRPC5, TRPC6, and TRPV6.","method":"Whole-cell patch clamp, pertussis toxin inhibition, co-expression with M2 receptor and constitutively active Gα constructs in HEK293 cells","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — electrophysiology with pertussis toxin validation, single lab, single method","pmids":["18854172"],"is_preprint":false},{"year":2015,"finding":"Gαi2 directly binds to TRPC4 when the channel is open, as demonstrated by FRET between TRPC4β-CFP and constitutively active Gαi2-YFP (~15% efficiency vs. ~5% for WT Gαi2); carbachol application via M2 receptor increases FRET efficiency between TRPC4 and Gαi2; Gβγ shows low FRET with TRPC4.","method":"FRET (CFP/YFP-tagged constructs), whole-cell patch clamp, Ca2+ imaging with YC6.1","journal":"American journal of physiology. Cell physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — FRET and electrophysiology combined, demonstrating direct interaction in open state, single lab","pmids":["25788576"],"is_preprint":false},{"year":2007,"finding":"The first ankyrin-like repeat of TRPC4/TRPC5 is the minimum structural domain required for homo- and heteromeric channel assembly; N-terminal fragments including the first ankyrin-like repeat potently suppress TRPC4/5 currents in a dominant-negative fashion; a TRPC5 mutant lacking the first ankyrin-like repeat fails to homo-multimerise and forms non-functional channels.","method":"FRET, TIRF microscopy, dominant-negative electrophysiology in HEK293 cells, deletion mutant analysis","journal":"Cell calcium","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — FRET and dominant-negative electrophysiology with systematic domain deletions, single lab","pmids":["17624425"],"is_preprint":false},{"year":2008,"finding":"The N-terminus of TRPC4 self-associates and forms a tetramer; two distinct self-association domains exist in the N-terminus: the ankyrin repeat domain and the region downstream from the coiled-coil domain, both of which can self-associate independently.","method":"Size-exclusion chromatography, GST pull-down, yeast two-hybrid, circular dichroism","journal":"Cell calcium","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple in vitro biochemical approaches for domain mapping of tetramerization, single lab","pmids":["19070363"],"is_preprint":false},{"year":2014,"finding":"Deletion of TRPC4 (constitutive or lentiviral RNAi in lateral amygdala) decreases anxiety-like behavior; lateral amygdala neurons from TRPC4-/- mice lack potentiation responses through Gαq/11-coupled Group I metabotropic glutamate receptors and CCK2 receptors, establishing TRPC4 as a required component of these Gαq/11-mediated depolarizing responses in the amygdala.","method":"TRPC4-/- mice, lentiviral RNAi, brain slice electrophysiology, behavioral testing (elevated plus maze, open field)","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Moderate — KO mouse plus lentiviral RNAi with concordant slice electrophysiology and behavioral phenotypes","pmids":["24599464"],"is_preprint":false},{"year":2014,"finding":"In lateral septal (LS) neurons, TRPC4-containing channels mediate both below-threshold depolarization (BTD) and above-threshold plateau depolarization (ATPD) in response to group I mGluR agonist; both responses are absent in TRPC4-/- mice; ATPD requires coincident mGluR stimulation and depolarization, and depends on Na+ and Ca2+ influx with dynamic intracellular Ca2+ changes.","method":"TRPC4-/- mice, whole-cell slice recordings, pharmacological manipulation of intracellular Ca2+ and ion gradients","journal":"Pflugers Archiv : European journal of physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse electrophysiology with mechanistic ion substitution experiments, single lab","pmids":["24121765"],"is_preprint":false},{"year":2022,"finding":"TRPC4 acts as a coincidence sensor for Gq/11 and Gi/o signals in lateral septal neurons: group I mGluR (Gq/11) plus GABAB (Gi/o) co-activation is required for strong TRPC4-mediated plateau depolarization; GIRK channels mediate subsequent hyperpolarization; the combination of TRPC4 and GIRK conductances encodes the relative strengths of Gq/11 vs. Gi/o inputs as distinct firing patterns.","method":"Whole-cell slice recordings in lateral septal neurons, receptor agonist/antagonist pharmacology, TRPC4-/- mice, computer modeling","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Moderate — KO mouse electrophysiology with defined receptor pharmacology and computational validation, single lab","pmids":["35544691"],"is_preprint":false},{"year":2013,"finding":"Kisspeptin activation of TRPC4α channels in GnRH neurons requires PIP2 hydrolysis (application of DiC8-PIP2 inhibits current; wortmannin prolongs activation), and cSrc tyrosine kinase activity (inhibition by genistein or PP2 blocks activation); channel activation is not store-operated (thapsigargin and IP3 have no effect) and PKC-independent.","method":"Whole-cell patch clamp in GnRH neurons, single-cell RT-PCR (TRPC4α identification), pharmacological dissection with kinase inhibitors, PIP2 dialysis, PKC activators/inhibitors","journal":"Endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — electrophysiology with multiple pharmacological tools, native neurons, single lab","pmids":["23744639"],"is_preprint":false},{"year":2009,"finding":"Cell-cell contact formation regulates TRPC4 surface expression and Ca2+ signaling in endothelial cells; TRPC4 co-precipitates with β-catenin and VE-cadherin; β-catenin promotes TRPC4 function in a cell-cell contact-dependent manner; EGF recruits TRPC4 to the plasma membrane in proliferating cells but causes retrieval in quiescent barrier-forming cells.","method":"Co-immunoprecipitation, siRNA knockdown, dominant-negative expression, Ca2+ imaging, fluorescent fusion protein localization in HMEC-1 and HEK293 cells","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with functional knockdown and overexpression experiments, single lab","pmids":["19996314"],"is_preprint":false},{"year":2007,"finding":"TRPC4 knockdown by siRNA or antisense in DRG neurons significantly reduces neurite length; this is rescued by overexpression of human TRPC4, establishing a required role for TRPC4 in neurite outgrowth/axonal regeneration.","method":"siRNA and antisense knockdown, neurite length measurement, rescue by hTRPC4 overexpression in DRG neurons and ND7/23 cells","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA/antisense KD with rescue experiment, single lab","pmids":["17928298"],"is_preprint":false},{"year":2004,"finding":"ATP-induced CREB phosphorylation precedes and is required for TRPC4 protein upregulation in human pulmonary artery smooth muscle cells (PASMCs); transfection of a non-phosphorylatable CREB mutant abolishes ATP-mediated TRPC4 expression; TRPC4 siRNA attenuates ATP-enhanced capacitative Ca2+ entry and inhibits ATP-induced PASMC proliferation, placing CREB upstream of TRPC4 in a mitogenic signaling pathway.","method":"CREB phosphorylation assay, non-phosphorylatable CREB mutant transfection, siRNA knockdown, CCE measurement, [3H]thymidine incorporation","journal":"American journal of physiology. Cell physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistatic ordering of CREB→TRPC4 with dominant-negative mutant and siRNA functional readout, single lab","pmids":["15229105"],"is_preprint":false},{"year":2015,"finding":"(-)-Englerin A is a potent, selective, direct activator of TRPC4 and TRPC5 channels; TRPC4 expression is necessary and sufficient for englerin A-induced Ca2+ influx, membrane depolarization, and growth inhibition; englerin A-induced current and growth inhibition are blocked by the TRPC4/C5 inhibitor ML204.","method":"Electrophysiology, Ca2+ imaging, genetic knockdown and overexpression, ML204 pharmacological rescue","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic necessity and sufficiency experiments combined with pharmacological rescue and electrophysiology, multiple cell lines","pmids":["26098886"],"is_preprint":false},{"year":2017,"finding":"(-)-Englerin A cytotoxicity in synovial sarcoma cells is mediated by heteromeric TRPC4/TRPC1 channels; depletion of TRPC1 converts the current to homomeric TRPC4 biophysical properties; Na+ loading through the channel mediates cytotoxicity; depletion of either TRPC1 or TRPC4 suppresses EA cytotoxicity.","method":"Whole-cell patch clamp, siRNA depletion of TRPC1 and TRPC4, Na+-loading with gramicidin-A, selective pharmacological inhibitors (Pico145 vs. ML204)","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biophysical and pharmacological dissection of heteromeric vs. homomeric channels with genetic validation, single lab","pmids":["29209034"],"is_preprint":false},{"year":2015,"finding":"Intracellular spermine blocks TRPC4 (and TRPC5) but not TRPC1/4, TRPC1/5, or TRPC3 channels; the blocking mechanism is electrostatic interaction with glutamate residues E728 and E729 at the C-terminus of TRPC4.","method":"Whole-cell patch clamp with intracellular spermine, site-directed mutagenesis of E728/E729","journal":"Pflugers Archiv : European journal of physiology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — mutagenesis identifies critical residues for block mechanism, electrophysiology, single lab","pmids":["26631167"],"is_preprint":false},{"year":2001,"finding":"TRPC4 is necessary for activation of CFTR Cl- current in mouse aortic endothelial cells; a phosphorylating cocktail activates CFTR-like Cl- current in trp4+/+ but not in trp4-/- cells, despite unchanged CFTR expression, suggesting TRP4 provides a scaffold for functional CFTR channel formation.","method":"TRPC4 knockout mouse endothelial cells, whole-cell patch clamp, RT-PCR for CFTR expression","journal":"BMC physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO endothelial cells with electrophysiology, mechanistic interpretation supported by unchanged CFTR expression","pmids":["11356184"],"is_preprint":false},{"year":2007,"finding":"TRPC4 knockdown reduces EGF-induced store-operated channel (SOC) activation and Ca2+ entry in human corneal epithelial cells; TRPC4 siRNA (89% mRNA knockdown) eliminates EGF-induced SOC activity and reduces EGF-induced proliferation by 54%, establishing TRPC4 as a component of SOC required for EGF mitogenic responses.","method":"siRNA knockdown, whole-cell patch clamp, Ca2+ fluorescence imaging, [3H]thymidine incorporation","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA with multiple functional readouts, single lab","pmids":["16033767"],"is_preprint":false},{"year":2007,"finding":"NO/cGMP/PKG-1α signaling inhibits TRPC4-SOC activity in mesangial cells; PKG-phosphorylated VASP (P-Ser239) co-immunoprecipitates with TRPC4, while unphosphorylated VASP does not, identifying phospho-VASP as a state-dependent interaction partner mediating PKG-dependent inhibition of TRPC4.","method":"Co-immunoprecipitation, immunocytochemistry, Ca2+ imaging (fura-2), PKG-1α inhibitor DT-3, 8-Br-cGMP treatment, Western blot for P-VASP","journal":"American journal of physiology. Renal physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP demonstrating phosphorylation-dependent interaction with functional Ca2+ readout, single lab","pmids":["17913834"],"is_preprint":false},{"year":2018,"finding":"PC1 (polycystin-1) activates TRPC4 through Gαi3; Gαi3 selectively binds to the G-protein-binding domain of PC1 C-terminus; PC1 cleavage dissociates Gαi3, increasing TRPC4 activity; Ca2+ influx through TRPC4 activates STAT1 to regulate cell proliferation/death; PC1/TRPC4/STAT1 downregulation disrupts endothelial cell migration and increases permeability.","method":"Co-immunoprecipitation, TRPC4 activity measurements, STAT1 activation assay, cell migration and permeability assays, siRNA knockdown","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with functional downstream signaling validation, single lab","pmids":["29472562"],"is_preprint":false},{"year":2014,"finding":"A TRPC4 membrane-targeting domain (residues 23-29 in the N-terminus) distinct from the tetramerization domain (requiring residues downstream of aa99 in N-term and upstream of aa730 in C-term) is identified; deletion of the 23-29 region causes ER retention; FRET mapping distinguishes assembly domains from trafficking domains.","method":"Deletion mutant construction, FRET, co-expression with WT TRPC4, confocal microscopy in HEK293 cells","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — FRET-based domain mapping with deletion mutants, single lab","pmids":["25349210"],"is_preprint":false},{"year":2019,"finding":"PI(4,5)P2 dephosphorylation (by voltage-sensing phosphatase DrVSP) inhibits TRPC4α, TRPC4β, TRPC5 homotetramers and TRPC1/4α, TRPC1/4β, TRPC1/5 hetetrotramers; sensitivity to PI(4,5)P2 depletion increases TRPC4β < TRPC4α < TRPC5 in homotetramers; TRPC1 incorporation equalizes PI(4,5)P2 sensitivity; putative PI(4,5)P2 binding sites are identified at K419, K664/R511, K518, and H630 by mutagenesis.","method":"Danio rerio VSP-based PI(4,5)P2 depletion, patch clamp, FRET with PI(4,5)P2 sensor, mutagenesis of basic residues","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis mapping of PIP2 binding combined with functional depletion approach and FRET, single lab","pmids":["30755645"],"is_preprint":false},{"year":2002,"finding":"TRPC4 channels in GI smooth muscle are Ca2+-inhibited nonselective cation channels; TRPC4β single-channel conductance is 17.5 pS; calmidazolium increases inward current; currents are sensitive to lanthanum, niflumic acid, and DIDS; TRPC4β properties match the ICC pacemaker current.","method":"Heterologous expression, whole-cell and single-channel patch clamp, BAPTA dialysis, N-methyl-D-glucamine replacement experiments","journal":"American journal of physiology. Cell physiology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — detailed biophysical characterization with ion substitution and pharmacology, single lab","pmids":["12388058"],"is_preprint":false},{"year":2004,"finding":"TRPC4 co-interacts with NHERF-2 in rat descending vasa recta (DVR); TRPC4 co-immunoprecipitates with NHERF-2 from renal medullary lysates and proteins co-localize in DVR endothelial cells and pericytes; TRPC5 is not detected in DVR.","method":"RT-PCR, immunohistochemistry, co-immunoprecipitation from native renal medullary tissue","journal":"American journal of physiology. Cell physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP from native tissue with corroborating colocalization, single lab","pmids":["15590898"],"is_preprint":false},{"year":2022,"finding":"Tricyclic antidepressants (TCAs) directly inhibit TRPC4 channels in heterologous expression (HEK293) and in native murine colonic myocytes; TCA inhibition of muscarinic cationic current (mIcat) is reduced in TRPC4-knockout mice; TCA treatment inhibits colonic motility in human tissue strips, connecting TRPC4 inhibition to TCA-induced constipation.","method":"Patch clamp in HEK293 cells and native colonic myocytes, TRPC4-KO mice, muscle contraction recordings in human colonic strips","journal":"Journal of cellular and molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — heterologous and native electrophysiology with KO validation and human tissue functional readout, single lab","pmids":["35560982"],"is_preprint":false},{"year":2020,"finding":"TRPC4 and TRPC5 channels support persistent firing in CA1 pyramidal neurons; extracellular application of TRPC4 blocker ML204, TRPC5 blocker clemizole, or pan-TRPC4/5 blocker Pico145 significantly inhibits cholinergically-induced persistent firing; intracellular application of TRPC4 or TRPC5 antibodies also reduces persistent firing.","method":"Whole-cell patch clamp in CA1 neurons, selective pharmacological blockers applied extracellularly and intracellularly","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple pharmacological tools with two application routes confirming TRPC4 involvement in persistent firing, single lab","pmids":["32033274"],"is_preprint":false},{"year":2013,"finding":"TRPC1 and TRPC4 are required for normal myotube size during human post-natal myogenesis; siRNA knockdown or dominant-negative TRPC overexpression reduces SOCE, impairs MEF2 expression, and reduces myotube size; overexpression of STIM1 with TRPC4 or TRPC1 increases SOCE and produces hypertrophic myotubes; normalization of SOCE by extracellular Ca2+, STIM1, or Orai1 overexpression does not rescue the fusion defect without TRPC channel re-expression, indicating TRPC-specific signaling requirements.","method":"siRNA knockdown, dominant-negative overexpression, SOCE measurement, MEF2 expression assay, myotube size quantification","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA/dominant-negative with rescue experiments and specific downstream marker (MEF2), single lab","pmids":["23549783"],"is_preprint":false},{"year":2017,"finding":"TRPC1 and TRPC4 interact preferentially with STIM1L (muscle-specific long isoform) over STIM1 upon store depletion; STIM1L and TRPC1/4 knockdown produce similar reductions in SOCE (~50%) and similar delays in Ca2+ entry onset; STIM1L knockdown produces smaller myotubes similar to TRPC1/4 knockdown.","method":"Co-immunoprecipitation (interaction preference), siRNA knockdown, SOCE measurement, myotube differentiation assay","journal":"Biochimica et biophysica acta. Molecular cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP showing isoform-selective interaction with functional knockdown phenotype, single lab","pmids":["28185894"],"is_preprint":false},{"year":2013,"finding":"TRPC1 and TRPC4 are required for cystitis-induced sensory neuron sprouting into the bladder mucosa; cyclophosphamide-treated Trpc1/c4-/- mice show no increased bladder innervation and diminished bladder overactivity, establishing TRPC1/C4 as necessary for injury-induced neuronal sprouting.","method":"Double knockout mice, cyclophosphamide cystitis model, immunohistochemistry for nerve fiber density, urodynamics","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — double KO mouse with quantified anatomical and functional endpoints, single lab","pmids":["23922735"],"is_preprint":false},{"year":2014,"finding":"TRPC4 upregulation increases intracellular Ca2+ concentration, which activates the Ca2+/CaMKKβ/AMPK pathway leading to mTOR inhibition and autophagy induction in vascular endothelial cells; TRPC4 siRNA abrogates TMS-induced autophagy.","method":"DNA microarray, siRNA knockdown of TRPC4, TRPC4 overexpression, Ca2+ imaging, CaMKKβ/AMPK/mTOR pathway analysis","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA and overexpression with downstream pathway measurements, single lab","pmids":["25476892"],"is_preprint":false},{"year":2011,"finding":"A gain-of-function SNP TRPC4-I957V is associated with reduced myocardial infarction risk; functional studies show TRPC4-I957V has increased channel activity and Ca2+ signals in response to muscarinic agonists and direct G-protein activation; molecular modeling suggests I957V allows firmer interaction between TRPC4 and a tyrosine kinase that phosphorylates Y959, facilitating plasma membrane insertion.","method":"Patch clamp and intracellular Ca2+ measurements in transfected HEK293/CHO cells, site-directed mutagenesis, molecular modeling","journal":"Cardiovascular research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — electrophysiology and Ca2+ measurements with mutagenesis and structural modeling, single lab","pmids":["21427121"],"is_preprint":false}],"current_model":"TRPC4 is a Ca2+-permeable nonselective cation channel that forms homo- or heteromeric (with TRPC1, TRPC3, TRPC5) tetramers with distinct biophysical properties; it is activated downstream of Gi/o proteins (primarily Gαi2 via direct C-terminal SESTD interaction) coincident with PLCδ1 and PIP2 depletion, rather than by store depletion or DAG alone; PIP2 tonically inhibits TRPC4α (but not TRPC4β) through its C-terminal PDZ-binding motif-actin cytoskeleton linkage via NHERF/ERM; dynamic NHERF1/2 dissociation upon PIP2 hydrolysis gates DAG sensitivity; calmodulin binds two C-terminal domains and, as revealed by cryo-EM structures, fixes the channel in a closed conformation via the rib helix; conserved S4-S5 linker glycine G503 and S6 residue S623 form a gating couple; the channel's plasma membrane targeting requires an N-terminal trafficking domain (aa 23-29) and PDZ-motif-dependent interaction with NHERF/EBP50 and ZO-1; in native tissues TRPC4 underlies store-operated Ca2+ entry in vascular endothelium (required for vasorelaxation and lung microvascular permeability), the muscarinic cation current (mICAT) in intestinal and detrusor smooth muscle, pacemaker activity in interstitial cells of Cajal, Gq/11-Gi/o coincidence-detector plateau depolarizations in lateral septal and amygdala neurons, and heteromultimeric TRPC1/4/5 channels supporting hippocampal synaptic transmission and working memory."},"narrative":{"mechanistic_narrative":"TRPC4 is a Ca2+-permeable nonselective cation channel that converts G-protein-coupled receptor signaling into membrane depolarization and Ca2+ entry across vascular, smooth muscle, and neuronal tissues [PMID:11175743, PMID:10837492, PMID:19549525]. The channel is activated by Gq/11-coupled receptors and receptor tyrosine kinases independently of store depletion [PMID:10837492], but its principal direct trigger is the Gi/o family — particularly Gαi2, which binds the C-terminal SESTD/CIRB region (via residues K715/R716) when the channel is open [PMID:22457348, PMID:18854172, PMID:25788576]; full activation requires coincident PLCδ1 engagement and hydrolysis of PIP2 rather than store depletion or DAG alone [PMID:26755577]. PIP2 exerts isoform-specific tonic inhibition of TRPC4α (but not TRPC4β) through the C-terminal PDZ-binding TRL motif that tethers the channel to the actin cytoskeleton via NHERF/EBP50 and ERM proteins; dynamic dissociation of NHERF upon PIP2 hydrolysis is a prerequisite for DAG-mediated gating, making the scaffold itself a negative regulator [PMID:18230622, PMID:27994151, PMID:30755645]. Calmodulin binds two C-terminal domains in a Ca2+-dependent manner and, as resolved by cryo-EM, locks the channel closed by ordering the rib helix, while structural studies define the tetrameric six-transmembrane fold, the disulfide-stabilized pore loop, and a conserved S4-S5 linker glycine (G503) that couples to S6 residue S623 during gating [PMID:11311128, PMID:30082700, PMID:33236980, PMID:23677990]. TRPC4 assembles as homotetramers or heteromultimers exclusively with TRPC1, TRPC3, and TRPC5, with the first ankyrin-like repeat and N-terminal self-association domains driving assembly and a distinct N-terminal domain (aa 23-29) plus the PDZ motif directing plasma-membrane targeting [PMID:16537542, PMID:11713258, PMID:28790178, PMID:17624425, PMID:19070363, PMID:25349210]. In native tissues TRPC4 underlies store-operated Ca2+ entry in vascular endothelium required for vasorelaxation and microvascular permeability [PMID:11175743, PMID:12114324], the muscarinic cation current (mICAT) coupling muscarinic receptors to intestinal smooth muscle contraction [PMID:19549525], and Gq/11–Gi/o coincidence-detector plateau depolarizations in lateral septal and amygdala neurons as well as heteromeric TRPC1/4/5 channels supporting hippocampal synaptic transmission and working memory [PMID:28790178, PMID:24599464, PMID:35544691]. A gain-of-function TRPC4-I957V variant is associated with reduced myocardial infarction risk [PMID:21427121].","teleology":[{"year":2000,"claim":"Establishing how TRPC4 is gated answered whether it is a store-operated channel or a receptor-operated one — TRPC4 was shown to be activated by Gq/11 receptors and RTKs independent of store depletion, defining it as a receptor-operated cation channel.","evidence":"Heterologous expression in HEK293 with patch clamp, Mn2+ quench, and GTPγS infusion","pmids":["10837492"],"confidence":"High","gaps":["Did not identify which G-protein subunit directly couples to the channel","Native physiological agonists not defined"]},{"year":2001,"claim":"Knockout and antisense studies resolved whether TRPC4 contributes to native store-operated currents, establishing it as an indispensable component of endothelial SOC required for agonist-induced Ca2+ entry and vasorelaxation.","evidence":"TRPC4-/- mice and bovine adrenal antisense knockdown with electrophysiology, Ca2+ imaging, and vascular tension assays","pmids":["11175743","10816590"],"confidence":"High","gaps":["Apparent conflict with the receptor-operated gating model not reconciled","Molecular composition of the native SOC complex undefined"]},{"year":2001,"claim":"Splice-variant analysis explained why TRPC4 activity varies — the TRPC4α C-terminus acts as an autoinhibitory domain whose truncation restores activity, and FRET confirmed homo- and heteromultimer assembly of α and β.","evidence":"Heterologous expression, whole-cell patch clamp, FRET, GFP-fusion trafficking in HEK293 cells","pmids":["11713258"],"confidence":"High","gaps":["Molecular basis of the autoinhibition (lipid vs. scaffold) not yet identified","Physiological relevance of α/β ratio in native tissue unknown"]},{"year":2000,"claim":"Identifying scaffold partners addressed how TRPC4 is organized and localized — NHERF/EBP50 binds the channel and PLCβ, linking it to the ERM-actin cytoskeleton and forming a signaling complex in native brain.","evidence":"Reciprocal Co-IP from transfected cells and mouse brain plus GST pull-down","pmids":["10980202"],"confidence":"High","gaps":["Functional consequence of the complex for gating not established at this stage"]},{"year":2001,"claim":"Mapping calmodulin binding addressed Ca2+-dependent feedback — TRPC4 binds CaM at two C-terminal domains in a Ca2+-dependent manner with defined affinities.","evidence":"CaM-Sepharose affinity, GST pull-down, dansyl-CaM peptide fluorimetry","pmids":["11311128"],"confidence":"High","gaps":["Functional effect of CaM binding on channel gating not shown until structural work","Stoichiometry in the assembled channel unknown"]},{"year":2002,"claim":"Knockout studies extended TRPC4's endothelial role to barrier function, showing TRPC4-dependent Ca2+ entry drives actin stress-fiber formation, cell retraction, and increased lung microvascular permeability, and to pacemaker ICC where TRPC4 localizes to caveolae.","evidence":"TRPC4-/- lung endothelial cells, isolated-perfused lung filtration measurement, caveolae fractionation and Ca2+ imaging in ICC","pmids":["12114324","11897792"],"confidence":"High","gaps":["Direct demonstration that caveolar localization gates ICC pacemaking lacking","Link between Ca2+ entry and cytoskeletal contraction mechanistically incomplete"]},{"year":2002,"claim":"Surface-targeting studies defined how TRPC4 reaches the plasma membrane — the C-terminal TRL PDZ motif and its EBP50/ERM interaction control membrane localization, with motif deletion causing perinuclear/Golgi retention.","evidence":"Immunofluorescence, surface biotinylation, EBP50 truncation mutants in HEK293; biophysical channel characterization in GI smooth muscle","pmids":["12154080","12388058"],"confidence":"High","gaps":["Trafficking machinery downstream of the motif not identified","Relationship of TRL-dependent targeting to PIP2 inhibition not yet linked"]},{"year":2005,"claim":"Cytoskeletal anchoring partners addressed how the channel's gating machinery is assembled at the membrane — protein 4.1 and ZO-1 bind TRPC4, and protein 4.1 interaction is essential for endothelial store-operated channel gating.","evidence":"Co-IP, domain deletion, peptide competition, electrophysiology in endothelial and astrocyte systems","pmids":["16254212","15540229"],"confidence":"High","gaps":["How spectrin-actin coupling mechanically gates the pore is unresolved","ZO-1 and protein 4.1 contributions to native gating in other tissues untested"]},{"year":2006,"claim":"Heteromultimer studies established TRPC4's partner specificity beyond homotetramers, showing TRPC3-TRPC4 form a redox-sensitive heteromeric channel in native endothelium.","evidence":"Co-IP, FRET, dominant-negative electrophysiology in porcine aortic endothelial cells","pmids":["16537542"],"confidence":"High","gaps":["Physiological stimulus engaging the redox-sensitive heteromer in vivo undefined"]},{"year":2007,"claim":"Assembly-domain mapping answered which structural element drives tetramerization — the first ankyrin-like repeat is the minimal domain for homo/heteromeric assembly and acts dominant-negatively when expressed alone.","evidence":"FRET, TIRF, dominant-negative electrophysiology, deletion mutants in HEK293","pmids":["17624425"],"confidence":"Medium","gaps":["Single lab; in vivo relevance of N-terminal assembly fragments not tested"]},{"year":2008,"claim":"Identifying the direct G-protein activator answered the long-standing gating question — only Gαi among tested Gα proteins directly activates TRPC4, with M2-receptor coupling blocked by pertussis toxin, distinguishing it from TRPC5/6/V6.","evidence":"Whole-cell patch clamp, pertussis toxin, constitutively active Gα and M2 co-expression in HEK293","pmids":["18854172"],"confidence":"Medium","gaps":["Single method, single lab","Direct binding not yet demonstrated, only functional selectivity"]},{"year":2008,"claim":"Lipid-regulation studies revealed isoform-specific PIP2 control — PIP2 inhibits TRPC4α (not β) through the PDZ-motif/actin linkage, and PIP2 breakdown is a required activation step alongside Ca2+ and Gi/o.","evidence":"Patch clamp with intracellular PIP2, in vitro lipid binding, cytochalasin D, PDZ-deletion mutants","pmids":["18230622"],"confidence":"High","gaps":["Direct PIP2 binding residues not yet mapped","How cytoskeletal tethering transduces lipid signal to the gate unclear"]},{"year":2012,"claim":"Mapping the Gαi interface answered how Gi/o coupling occurs structurally — Gαi2 directly binds the C-terminal SESTD domain via K715/R716 and fully activates the channel.","evidence":"Co-IP, site-directed mutagenesis, constitutively active Gαi2, patch clamp, Ca2+ imaging","pmids":["22457348"],"confidence":"High","gaps":["Stoichiometry of Gαi2:channel interaction undefined","Coupling to receptor identity in different native tissues incompletely mapped"]},{"year":2013,"claim":"Gating-residue and gain-of-function studies defined the pore-coupling mechanism — S4-S5 linker glycine G503 couples to S6 S623, and a TRPC4-I957V variant associated with reduced MI risk shows enhanced activity.","evidence":"Site-directed mutagenesis with gain-of-function electrophysiology and homology modeling; HEK293/CHO functional studies of the SNP","pmids":["23677990","21427121"],"confidence":"Medium","gaps":["G503-S623 coupling inferred from mutagenesis prior to structure","I957V genetic association is correlative for disease risk"]},{"year":2016,"claim":"Coincidence-detection studies integrated the activation logic — TRPC4 requires coincident Gi/o and PLCδ1 signaling with PIP2, and dynamic NHERF1/2 dissociation upon PIP2 hydrolysis is the prerequisite for DAG sensitivity, making the scaffold a direct negative regulator.","evidence":"Patch clamp, PLCδ1 siRNA, dominant-negative and constitutively active constructs, FRET conformational analysis, PDZ-motif mutagenesis","pmids":["26755577","27994151"],"confidence":"High","gaps":["Exact conformational path from C-terminus to pore not resolved at this stage","Generalizability of the PLCδ1 requirement across native tissues untested"]},{"year":2017,"claim":"Native interactome and triple-KO behavior established TRPC4's neuronal partner identity and physiology — TRPC1/4/5 assemble exclusively with one another in brain and support hippocampal synaptic transmission and spatial working memory.","evidence":"Quantitative mass spectrometry interactome, triple-KO hippocampal slice electrophysiology, in vivo LFP, behavior","pmids":["28790178"],"confidence":"High","gaps":["Subunit stoichiometry of native TRPC1/4/5 channels undefined","Receptor coupling driving synaptic TRPC currents not fully mapped"]},{"year":2018,"claim":"Cryo-EM structures answered the architecture question — TRPC4 is a tetrameric six-transmembrane channel with a disulfide-stabilized pore loop, defined selectivity filter and lower gate, and cytoplasmic hubs for protein interactions.","evidence":"Cryo-EM of mouse (3.3 Å) and zebrafish (3.6 Å) TRPC4","pmids":["30082700","29717981"],"confidence":"High","gaps":["Closed/apo states only; activated-state structure not captured","Direct visualization of lipid and G-protein binding not yet achieved"]},{"year":2020,"claim":"Ligand-bound cryo-EM revealed the CaM-mediated closing mechanism and a drug-binding pocket — CaM binds the rib helix to fix the channel closed, and pyridazinone inhibitors bind a voltage-sensing-like-domain cavity that propagates to the pore.","evidence":"Cryo-EM of TRPC4-CaM and TRPC4-inhibitor complexes","pmids":["33236980"],"confidence":"High","gaps":["How physiological Ca2+/CaM dynamics tune gating in vivo not established","Structure of the activating PIP2/Gαi-bound state still absent"]},{"year":2022,"claim":"Neuronal recordings refined TRPC4 as a bidirectional coincidence sensor — in lateral septal neurons it integrates Gq/11 (mGluR) and Gi/o (GABAB) inputs into plateau depolarizations, with GIRK encoding the relative input strengths as firing patterns.","evidence":"Whole-cell slice recordings, receptor pharmacology, TRPC4-/- mice, computational modeling","pmids":["35544691"],"confidence":"High","gaps":["Molecular determinants distinguishing coincidence detection across neuron types unclear","In vivo behavioral correlate of the encoding scheme untested"]},{"year":null,"claim":"How the activating conformation is achieved at the structural level — the simultaneous engagement of Gαi2, PIP2 depletion, PLCδ1, NHERF dissociation, and Ca2+/CaM converging on the G503-S623 gate — remains unresolved, as all available high-resolution structures are closed or inhibitor-bound.","evidence":"No activated-state structure or integrated reconstitution in the available corpus","pmids":[],"confidence":"High","gaps":["No open-state structure of TRPC4","No reconstitution combining G-protein, lipid, and scaffold inputs","Subunit stoichiometry of native heteromers undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[0,1,7,48]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[12,28,29,34]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[8,47]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[11,12,21,23,46]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[2,8,10]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[46]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[11]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,12,13,34]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[18,32,33,34,51]},{"term_id":"R-HSA-397014","term_label":"Muscle contraction","supporting_discovery_ids":[7]},{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[0,3,4]}],"complexes":["TRPC1/4/5 heteromeric channel","TRPC3/4 redox-sensitive heteromeric channel","TRPC1/4 heteromeric channel","TRPC4-NHERF/EBP50 scaffold complex"],"partners":["TRPC1","TRPC5","TRPC3","NHERF1","GNAI2","CALM1","STIM1","SESTD1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9UBN4","full_name":"Short transient receptor potential channel 4","aliases":["Trp-related protein 4","hTrp-4","hTrp4"],"length_aa":977,"mass_kda":112.1,"function":"Forms a receptor-activated non-selective calcium permeant cation channel (PubMed:11042129, PubMed:11713258, PubMed:16144838, PubMed:39478185). Acts as a cell-cell contact-dependent endothelial calcium entry channel (PubMed:19996314). Forms a homomeric ion channel or a heteromeric ion channel with TRPC1; the heteromeric ion channel has reduced calcium permeability compared to the homomeric channel (PubMed:39478185). Also permeable to monovalent ions including sodium, lithium and cesium ions (PubMed:39478185) Forms a receptor-activated non-selective calcium permeant cation channel","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q9UBN4/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TRPC4","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/TRPC4","total_profiled":1310},"omim":[{"mim_id":"621011","title":"SEC14 AND SPECTRIN DOMAINS-CONTAINING PROTEIN 1; SESTD1","url":"https://www.omim.org/entry/621011"},{"mim_id":"612489","title":"RING FINGER PROTEIN 24; RNF24","url":"https://www.omim.org/entry/612489"},{"mim_id":"608945","title":"FRAS1-RELATED EXTRACELLULAR MATRIX PROTEIN 2; FREM2","url":"https://www.omim.org/entry/608945"},{"mim_id":"608430","title":"TRPC4-ASSOCIATED PROTEIN; TRPC4AP","url":"https://www.omim.org/entry/608430"},{"mim_id":"605921","title":"STROMAL INTERACTION MOLECULE 1; STIM1","url":"https://www.omim.org/entry/605921"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"endometrium 1","ntpm":11.2},{"tissue":"smooth muscle","ntpm":11.8}],"url":"https://www.proteinatlas.org/search/TRPC4"},"hgnc":{"alias_symbol":["HTRP4","TRP4"],"prev_symbol":[]},"alphafold":{"accession":"Q9UBN4","domains":[{"cath_id":"-","chopping":"391-494","consensus_level":"medium","plddt":90.0352,"start":391,"end":494},{"cath_id":"-","chopping":"509-646","consensus_level":"high","plddt":92.8365,"start":509,"end":646}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UBN4","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UBN4-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UBN4-F1-predicted_aligned_error_v6.png","plddt_mean":74.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TRPC4","jax_strain_url":"https://www.jax.org/strain/search?query=TRPC4"},"sequence":{"accession":"Q9UBN4","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9UBN4.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9UBN4/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UBN4"}},"corpus_meta":[{"pmid":"11175743","id":"PMC_11175743","title":"Lack of an endothelial store-operated Ca2+ current impairs agonist-dependent vasorelaxation in TRP4-/- mice.","date":"2001","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/11175743","citation_count":480,"is_preprint":false},{"pmid":"10837492","id":"PMC_10837492","title":"Receptor-mediated regulation of the nonselective cation channels TRPC4 and TRPC5.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10837492","citation_count":348,"is_preprint":false},{"pmid":"12114324","id":"PMC_12114324","title":"Impairment of store-operated Ca2+ entry in TRPC4(-/-) mice interferes with increase in lung microvascular permeability.","date":"2002","source":"Circulation research","url":"https://pubmed.ncbi.nlm.nih.gov/12114324","citation_count":311,"is_preprint":false},{"pmid":"10980202","id":"PMC_10980202","title":"Association of mammalian trp4 and phospholipase C isozymes with a PDZ domain-containing protein, NHERF.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10980202","citation_count":195,"is_preprint":false},{"pmid":"20696377","id":"PMC_20696377","title":"C. elegans TRP family protein TRP-4 is a pore-forming subunit of a native mechanotransduction channel.","date":"2010","source":"Neuron","url":"https://pubmed.ncbi.nlm.nih.gov/20696377","citation_count":186,"is_preprint":false},{"pmid":"16537542","id":"PMC_16537542","title":"TRPC3 and TRPC4 associate to form a redox-sensitive cation channel. Evidence for expression of native TRPC3-TRPC4 heteromeric channels in endothelial cells.","date":"2006","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16537542","citation_count":177,"is_preprint":false},{"pmid":"21795696","id":"PMC_21795696","title":"Identification of ML204, a novel potent antagonist that selectively modulates native TRPC4/C5 ion channels.","date":"2011","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21795696","citation_count":177,"is_preprint":false},{"pmid":"10816590","id":"PMC_10816590","title":"TRP4 (CCE1) protein is part of native calcium release-activated Ca2+-like channels in adrenal cells.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10816590","citation_count":164,"is_preprint":false},{"pmid":"25707820","id":"PMC_25707820","title":"(-)-Englerin A is a potent and selective activator of TRPC4 and TRPC5 calcium channels.","date":"2015","source":"Angewandte Chemie (International ed. in English)","url":"https://pubmed.ncbi.nlm.nih.gov/25707820","citation_count":163,"is_preprint":false},{"pmid":"11897792","id":"PMC_11897792","title":"Calcium oscillation linked to pacemaking of interstitial cells of Cajal: requirement of calcium influx and localization of TRP4 in caveolae.","date":"2002","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11897792","citation_count":158,"is_preprint":false},{"pmid":"19549525","id":"PMC_19549525","title":"Deletion of TRPC4 and TRPC6 in mice impairs smooth muscle contraction and intestinal motility in vivo.","date":"2009","source":"Gastroenterology","url":"https://pubmed.ncbi.nlm.nih.gov/19549525","citation_count":146,"is_preprint":false},{"pmid":"17593972","id":"PMC_17593972","title":"Corticolimbic expression of TRPC4 and TRPC5 channels in the rodent brain.","date":"2007","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/17593972","citation_count":143,"is_preprint":false},{"pmid":"18230622","id":"PMC_18230622","title":"Isoform-specific inhibition of TRPC4 channel by phosphatidylinositol 4,5-bisphosphate.","date":"2008","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/18230622","citation_count":139,"is_preprint":false},{"pmid":"12765689","id":"PMC_12765689","title":"TRPC4 and TRPC5: receptor-operated Ca2+-permeable nonselective cation channels.","date":"2003","source":"Cell calcium","url":"https://pubmed.ncbi.nlm.nih.gov/12765689","citation_count":133,"is_preprint":false},{"pmid":"30082700","id":"PMC_30082700","title":"Structure of the mouse TRPC4 ion channel.","date":"2018","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/30082700","citation_count":113,"is_preprint":false},{"pmid":"29385160","id":"PMC_29385160","title":"Treatment with HC-070, a potent inhibitor of TRPC4 and TRPC5, leads to anxiolytic and antidepressant effects in mice.","date":"2018","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/29385160","citation_count":110,"is_preprint":false},{"pmid":"28790178","id":"PMC_28790178","title":"Heteromeric channels formed by TRPC1, TRPC4 and TRPC5 define hippocampal synaptic transmission and working memory.","date":"2017","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/28790178","citation_count":109,"is_preprint":false},{"pmid":"11042129","id":"PMC_11042129","title":"Cloning and expression of the human transient receptor potential 4 (TRP4) gene: localization and functional expression of human TRP4 and TRP3.","date":"2000","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/11042129","citation_count":107,"is_preprint":false},{"pmid":"12154080","id":"PMC_12154080","title":"The PDZ-interacting domain of TRPC4 controls its localization and surface expression in HEK293 cells.","date":"2002","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/12154080","citation_count":99,"is_preprint":false},{"pmid":"27994151","id":"PMC_27994151","title":"Dynamic NHERF interaction with TRPC4/5 proteins is required for channel gating by diacylglycerol.","date":"2016","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/27994151","citation_count":96,"is_preprint":false},{"pmid":"24599464","id":"PMC_24599464","title":"Decreased anxiety-like behavior and Gαq/11-dependent responses in the amygdala of mice lacking TRPC4 channels.","date":"2014","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/24599464","citation_count":92,"is_preprint":false},{"pmid":"26098886","id":"PMC_26098886","title":"Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation.","date":"2015","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/26098886","citation_count":91,"is_preprint":false},{"pmid":"26069213","id":"PMC_26069213","title":"A background Ca2+ entry pathway mediated by TRPC1/TRPC4 is critical for development of pathological cardiac remodelling.","date":"2015","source":"European heart journal","url":"https://pubmed.ncbi.nlm.nih.gov/26069213","citation_count":89,"is_preprint":false},{"pmid":"29717981","id":"PMC_29717981","title":"Electron cryo-microscopy structure of the canonical TRPC4 ion channel.","date":"2018","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/29717981","citation_count":86,"is_preprint":false},{"pmid":"15229105","id":"PMC_15229105","title":"ATP-induced mitogenesis is mediated by cyclic AMP response element-binding protein-enhanced TRPC4 expression and activity in human pulmonary artery smooth muscle cells.","date":"2004","source":"American journal of physiology. Cell physiology","url":"https://pubmed.ncbi.nlm.nih.gov/15229105","citation_count":86,"is_preprint":false},{"pmid":"11713258","id":"PMC_11713258","title":"Functional differences between TRPC4 splice variants.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11713258","citation_count":85,"is_preprint":false},{"pmid":"22457348","id":"PMC_22457348","title":"Selective Gαi subunits as novel direct activators of transient receptor potential canonical (TRPC)4 and TRPC5 channels.","date":"2012","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/22457348","citation_count":85,"is_preprint":false},{"pmid":"12388058","id":"PMC_12388058","title":"TRPC4 currents have properties similar to the pacemaker current in interstitial cells of Cajal.","date":"2002","source":"American journal of physiology. Cell physiology","url":"https://pubmed.ncbi.nlm.nih.gov/12388058","citation_count":80,"is_preprint":false},{"pmid":"11830588","id":"PMC_11830588","title":"The role of endogenous human Trp4 in regulating carbachol-induced calcium oscillations in HEK-293 cells.","date":"2002","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11830588","citation_count":78,"is_preprint":false},{"pmid":"16254212","id":"PMC_16254212","title":"Activation of the endothelial store-operated ISOC Ca2+ channel requires interaction of protein 4.1 with TRPC4.","date":"2005","source":"Circulation research","url":"https://pubmed.ncbi.nlm.nih.gov/16254212","citation_count":78,"is_preprint":false},{"pmid":"17928298","id":"PMC_17928298","title":"TRPC4 in rat dorsal root ganglion neurons is increased after nerve injury and is necessary for neurite outgrowth.","date":"2007","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17928298","citation_count":72,"is_preprint":false},{"pmid":"16033767","id":"PMC_16033767","title":"TRPC4 knockdown suppresses epidermal growth factor-induced store-operated channel activation and growth in human corneal epithelial cells.","date":"2005","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16033767","citation_count":70,"is_preprint":false},{"pmid":"11856742","id":"PMC_11856742","title":"TRPC4 can be activated by G-protein-coupled receptors and provides sufficient Ca(2+) to trigger exocytosis in neuroendocrine cells.","date":"2002","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11856742","citation_count":69,"is_preprint":false},{"pmid":"15902430","id":"PMC_15902430","title":"Receptor-operated cation channels formed by TRPC4 and TRPC5.","date":"2005","source":"Naunyn-Schmiedeberg's archives of pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/15902430","citation_count":65,"is_preprint":false},{"pmid":"29634917","id":"PMC_29634917","title":"Ca2+ handling remodeling and STIM1L/Orai1/TRPC1/TRPC4 upregulation in monocrotaline-induced right ventricular hypertrophy.","date":"2018","source":"Journal of molecular and cellular cardiology","url":"https://pubmed.ncbi.nlm.nih.gov/29634917","citation_count":65,"is_preprint":false},{"pmid":"11311128","id":"PMC_11311128","title":"The transient receptor potential, TRP4, cation channel is a novel member of the family of calmodulin binding proteins.","date":"2001","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/11311128","citation_count":64,"is_preprint":false},{"pmid":"19812031","id":"PMC_19812031","title":"Regulation of TRPC1 and TRPC4 cation channels requires an alpha1-syntrophin-dependent complex in skeletal mouse myotubes.","date":"2009","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19812031","citation_count":64,"is_preprint":false},{"pmid":"11163362","id":"PMC_11163362","title":"Alternative splice variants of hTrp4 differentially interact with the C-terminal portion of the inositol 1,4,5-trisphosphate receptors.","date":"2001","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/11163362","citation_count":63,"is_preprint":false},{"pmid":"15044151","id":"PMC_15044151","title":"TRPC4 forms store-operated Ca2+ channels in mouse mesangial cells.","date":"2004","source":"American journal of physiology. Cell physiology","url":"https://pubmed.ncbi.nlm.nih.gov/15044151","citation_count":62,"is_preprint":false},{"pmid":"26755577","id":"PMC_26755577","title":"Critical roles of Gi/o proteins and phospholipase C-δ1 in the activation of receptor-operated TRPC4 channels.","date":"2016","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/26755577","citation_count":61,"is_preprint":false},{"pmid":"19307462","id":"PMC_19307462","title":"Interaction between TRPC1/TRPC4 assembly and STIM1 contributes to store-operated Ca2+ entry in mesangial cells.","date":"2009","source":"Experimental biology and medicine (Maywood, N.J.)","url":"https://pubmed.ncbi.nlm.nih.gov/19307462","citation_count":59,"is_preprint":false},{"pmid":"33236980","id":"PMC_33236980","title":"Structural basis of TRPC4 regulation by calmodulin and pharmacological agents.","date":"2020","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/33236980","citation_count":58,"is_preprint":false},{"pmid":"2656261","id":"PMC_2656261","title":"Interpathway regulation of the TRP4 gene of yeast.","date":"1989","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/2656261","citation_count":56,"is_preprint":false},{"pmid":"26317356","id":"PMC_26317356","title":"Acute Treatment with a Novel TRPC4/C5 Channel Inhibitor Produces Antidepressant and Anxiolytic-Like Effects in Mice.","date":"2015","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/26317356","citation_count":51,"is_preprint":false},{"pmid":"15540229","id":"PMC_15540229","title":"Canonical transient receptor potential channel 4 (TRPC4) co-localizes with the scaffolding protein ZO-1 in human fetal astrocytes in culture.","date":"2005","source":"Glia","url":"https://pubmed.ncbi.nlm.nih.gov/15540229","citation_count":50,"is_preprint":false},{"pmid":"23744639","id":"PMC_23744639","title":"Kisspeptin activation of TRPC4 channels in female GnRH neurons requires PIP2 depletion and cSrc kinase activation.","date":"2013","source":"Endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/23744639","citation_count":47,"is_preprint":false},{"pmid":"23549783","id":"PMC_23549783","title":"During post-natal human myogenesis, normal myotube size requires TRPC1- and TRPC4-mediated Ca²⁺ entry.","date":"2013","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/23549783","citation_count":47,"is_preprint":false},{"pmid":"16404161","id":"PMC_16404161","title":"TRPC4 is an essential component of the nonselective cation channel activated by muscarinic stimulation in mouse visceral smooth muscle cells.","date":"2005","source":"Molecules and cells","url":"https://pubmed.ncbi.nlm.nih.gov/16404161","citation_count":46,"is_preprint":false},{"pmid":"19996314","id":"PMC_19996314","title":"Cell-cell contact formation governs Ca2+ signaling by TRPC4 in the vascular endothelium: evidence for a regulatory TRPC4-beta-catenin interaction.","date":"2009","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19996314","citation_count":45,"is_preprint":false},{"pmid":"28185894","id":"PMC_28185894","title":"TRPC1 and TRPC4 channels functionally interact with STIM1L to promote myogenesis and maintain fast repetitive Ca2+ release in human myotubes.","date":"2017","source":"Biochimica et biophysica acta. Molecular cell research","url":"https://pubmed.ncbi.nlm.nih.gov/28185894","citation_count":44,"is_preprint":false},{"pmid":"2428012","id":"PMC_2428012","title":"The TRP4 gene of Saccharomyces cerevisiae: isolation and structural analysis.","date":"1986","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/2428012","citation_count":43,"is_preprint":false},{"pmid":"20164195","id":"PMC_20164195","title":"The phospholipid-binding protein SESTD1 is a novel regulator of the transient receptor potential channels TRPC4 and TRPC5.","date":"2010","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20164195","citation_count":43,"is_preprint":false},{"pmid":"25816897","id":"PMC_25816897","title":"Identification and optimization of 2-aminobenzimidazole derivatives as novel inhibitors of TRPC4 and TRPC5 channels.","date":"2015","source":"British journal of pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/25816897","citation_count":42,"is_preprint":false},{"pmid":"24756704","id":"PMC_24756704","title":"TRPC4- and TRPC4-containing channels.","date":"2014","source":"Handbook of experimental pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/24756704","citation_count":39,"is_preprint":false},{"pmid":"23677990","id":"PMC_23677990","title":"Conserved gating elements in TRPC4 and TRPC5 channels.","date":"2013","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/23677990","citation_count":39,"is_preprint":false},{"pmid":"22285229","id":"PMC_22285229","title":"Effect of non-steroidal anti-inflammatory drugs and new fenamate analogues on TRPC4 and TRPC5 channels.","date":"2012","source":"Biochemical pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/22285229","citation_count":38,"is_preprint":false},{"pmid":"24388923","id":"PMC_24388923","title":"A rat knockout model implicates TRPC4 in visceral pain sensation.","date":"2014","source":"Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/24388923","citation_count":36,"is_preprint":false},{"pmid":"22878724","id":"PMC_22878724","title":"The roles of G proteins in the activation of TRPC4 and TRPC5 transient receptor potential channels.","date":"2012","source":"Channels (Austin, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/22878724","citation_count":35,"is_preprint":false},{"pmid":"30463370","id":"PMC_30463370","title":"Emerging Roles of Diacylglycerol-Sensitive TRPC4/5 Channels.","date":"2018","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/30463370","citation_count":33,"is_preprint":false},{"pmid":"31222490","id":"PMC_31222490","title":"TRPC1 as a negative regulator for TRPC4 and TRPC5 channels.","date":"2019","source":"Pflugers Archiv : European journal of physiology","url":"https://pubmed.ncbi.nlm.nih.gov/31222490","citation_count":33,"is_preprint":false},{"pmid":"29209034","id":"PMC_29209034","title":"Na+ entry through heteromeric TRPC4/C1 channels mediates (-)Englerin A-induced cytotoxicity in synovial sarcoma cells.","date":"2017","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/29209034","citation_count":33,"is_preprint":false},{"pmid":"26432495","id":"PMC_26432495","title":"Regulation of neuropathic pain behavior by amygdaloid TRPC4/C5 channels.","date":"2015","source":"Neuroscience letters","url":"https://pubmed.ncbi.nlm.nih.gov/26432495","citation_count":33,"is_preprint":false},{"pmid":"36834672","id":"PMC_36834672","title":"The Molecular Heterogeneity of Store-Operated Ca2+ Entry in Vascular Endothelial Cells: The Different roles of Orai1 and TRPC1/TRPC4 Channels in the Transition from Ca2+-Selective to Non-Selective Cation Currents.","date":"2023","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/36834672","citation_count":32,"is_preprint":false},{"pmid":"18854172","id":"PMC_18854172","title":"The specific activation of TRPC4 by Gi protein subtype.","date":"2008","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/18854172","citation_count":31,"is_preprint":false},{"pmid":"17031666","id":"PMC_17031666","title":"Evidence that TRPC4 supports the calcium selective I(CRAC)-like current in human gingival keratinocytes.","date":"2006","source":"Pflugers Archiv : European journal of physiology","url":"https://pubmed.ncbi.nlm.nih.gov/17031666","citation_count":30,"is_preprint":false},{"pmid":"15590898","id":"PMC_15590898","title":"Expression of TRPC4 channel protein that interacts with NHERF-2 in rat descending vasa recta.","date":"2004","source":"American journal of physiology. Cell physiology","url":"https://pubmed.ncbi.nlm.nih.gov/15590898","citation_count":29,"is_preprint":false},{"pmid":"17345099","id":"PMC_17345099","title":"Distribution of TRPC4 in developing and adult murine brain.","date":"2007","source":"Cell and tissue research","url":"https://pubmed.ncbi.nlm.nih.gov/17345099","citation_count":29,"is_preprint":false},{"pmid":"12672226","id":"PMC_12672226","title":"A novel N-terminal cyclic dynorphin A analogue cyclo(N,5)[Trp(3),Trp(4),Glu(5)] dynorphin A-(1-11)NH(2) that lacks the basic N-terminus.","date":"2003","source":"Journal of medicinal chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12672226","citation_count":29,"is_preprint":false},{"pmid":"17913834","id":"PMC_17913834","title":"Association of VASP with TRPC4 in PKG-mediated inhibition of the store-operated calcium response in mesangial cells.","date":"2007","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/17913834","citation_count":27,"is_preprint":false},{"pmid":"30038709","id":"PMC_30038709","title":"TRPC4/TRPC5 channels mediate adverse reaction to the cancer cell cytotoxic agent (-)-Englerin A.","date":"2018","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/30038709","citation_count":27,"is_preprint":false},{"pmid":"32289348","id":"PMC_32289348","title":"Sensory Neuron-Expressed TRPC4 Is a Target for the Relief of Psoriasiform Itch and Skin Inflammation in Mice.","date":"2020","source":"The Journal of investigative dermatology","url":"https://pubmed.ncbi.nlm.nih.gov/32289348","citation_count":27,"is_preprint":false},{"pmid":"17624425","id":"PMC_17624425","title":"The first ankyrin-like repeat is the minimum indispensable key structure for functional assembly of homo- and heteromeric TRPC4/TRPC5 channels.","date":"2007","source":"Cell calcium","url":"https://pubmed.ncbi.nlm.nih.gov/17624425","citation_count":27,"is_preprint":false},{"pmid":"21427121","id":"PMC_21427121","title":"A gain-of-function SNP in TRPC4 cation channel protects against myocardial infarction.","date":"2011","source":"Cardiovascular research","url":"https://pubmed.ncbi.nlm.nih.gov/21427121","citation_count":26,"is_preprint":false},{"pmid":"25476892","id":"PMC_25476892","title":"Novel role for TRPC4 in regulation of macroautophagy by a small molecule in vascular endothelial cells.","date":"2014","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/25476892","citation_count":23,"is_preprint":false},{"pmid":"23922735","id":"PMC_23922735","title":"Crucial role of TRPC1 and TRPC4 in cystitis-induced neuronal sprouting and bladder overactivity.","date":"2013","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/23922735","citation_count":22,"is_preprint":false},{"pmid":"30755645","id":"PMC_30755645","title":"Differential PI(4,5)P2 sensitivities of TRPC4, C5 homomeric and TRPC1/4, C1/5 heteromeric channels.","date":"2019","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/30755645","citation_count":22,"is_preprint":false},{"pmid":"1697266","id":"PMC_1697266","title":"Three GCN4 responsive elements act synergistically as upstream and as TATA-like elements in the yeast TRP4 promoter.","date":"1990","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/1697266","citation_count":22,"is_preprint":false},{"pmid":"24352411","id":"PMC_24352411","title":"Differential regulation of TRPC4 in the vasopressin magnocellular system by water deprivation and hepatic cirrhosis in the rat.","date":"2013","source":"American journal of physiology. Regulatory, integrative and comparative physiology","url":"https://pubmed.ncbi.nlm.nih.gov/24352411","citation_count":20,"is_preprint":false},{"pmid":"29472562","id":"PMC_29472562","title":"Gαi-mediated TRPC4 activation by polycystin-1 contributes to endothelial function via STAT1 activation.","date":"2018","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/29472562","citation_count":19,"is_preprint":false},{"pmid":"20574736","id":"PMC_20574736","title":"Association study of TRPC4 as a candidate gene for generalized epilepsy with photosensitivity.","date":"2010","source":"Neuromolecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/20574736","citation_count":18,"is_preprint":false},{"pmid":"12003493","id":"PMC_12003493","title":"Replacement of the yeast TRP4 3' untranslated region by a hammerhead ribozyme results in a stable and efficiently exported mRNA that lacks a poly(A) tail.","date":"2002","source":"RNA (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/12003493","citation_count":18,"is_preprint":false},{"pmid":"11356184","id":"PMC_11356184","title":"Functional interaction between TRP4 and CFTR in mouse aorta endothelial cells.","date":"2001","source":"BMC physiology","url":"https://pubmed.ncbi.nlm.nih.gov/11356184","citation_count":17,"is_preprint":false},{"pmid":"32033274","id":"PMC_32033274","title":"Involvement of TRPC4 and 5 Channels in Persistent Firing in Hippocampal CA1 Pyramidal Cells.","date":"2020","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/32033274","citation_count":17,"is_preprint":false},{"pmid":"2875777","id":"PMC_2875777","title":"Synthesis and biological activity of analogs of dynorphin-A(1-13) substituted in positions 2 and 4: design of [Ala2,Trp4]-Dyn-A(1-13) as a putative selective opioid antagonist.","date":"1986","source":"Canadian journal of physiology and pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/2875777","citation_count":17,"is_preprint":false},{"pmid":"24121765","id":"PMC_24121765","title":"Dual depolarization responses generated within the same lateral septal neurons by TRPC4-containing channels.","date":"2014","source":"Pflugers Archiv : European journal of physiology","url":"https://pubmed.ncbi.nlm.nih.gov/24121765","citation_count":17,"is_preprint":false},{"pmid":"27287524","id":"PMC_27287524","title":"Muscarinic Receptor Induced Contractions of the Detrusor are Mediated by Activation of TRPC4 Channels.","date":"2016","source":"The Journal of urology","url":"https://pubmed.ncbi.nlm.nih.gov/27287524","citation_count":17,"is_preprint":false},{"pmid":"18538797","id":"PMC_18538797","title":"Cyclic stretch decreases TRPC4 protein and capacitative calcium entry in rat vascular smooth muscle cells.","date":"2008","source":"Life sciences","url":"https://pubmed.ncbi.nlm.nih.gov/18538797","citation_count":16,"is_preprint":false},{"pmid":"1425591","id":"PMC_1425591","title":"Sequence-specific initiator elements focus initiation of transcription to distinct sites in the yeast TRP4 promoter.","date":"1992","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/1425591","citation_count":16,"is_preprint":false},{"pmid":"35560982","id":"PMC_35560982","title":"Inhibition of TRPC4 channel activity in colonic myocytes by tricyclic antidepressants disrupts colonic motility causing constipation.","date":"2022","source":"Journal of cellular and molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/35560982","citation_count":15,"is_preprint":false},{"pmid":"19070363","id":"PMC_19070363","title":"The self-association of two N-terminal interaction domains plays an important role in the tetramerization of TRPC4.","date":"2008","source":"Cell calcium","url":"https://pubmed.ncbi.nlm.nih.gov/19070363","citation_count":15,"is_preprint":false},{"pmid":"25349210","id":"PMC_25349210","title":"Identification of a membrane-targeting domain of the transient receptor potential canonical (TRPC)4 channel unrelated to its formation of a tetrameric structure.","date":"2014","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/25349210","citation_count":15,"is_preprint":false},{"pmid":"35157953","id":"PMC_35157953","title":"Magnolol and honokiol target TRPC4 to regulate extracellular calcium influx and relax intestinal smooth muscle.","date":"2022","source":"Journal of ethnopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/35157953","citation_count":14,"is_preprint":false},{"pmid":"12381092","id":"PMC_12381092","title":"Cloning and functional expression of a novel splice variant of rat TRPC4.","date":"2002","source":"Circulation journal : official journal of the Japanese Circulation Society","url":"https://pubmed.ncbi.nlm.nih.gov/12381092","citation_count":14,"is_preprint":false},{"pmid":"37913983","id":"PMC_37913983","title":"Molecular mechanisms of cholinergic neurotransmission in visceral smooth muscles with a focus on receptor-operated TRPC4 channel and impairment of gastrointestinal motility by general anaesthetics and anxiolytics.","date":"2023","source":"Neuropharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/37913983","citation_count":13,"is_preprint":false},{"pmid":"25788576","id":"PMC_25788576","title":"Close spatio-association of the transient receptor potential canonical 4 (TRPC4) channel with Gαi in TRPC4 activation process.","date":"2015","source":"American journal of physiology. Cell physiology","url":"https://pubmed.ncbi.nlm.nih.gov/25788576","citation_count":13,"is_preprint":false},{"pmid":"28066150","id":"PMC_28066150","title":"Calcium permeability of transient receptor potential canonical (TRPC) 4 channels measured by TRPC4-GCaMP6s.","date":"2016","source":"The Korean journal of physiology & pharmacology : official journal of the Korean Physiological Society and the Korean Society of Pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/28066150","citation_count":13,"is_preprint":false},{"pmid":"35544691","id":"PMC_35544691","title":"TRPC4 and GIRK channels underlie neuronal coding of firing patterns that reflect Gq/11-Gi/o coincidence signals of variable strengths.","date":"2022","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/35544691","citation_count":12,"is_preprint":false},{"pmid":"17217052","id":"PMC_17217052","title":"Ionic channels formed by TRPC4.","date":"2007","source":"Handbook of experimental pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/17217052","citation_count":12,"is_preprint":false},{"pmid":"26631167","id":"PMC_26631167","title":"Intracellular spermine blocks TRPC4 channel via electrostatic interaction with C-terminal negative amino acids.","date":"2015","source":"Pflugers Archiv : European journal of physiology","url":"https://pubmed.ncbi.nlm.nih.gov/26631167","citation_count":12,"is_preprint":false},{"pmid":"38636714","id":"PMC_38636714","title":"TRPC4 aggravates hypoxic pulmonary hypertension by promoting pulmonary endothelial cell apoptosis.","date":"2024","source":"Free radical biology & medicine","url":"https://pubmed.ncbi.nlm.nih.gov/38636714","citation_count":11,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":51719,"output_tokens":14312,"usd":0.184919,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":26786,"output_tokens":6489,"usd":0.148078,"stage2_stop_reason":"end_turn"},"total_usd":0.332997,"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\": 2001,\n      \"finding\": \"TRP4-deficient (TRPC4-/-) mice lack a store-operated Ca2+ current in vascular endothelial cells, establishing that TRP4 is an indispensable component of store-operated channels in native endothelial cells and that these channels are required for agonist-induced Ca2+ entry and vasorelaxation.\",\n      \"method\": \"Knockout mouse model, electrophysiology, Ca2+ imaging, vascular tension measurements\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO mouse with defined cellular and vascular phenotype, replicated across multiple functional readouts\",\n      \"pmids\": [\"11175743\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Murine TRPC4 forms a nonselective cation channel activated by Gq/11-coupled receptors and receptor tyrosine kinases independently of intracellular Ca2+ store depletion; single-channel conductance is 42 pS at -60 mV; store depletion alone fails to activate the channel.\",\n      \"method\": \"Heterologous expression in HEK293 cells, whole-cell and inside-out patch clamp, Mn2+ quench fluorimetry, GTPγS infusion\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — rigorous electrophysiology with multiple activation paradigms, replicated by independent labs\",\n      \"pmids\": [\"10837492\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Murine TRP4 and phospholipase Cβ1/β2 interact with the first PDZ domain of the scaffolding protein NHERF (EBP50), and TRP4/PLCβ1/NHERF complexes co-immunoprecipitate from HEK293-Trp4 cells and adult mouse brain, linking the channel to the actin cytoskeleton via the ERM-NHERF interaction.\",\n      \"method\": \"Co-immunoprecipitation from transfected cells and native brain tissue, GST pull-down\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP in heterologous and native systems, multiple binding partners confirmed\",\n      \"pmids\": [\"10980202\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"TRP4 protein is abundantly expressed in bovine adrenal cortex cells and contributes essentially to native CRAC-like (store-operated) currents; antisense reduction of TRP4 protein significantly reduces both endogenous CRAC-like currents and native TRP4 protein.\",\n      \"method\": \"Antisense knockdown, Northern blot, immunoblot, immunohistochemistry, electrophysiology\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — antisense KD with correlated electrophysiological and protein readouts in native cells\",\n      \"pmids\": [\"10816590\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"TRPC4-/- lung endothelial cells show drastically reduced agonist (thrombin/PAR-1)-induced Ca2+ influx, lack actin stress fiber formation and cell retraction, and isolated-perfused TRPC4-/- lungs show markedly attenuated increases in microvascular permeability, establishing TRPC4-dependent Ca2+ entry as a key determinant of increased microvascular permeability.\",\n      \"method\": \"TRPC4 knockout mouse model, Ca2+ imaging, actin staining, isolated-perfused lung microvascular filtration coefficient measurement\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO mouse with multiple orthogonal cellular and organ-level phenotypic readouts\",\n      \"pmids\": [\"12114324\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"TRP4 directly binds calmodulin (CaM) in a Ca2+-dependent manner through two C-terminal domains (residues 688-759 and 786-848); half-maximal CaM binding occurs at 16.6 µM (domain 1) and 27.9 µM (domain 2) Ca2+; synthetic peptides from these regions bind dansyl-CaM with Kd 94-189 nM.\",\n      \"method\": \"CaM-Sepharose affinity chromatography, GST pull-down, synthetic peptide binding assay with dansyl-CaM fluorimetry\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro biochemical binding assays with domain mapping and Kd determination, single lab\",\n      \"pmids\": [\"11311128\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"TRPC3 and TRPC4 associate to form a redox-sensitive heteromeric cation channel complex in porcine aortic endothelial cells; co-IP demonstrates physical association; FRET shows close proximity between TRPC4 N-terminus and TRPC3 C-terminus; dominant-negative TRPC4 suppresses TRPC3-related currents and the native redox-sensitive conductance.\",\n      \"method\": \"Co-immunoprecipitation, FRET, whole-cell electrophysiology, dominant-negative expression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — four orthogonal lines of evidence including native tissue Co-IP, FRET, and dominant-negative functional suppression\",\n      \"pmids\": [\"16537542\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"In intestinal smooth muscle cells, TRPC4 forms a 55 pS cation channel underlying >80% of muscarinic receptor-induced cation current (mICAT); TRPC4-deficient myocytes show greatly reduced carbachol-induced membrane depolarization; TRPC4/C6 double KO slows intestinal transit in vivo, establishing TRPC4 and TRPC6 as the molecular basis of mICAT coupling muscarinic receptors to smooth muscle contraction.\",\n      \"method\": \"Single and double knockout mice, whole-cell patch clamp, muscle contraction assays, intestinal transit measurement\",\n      \"journal\": \"Gastroenterology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO mice, single-channel electrophysiology, multiple functional readouts in vitro and in vivo\",\n      \"pmids\": [\"19549525\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"TRPC4α (but not TRPC4β) is strongly inhibited by intracellularly applied PIP2 in an isoform-specific manner; PIP2 binds to the C-terminus of TRPC4α but not TRPC4β in vitro; inhibition requires association with actin cytoskeleton via the C-terminal PDZ-binding motif (Thr-Thr-Arg-Leu) that links TRPC4 to F-actin through NHERF and ezrin; PIP2 breakdown is a required step in TRPC4α activation requiring additional Ca2+ and Gi/o proteins.\",\n      \"method\": \"Whole-cell patch clamp with intracellular PIP2 application, in vitro lipid-binding assay, cytochalasin D treatment, PDZ-motif deletion mutants\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstituted lipid-protein binding assay plus electrophysiology with mutagenesis, single lab with multiple orthogonal approaches\",\n      \"pmids\": [\"18230622\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Human TRPC4α contains a C-terminal autoinhibitory domain: TRPC4β is robustly activated by receptor stimulation across species, whereas TRPC4α shows poor activation; C-terminal truncation of TRPC4α fully restores channel activity; TRPC4α exerts a dominant-negative effect on TRPC4β with cooperativity >2 in heteromultimers; FRET confirms homomultimer and heteromultimer assembly of TRPC4α and TRPC4β.\",\n      \"method\": \"Heterologous expression, whole-cell patch clamp, FRET in living cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — electrophysiology with deletion mutants, FRET for assembly, multiple isoforms and ratios tested\",\n      \"pmids\": [\"11713258\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Protein 4.1 interacts with TRPC4 and the spectrin-actin membrane skeleton; deletion of the protein 4.1 binding domain on TRPC4 or peptide competition to this domain prevents activation of the endothelial ISOC (store-operated) channel, establishing protein 4.1 interaction with TRPC4 as an essential component of the ISOC gating mechanism.\",\n      \"method\": \"Co-immunoprecipitation, domain deletion, peptide competition, whole-cell electrophysiology\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain deletion and peptide competition combined with electrophysiology, multiple orthogonal approaches\",\n      \"pmids\": [\"16254212\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"The PDZ-interacting TRL motif at the TRPC4 C-terminus controls its plasma membrane localization; deletion of TRL causes accumulation in cell outgrowths and reduces plasma membrane expression ~2.4-fold; co-expression with an EBP50 mutant lacking the ERM-binding site retains TRPC4 in a perinuclear (Golgi) compartment.\",\n      \"method\": \"Immunofluorescence microscopy, cell surface biotinylation, co-expression with EBP50 truncation mutants in HEK293 cells\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization experiments with surface biotinylation quantification and deletion mutants, clear functional consequence on membrane targeting\",\n      \"pmids\": [\"12154080\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Gαi subunits, particularly Gαi2, are primary and direct activators of TRPC4, acting through direct interaction with the conserved C-terminal SESTD domain; Gαi2 activation by muscarinic M2 receptors or constitutively active Gαi2 mutants fully activates the channel; two amino acids (K715 and R716) in the TRPC4 C-terminus mediate the interaction with Gαi2.\",\n      \"method\": \"Co-immunoprecipitation, whole-cell patch clamp, constitutively active Gα mutant expression, site-directed mutagenesis, Ca2+ imaging\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with mutagenesis mapping of interaction residues plus functional electrophysiology, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"22457348\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TRPC4 activation requires coincident stimulation of Gi/o proteins and PLCδ1 (preferentially over PLCβ); PIP2 is required for biphasic TRPC4 activation; reducing PIP2 via phosphatases abolishes biphasic kinetics; dominant-negative PLCδ1 or constitutively active RhoA almost completely eliminates TRPC4 activation; this mechanism differs from the closely related TRPC5.\",\n      \"method\": \"Whole-cell patch clamp, siRNA knockdown of PLCδ1, dominant-negative constructs, constitutively active RhoA, PIP2 manipulation in HEK293 and A-498 renal carcinoma cells\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple genetic and pharmacological perturbations with electrophysiological readouts in heterologous and native cells\",\n      \"pmids\": [\"26755577\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Dynamic interaction of NHERF1/2 with the C-terminal PDZ-binding motif of TRPC4/5 suppresses DAG sensitivity; PIP2 depletion evokes a C-terminal conformational change leading to NHERF dissociation, which is a prerequisite for DAG-mediated channel activation; PKC inhibition or PDZ-motif mutation confers DAG sensitivity, establishing NHERF as a direct negative regulator of TRPC4/5 DAG sensitivity.\",\n      \"method\": \"Whole-cell patch clamp, FRET-based conformational analysis, PKC inhibition, site-directed mutagenesis of PDZ-binding motif\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — FRET conformational readout combined with electrophysiology and mutagenesis, multiple orthogonal approaches in single study\",\n      \"pmids\": [\"27994151\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Cryo-EM structure of mouse TRPC4 at 3.3 Å resolution reveals a unique architecture with a long pore loop stabilized by a disulfide bond, a unique cytosolic N-terminal domain forming extensive aromatic contacts with the TRP and C-terminal domains, and a tetrameric six-transmembrane fold; structural features provide molecular basis for TRPC4 ion selectivity.\",\n      \"method\": \"Cryo-electron microscopy, 3.3 Å resolution structure determination\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution cryo-EM structure with functional domain interpretation\",\n      \"pmids\": [\"30082700\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Cryo-EM structure of zebrafish TRPC4 at 3.6 Å resolution in unliganded closed state reveals molecular architecture of the cation-conducting pore including selectivity filter and lower gate; cytoplasmic domain contains two key hubs for modulating protein interactions.\",\n      \"method\": \"Cryo-electron microscopy, 3.6 Å resolution structure determination\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution cryo-EM structure identifying pore elements and cytoplasmic regulatory hubs\",\n      \"pmids\": [\"29717981\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Cryo-EM structures of TRPC4 in complex with calmodulin and three pyridazinone inhibitors reveal that all inhibitors bind to the same cavity in the voltage-sensing-like domain; structural changes propagate from the ligand-binding site to the central ion-conducting pore; CaM binds to the rib helix of TRPC4, ordering a previously disordered region and fixing the channel in a closed conformation — a novel CaM-induced regulatory mechanism.\",\n      \"method\": \"Cryo-EM structural determination of TRPC4-CaM and TRPC4-inhibitor complexes\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple high-resolution cryo-EM structures with distinct ligands identifying binding site and conformational changes\",\n      \"pmids\": [\"33236980\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TRPC1, TRPC4, and TRPC5 assemble exclusively into heteromultimers with each other (not other TRP family members) in mouse brain, as shown by quantitative mass spectrometry; TRPC1/4/5 triple-KO hippocampal neurons show significantly reduced action potential-triggered EPSCs and impaired hippocampal network cross-frequency coupling and spatial working memory, establishing heteromultimeric TRPC1/4/5 channels as regulators of hippocampal synaptic transmission.\",\n      \"method\": \"Quantitative high-resolution mass spectrometry for interactome, triple-knockout mouse model, hippocampal slice electrophysiology, in vivo LFP recording, behavioral testing\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — MS-based complex identification combined with KO electrophysiology and behavior, multiple orthogonal methods\",\n      \"pmids\": [\"28790178\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TRPC1/TRPC4 double-KO mice show reduced background Ca2+ entry (BGCE) in cardiomyocytes, lower diastolic and systolic Ca2+ concentrations, and are protected against neurohumoral-induced and pressure overload-induced cardiac hypertrophy and fibrosis; TRPC1 or TRPC4 single-KO mice do not show protection, establishing a cooperative TRPC1/C4 constitutively active BGCE pathway as a driver of pathological cardiac remodeling.\",\n      \"method\": \"Multiple knockout mouse models, fluorescence Ca2+ imaging of electrically paced cardiomyocytes, Mn2+ quench microfluorimetry, cardiac hypertrophy models (neurohumoral, pressure overload)\",\n      \"journal\": \"European heart journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — systematic analysis of multiple KO models with mechanistic Ca2+ entry measurements and multiple hypertrophy endpoints\",\n      \"pmids\": [\"26069213\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"The alpha1 and beta2 splice variants of human TRPC4 differ in regulation: hTRPC4β forms receptor-operated cation channels when expressed in HEK293 cells, while hTRPC4α is poorly activated by H1 receptor stimulation despite correct plasma membrane targeting; the C-terminal region of hTRPC4α acts as an autoinhibitory domain, as C-terminal truncation fully restores channel activity.\",\n      \"method\": \"Heterologous expression, whole-cell patch clamp, FRET, GFP-fusion protein trafficking\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — electrophysiology combined with FRET and truncation analysis demonstrating autoinhibitory domain\",\n      \"pmids\": [\"11713258\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"TRP4 is localized to caveolae of interstitial cells of Cajal (ICC); Ca2+ oscillations in ICC depend on Ca2+ influx through a non-selective, store-operated, SK&F 96365-sensitive cation channel; this links TRP4 localization in caveolae to pacemaker Ca2+ oscillations in ICC.\",\n      \"method\": \"Immunofluorescence/caveolae fractionation, Ca2+ imaging with fluo-4, pharmacological inhibition, c-Kit immunoreactivity for ICC identification\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization to caveolae plus correlated functional Ca2+ oscillation data, single lab\",\n      \"pmids\": [\"11897792\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Alpha-hTRP4 (but not beta-hTRP4) C-terminus associates in vitro with the C-terminal domain of InsP3 receptors types 1, 2, and 3, as demonstrated by yeast two-hybrid and GST pull-down; this interaction is regulated by alternative splicing (the 84 aa insert in alpha absent in beta contains the binding region).\",\n      \"method\": \"Yeast two-hybrid assay, GST pull-down experiments\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two orthogonal in vitro binding methods, single lab\",\n      \"pmids\": [\"11163362\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"TRPC4 co-localizes with and co-immunoprecipitates with ZO-1 in human fetal astrocytes via the C-terminal TRL PDZ-binding motif; deletion of TRL retains TRPC4 in a juxtanuclear compartment and reduces plasma membrane expression, demonstrating that TRPC4's PDZ-binding motif controls its surface localization in astrocytes.\",\n      \"method\": \"Co-immunoprecipitation, confocal microscopy, immunoelectron microscopy, cell surface biotinylation, GST fusion protein binding assay, TRL-motif deletion mutants\",\n      \"journal\": \"Glia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple methods confirming ZO-1 interaction and PDZ-motif-dependent membrane targeting, single lab\",\n      \"pmids\": [\"15540229\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"TRPC1 and TRPC4 channels are expressed at the sarcolemma of skeletal myotubes and associate with the alpha1-syntrophin–dystrophin complex (DAPC); siRNA silencing of alpha1-syntrophin dysregulates cation influx; deletion of the PDZ-containing domain of alpha1-syntrophin prevents restoration of normal cation entry; TRPC1 and TRPC4 co-immunoprecipitate with alpha1-syntrophin, establishing DAPC-anchored TRPC1/C4 as regulators of cation homeostasis in skeletal muscle.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, Ca2+ entry measurements, dominant-negative constructs\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and siRNA with functional Ca2+ readout, single lab\",\n      \"pmids\": [\"19812031\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"STIM1 interacts with TRPC4 (but not TRPC1) as demonstrated by co-immunoprecipitation in human mesangial cells; TRPC1/TRPC4 complexes constitute functional SOC subunits; STIM1 knockdown significantly reduces thapsigargin-stimulated membrane currents; simultaneous inhibition of STIM1 and TRPC1 produces no additive effect on SOC over single inhibition, suggesting they act in the same pathway.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, patch clamp, Ca2+ imaging\",\n      \"journal\": \"Experimental biology and medicine (Maywood, N.J.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus functional knockdown, single lab\",\n      \"pmids\": [\"19307462\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"SESTD1, a protein containing a SEC14-like lipid-binding domain and spectrin-type cytoskeleton interaction domains, associates with TRPC4 and TRPC5 via the channel's calmodulin- and IP3-receptor-binding (CIRB) domain; SESTD1 binds several phospholipid species in a Ca2+-dependent manner in vitro and is essential for efficient receptor-mediated activation of TRPC5.\",\n      \"method\": \"Yeast two-hybrid screen, co-immunoprecipitation, in vitro phospholipid binding, functional expression assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid + Co-IP + in vitro lipid binding + functional assay, single lab\",\n      \"pmids\": [\"20164195\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"A conserved glycine residue in the cytosolic S4-S5 linker (G503 in TRPC4) is a critical gating element; TRPC4G503S mutant is constitutively active with current-voltage relationships resembling fully activated WT; a second mutation S623A in the predicted S6 helix suppresses constitutive activation, indicating that the S4-S5 linker interacts with S6 during gating.\",\n      \"method\": \"Site-directed mutagenesis, whole-cell patch clamp, homology modeling\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis of gating residue with gain-of-function electrophysiology and rescue by second-site mutation, single lab\",\n      \"pmids\": [\"23677990\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Gαi protein (specifically Gαi subtype) directly activates TRPC4 channel; among Gα proteins tested, only Gαi activates TRPC4; co-expression with M2 muscarinic receptor induces TRPC4 activation by carbachol that is blocked by pertussis toxin; this selectivity distinguishes TRPC4 from TRPC5, TRPC6, and TRPV6.\",\n      \"method\": \"Whole-cell patch clamp, pertussis toxin inhibition, co-expression with M2 receptor and constitutively active Gα constructs in HEK293 cells\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — electrophysiology with pertussis toxin validation, single lab, single method\",\n      \"pmids\": [\"18854172\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Gαi2 directly binds to TRPC4 when the channel is open, as demonstrated by FRET between TRPC4β-CFP and constitutively active Gαi2-YFP (~15% efficiency vs. ~5% for WT Gαi2); carbachol application via M2 receptor increases FRET efficiency between TRPC4 and Gαi2; Gβγ shows low FRET with TRPC4.\",\n      \"method\": \"FRET (CFP/YFP-tagged constructs), whole-cell patch clamp, Ca2+ imaging with YC6.1\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — FRET and electrophysiology combined, demonstrating direct interaction in open state, single lab\",\n      \"pmids\": [\"25788576\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The first ankyrin-like repeat of TRPC4/TRPC5 is the minimum structural domain required for homo- and heteromeric channel assembly; N-terminal fragments including the first ankyrin-like repeat potently suppress TRPC4/5 currents in a dominant-negative fashion; a TRPC5 mutant lacking the first ankyrin-like repeat fails to homo-multimerise and forms non-functional channels.\",\n      \"method\": \"FRET, TIRF microscopy, dominant-negative electrophysiology in HEK293 cells, deletion mutant analysis\",\n      \"journal\": \"Cell calcium\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — FRET and dominant-negative electrophysiology with systematic domain deletions, single lab\",\n      \"pmids\": [\"17624425\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The N-terminus of TRPC4 self-associates and forms a tetramer; two distinct self-association domains exist in the N-terminus: the ankyrin repeat domain and the region downstream from the coiled-coil domain, both of which can self-associate independently.\",\n      \"method\": \"Size-exclusion chromatography, GST pull-down, yeast two-hybrid, circular dichroism\",\n      \"journal\": \"Cell calcium\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple in vitro biochemical approaches for domain mapping of tetramerization, single lab\",\n      \"pmids\": [\"19070363\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Deletion of TRPC4 (constitutive or lentiviral RNAi in lateral amygdala) decreases anxiety-like behavior; lateral amygdala neurons from TRPC4-/- mice lack potentiation responses through Gαq/11-coupled Group I metabotropic glutamate receptors and CCK2 receptors, establishing TRPC4 as a required component of these Gαq/11-mediated depolarizing responses in the amygdala.\",\n      \"method\": \"TRPC4-/- mice, lentiviral RNAi, brain slice electrophysiology, behavioral testing (elevated plus maze, open field)\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse plus lentiviral RNAi with concordant slice electrophysiology and behavioral phenotypes\",\n      \"pmids\": [\"24599464\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"In lateral septal (LS) neurons, TRPC4-containing channels mediate both below-threshold depolarization (BTD) and above-threshold plateau depolarization (ATPD) in response to group I mGluR agonist; both responses are absent in TRPC4-/- mice; ATPD requires coincident mGluR stimulation and depolarization, and depends on Na+ and Ca2+ influx with dynamic intracellular Ca2+ changes.\",\n      \"method\": \"TRPC4-/- mice, whole-cell slice recordings, pharmacological manipulation of intracellular Ca2+ and ion gradients\",\n      \"journal\": \"Pflugers Archiv : European journal of physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse electrophysiology with mechanistic ion substitution experiments, single lab\",\n      \"pmids\": [\"24121765\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TRPC4 acts as a coincidence sensor for Gq/11 and Gi/o signals in lateral septal neurons: group I mGluR (Gq/11) plus GABAB (Gi/o) co-activation is required for strong TRPC4-mediated plateau depolarization; GIRK channels mediate subsequent hyperpolarization; the combination of TRPC4 and GIRK conductances encodes the relative strengths of Gq/11 vs. Gi/o inputs as distinct firing patterns.\",\n      \"method\": \"Whole-cell slice recordings in lateral septal neurons, receptor agonist/antagonist pharmacology, TRPC4-/- mice, computer modeling\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse electrophysiology with defined receptor pharmacology and computational validation, single lab\",\n      \"pmids\": [\"35544691\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Kisspeptin activation of TRPC4α channels in GnRH neurons requires PIP2 hydrolysis (application of DiC8-PIP2 inhibits current; wortmannin prolongs activation), and cSrc tyrosine kinase activity (inhibition by genistein or PP2 blocks activation); channel activation is not store-operated (thapsigargin and IP3 have no effect) and PKC-independent.\",\n      \"method\": \"Whole-cell patch clamp in GnRH neurons, single-cell RT-PCR (TRPC4α identification), pharmacological dissection with kinase inhibitors, PIP2 dialysis, PKC activators/inhibitors\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — electrophysiology with multiple pharmacological tools, native neurons, single lab\",\n      \"pmids\": [\"23744639\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Cell-cell contact formation regulates TRPC4 surface expression and Ca2+ signaling in endothelial cells; TRPC4 co-precipitates with β-catenin and VE-cadherin; β-catenin promotes TRPC4 function in a cell-cell contact-dependent manner; EGF recruits TRPC4 to the plasma membrane in proliferating cells but causes retrieval in quiescent barrier-forming cells.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, dominant-negative expression, Ca2+ imaging, fluorescent fusion protein localization in HMEC-1 and HEK293 cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with functional knockdown and overexpression experiments, single lab\",\n      \"pmids\": [\"19996314\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"TRPC4 knockdown by siRNA or antisense in DRG neurons significantly reduces neurite length; this is rescued by overexpression of human TRPC4, establishing a required role for TRPC4 in neurite outgrowth/axonal regeneration.\",\n      \"method\": \"siRNA and antisense knockdown, neurite length measurement, rescue by hTRPC4 overexpression in DRG neurons and ND7/23 cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA/antisense KD with rescue experiment, single lab\",\n      \"pmids\": [\"17928298\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"ATP-induced CREB phosphorylation precedes and is required for TRPC4 protein upregulation in human pulmonary artery smooth muscle cells (PASMCs); transfection of a non-phosphorylatable CREB mutant abolishes ATP-mediated TRPC4 expression; TRPC4 siRNA attenuates ATP-enhanced capacitative Ca2+ entry and inhibits ATP-induced PASMC proliferation, placing CREB upstream of TRPC4 in a mitogenic signaling pathway.\",\n      \"method\": \"CREB phosphorylation assay, non-phosphorylatable CREB mutant transfection, siRNA knockdown, CCE measurement, [3H]thymidine incorporation\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistatic ordering of CREB→TRPC4 with dominant-negative mutant and siRNA functional readout, single lab\",\n      \"pmids\": [\"15229105\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"(-)-Englerin A is a potent, selective, direct activator of TRPC4 and TRPC5 channels; TRPC4 expression is necessary and sufficient for englerin A-induced Ca2+ influx, membrane depolarization, and growth inhibition; englerin A-induced current and growth inhibition are blocked by the TRPC4/C5 inhibitor ML204.\",\n      \"method\": \"Electrophysiology, Ca2+ imaging, genetic knockdown and overexpression, ML204 pharmacological rescue\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic necessity and sufficiency experiments combined with pharmacological rescue and electrophysiology, multiple cell lines\",\n      \"pmids\": [\"26098886\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"(-)-Englerin A cytotoxicity in synovial sarcoma cells is mediated by heteromeric TRPC4/TRPC1 channels; depletion of TRPC1 converts the current to homomeric TRPC4 biophysical properties; Na+ loading through the channel mediates cytotoxicity; depletion of either TRPC1 or TRPC4 suppresses EA cytotoxicity.\",\n      \"method\": \"Whole-cell patch clamp, siRNA depletion of TRPC1 and TRPC4, Na+-loading with gramicidin-A, selective pharmacological inhibitors (Pico145 vs. ML204)\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biophysical and pharmacological dissection of heteromeric vs. homomeric channels with genetic validation, single lab\",\n      \"pmids\": [\"29209034\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Intracellular spermine blocks TRPC4 (and TRPC5) but not TRPC1/4, TRPC1/5, or TRPC3 channels; the blocking mechanism is electrostatic interaction with glutamate residues E728 and E729 at the C-terminus of TRPC4.\",\n      \"method\": \"Whole-cell patch clamp with intracellular spermine, site-directed mutagenesis of E728/E729\",\n      \"journal\": \"Pflugers Archiv : European journal of physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis identifies critical residues for block mechanism, electrophysiology, single lab\",\n      \"pmids\": [\"26631167\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"TRPC4 is necessary for activation of CFTR Cl- current in mouse aortic endothelial cells; a phosphorylating cocktail activates CFTR-like Cl- current in trp4+/+ but not in trp4-/- cells, despite unchanged CFTR expression, suggesting TRP4 provides a scaffold for functional CFTR channel formation.\",\n      \"method\": \"TRPC4 knockout mouse endothelial cells, whole-cell patch clamp, RT-PCR for CFTR expression\",\n      \"journal\": \"BMC physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO endothelial cells with electrophysiology, mechanistic interpretation supported by unchanged CFTR expression\",\n      \"pmids\": [\"11356184\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"TRPC4 knockdown reduces EGF-induced store-operated channel (SOC) activation and Ca2+ entry in human corneal epithelial cells; TRPC4 siRNA (89% mRNA knockdown) eliminates EGF-induced SOC activity and reduces EGF-induced proliferation by 54%, establishing TRPC4 as a component of SOC required for EGF mitogenic responses.\",\n      \"method\": \"siRNA knockdown, whole-cell patch clamp, Ca2+ fluorescence imaging, [3H]thymidine incorporation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA with multiple functional readouts, single lab\",\n      \"pmids\": [\"16033767\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"NO/cGMP/PKG-1α signaling inhibits TRPC4-SOC activity in mesangial cells; PKG-phosphorylated VASP (P-Ser239) co-immunoprecipitates with TRPC4, while unphosphorylated VASP does not, identifying phospho-VASP as a state-dependent interaction partner mediating PKG-dependent inhibition of TRPC4.\",\n      \"method\": \"Co-immunoprecipitation, immunocytochemistry, Ca2+ imaging (fura-2), PKG-1α inhibitor DT-3, 8-Br-cGMP treatment, Western blot for P-VASP\",\n      \"journal\": \"American journal of physiology. Renal physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP demonstrating phosphorylation-dependent interaction with functional Ca2+ readout, single lab\",\n      \"pmids\": [\"17913834\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PC1 (polycystin-1) activates TRPC4 through Gαi3; Gαi3 selectively binds to the G-protein-binding domain of PC1 C-terminus; PC1 cleavage dissociates Gαi3, increasing TRPC4 activity; Ca2+ influx through TRPC4 activates STAT1 to regulate cell proliferation/death; PC1/TRPC4/STAT1 downregulation disrupts endothelial cell migration and increases permeability.\",\n      \"method\": \"Co-immunoprecipitation, TRPC4 activity measurements, STAT1 activation assay, cell migration and permeability assays, siRNA knockdown\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with functional downstream signaling validation, single lab\",\n      \"pmids\": [\"29472562\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"A TRPC4 membrane-targeting domain (residues 23-29 in the N-terminus) distinct from the tetramerization domain (requiring residues downstream of aa99 in N-term and upstream of aa730 in C-term) is identified; deletion of the 23-29 region causes ER retention; FRET mapping distinguishes assembly domains from trafficking domains.\",\n      \"method\": \"Deletion mutant construction, FRET, co-expression with WT TRPC4, confocal microscopy in HEK293 cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — FRET-based domain mapping with deletion mutants, single lab\",\n      \"pmids\": [\"25349210\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PI(4,5)P2 dephosphorylation (by voltage-sensing phosphatase DrVSP) inhibits TRPC4α, TRPC4β, TRPC5 homotetramers and TRPC1/4α, TRPC1/4β, TRPC1/5 hetetrotramers; sensitivity to PI(4,5)P2 depletion increases TRPC4β < TRPC4α < TRPC5 in homotetramers; TRPC1 incorporation equalizes PI(4,5)P2 sensitivity; putative PI(4,5)P2 binding sites are identified at K419, K664/R511, K518, and H630 by mutagenesis.\",\n      \"method\": \"Danio rerio VSP-based PI(4,5)P2 depletion, patch clamp, FRET with PI(4,5)P2 sensor, mutagenesis of basic residues\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis mapping of PIP2 binding combined with functional depletion approach and FRET, single lab\",\n      \"pmids\": [\"30755645\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"TRPC4 channels in GI smooth muscle are Ca2+-inhibited nonselective cation channels; TRPC4β single-channel conductance is 17.5 pS; calmidazolium increases inward current; currents are sensitive to lanthanum, niflumic acid, and DIDS; TRPC4β properties match the ICC pacemaker current.\",\n      \"method\": \"Heterologous expression, whole-cell and single-channel patch clamp, BAPTA dialysis, N-methyl-D-glucamine replacement experiments\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — detailed biophysical characterization with ion substitution and pharmacology, single lab\",\n      \"pmids\": [\"12388058\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"TRPC4 co-interacts with NHERF-2 in rat descending vasa recta (DVR); TRPC4 co-immunoprecipitates with NHERF-2 from renal medullary lysates and proteins co-localize in DVR endothelial cells and pericytes; TRPC5 is not detected in DVR.\",\n      \"method\": \"RT-PCR, immunohistochemistry, co-immunoprecipitation from native renal medullary tissue\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP from native tissue with corroborating colocalization, single lab\",\n      \"pmids\": [\"15590898\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Tricyclic antidepressants (TCAs) directly inhibit TRPC4 channels in heterologous expression (HEK293) and in native murine colonic myocytes; TCA inhibition of muscarinic cationic current (mIcat) is reduced in TRPC4-knockout mice; TCA treatment inhibits colonic motility in human tissue strips, connecting TRPC4 inhibition to TCA-induced constipation.\",\n      \"method\": \"Patch clamp in HEK293 cells and native colonic myocytes, TRPC4-KO mice, muscle contraction recordings in human colonic strips\",\n      \"journal\": \"Journal of cellular and molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — heterologous and native electrophysiology with KO validation and human tissue functional readout, single lab\",\n      \"pmids\": [\"35560982\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TRPC4 and TRPC5 channels support persistent firing in CA1 pyramidal neurons; extracellular application of TRPC4 blocker ML204, TRPC5 blocker clemizole, or pan-TRPC4/5 blocker Pico145 significantly inhibits cholinergically-induced persistent firing; intracellular application of TRPC4 or TRPC5 antibodies also reduces persistent firing.\",\n      \"method\": \"Whole-cell patch clamp in CA1 neurons, selective pharmacological blockers applied extracellularly and intracellularly\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple pharmacological tools with two application routes confirming TRPC4 involvement in persistent firing, single lab\",\n      \"pmids\": [\"32033274\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"TRPC1 and TRPC4 are required for normal myotube size during human post-natal myogenesis; siRNA knockdown or dominant-negative TRPC overexpression reduces SOCE, impairs MEF2 expression, and reduces myotube size; overexpression of STIM1 with TRPC4 or TRPC1 increases SOCE and produces hypertrophic myotubes; normalization of SOCE by extracellular Ca2+, STIM1, or Orai1 overexpression does not rescue the fusion defect without TRPC channel re-expression, indicating TRPC-specific signaling requirements.\",\n      \"method\": \"siRNA knockdown, dominant-negative overexpression, SOCE measurement, MEF2 expression assay, myotube size quantification\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA/dominant-negative with rescue experiments and specific downstream marker (MEF2), single lab\",\n      \"pmids\": [\"23549783\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TRPC1 and TRPC4 interact preferentially with STIM1L (muscle-specific long isoform) over STIM1 upon store depletion; STIM1L and TRPC1/4 knockdown produce similar reductions in SOCE (~50%) and similar delays in Ca2+ entry onset; STIM1L knockdown produces smaller myotubes similar to TRPC1/4 knockdown.\",\n      \"method\": \"Co-immunoprecipitation (interaction preference), siRNA knockdown, SOCE measurement, myotube differentiation assay\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP showing isoform-selective interaction with functional knockdown phenotype, single lab\",\n      \"pmids\": [\"28185894\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"TRPC1 and TRPC4 are required for cystitis-induced sensory neuron sprouting into the bladder mucosa; cyclophosphamide-treated Trpc1/c4-/- mice show no increased bladder innervation and diminished bladder overactivity, establishing TRPC1/C4 as necessary for injury-induced neuronal sprouting.\",\n      \"method\": \"Double knockout mice, cyclophosphamide cystitis model, immunohistochemistry for nerve fiber density, urodynamics\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — double KO mouse with quantified anatomical and functional endpoints, single lab\",\n      \"pmids\": [\"23922735\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TRPC4 upregulation increases intracellular Ca2+ concentration, which activates the Ca2+/CaMKKβ/AMPK pathway leading to mTOR inhibition and autophagy induction in vascular endothelial cells; TRPC4 siRNA abrogates TMS-induced autophagy.\",\n      \"method\": \"DNA microarray, siRNA knockdown of TRPC4, TRPC4 overexpression, Ca2+ imaging, CaMKKβ/AMPK/mTOR pathway analysis\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA and overexpression with downstream pathway measurements, single lab\",\n      \"pmids\": [\"25476892\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"A gain-of-function SNP TRPC4-I957V is associated with reduced myocardial infarction risk; functional studies show TRPC4-I957V has increased channel activity and Ca2+ signals in response to muscarinic agonists and direct G-protein activation; molecular modeling suggests I957V allows firmer interaction between TRPC4 and a tyrosine kinase that phosphorylates Y959, facilitating plasma membrane insertion.\",\n      \"method\": \"Patch clamp and intracellular Ca2+ measurements in transfected HEK293/CHO cells, site-directed mutagenesis, molecular modeling\",\n      \"journal\": \"Cardiovascular research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — electrophysiology and Ca2+ measurements with mutagenesis and structural modeling, single lab\",\n      \"pmids\": [\"21427121\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TRPC4 is a Ca2+-permeable nonselective cation channel that forms homo- or heteromeric (with TRPC1, TRPC3, TRPC5) tetramers with distinct biophysical properties; it is activated downstream of Gi/o proteins (primarily Gαi2 via direct C-terminal SESTD interaction) coincident with PLCδ1 and PIP2 depletion, rather than by store depletion or DAG alone; PIP2 tonically inhibits TRPC4α (but not TRPC4β) through its C-terminal PDZ-binding motif-actin cytoskeleton linkage via NHERF/ERM; dynamic NHERF1/2 dissociation upon PIP2 hydrolysis gates DAG sensitivity; calmodulin binds two C-terminal domains and, as revealed by cryo-EM structures, fixes the channel in a closed conformation via the rib helix; conserved S4-S5 linker glycine G503 and S6 residue S623 form a gating couple; the channel's plasma membrane targeting requires an N-terminal trafficking domain (aa 23-29) and PDZ-motif-dependent interaction with NHERF/EBP50 and ZO-1; in native tissues TRPC4 underlies store-operated Ca2+ entry in vascular endothelium (required for vasorelaxation and lung microvascular permeability), the muscarinic cation current (mICAT) in intestinal and detrusor smooth muscle, pacemaker activity in interstitial cells of Cajal, Gq/11-Gi/o coincidence-detector plateau depolarizations in lateral septal and amygdala neurons, and heteromultimeric TRPC1/4/5 channels supporting hippocampal synaptic transmission and working memory.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TRPC4 is a Ca2+-permeable nonselective cation channel that converts G-protein-coupled receptor signaling into membrane depolarization and Ca2+ entry across vascular, smooth muscle, and neuronal tissues [#0, #1, #7]. The channel is activated by Gq/11-coupled receptors and receptor tyrosine kinases independently of store depletion [#1], but its principal direct trigger is the Gi/o family — particularly Gαi2, which binds the C-terminal SESTD/CIRB region (via residues K715/R716) when the channel is open [#12, #28, #29]; full activation requires coincident PLCδ1 engagement and hydrolysis of PIP2 rather than store depletion or DAG alone [#13]. PIP2 exerts isoform-specific tonic inhibition of TRPC4α (but not TRPC4β) through the C-terminal PDZ-binding TRL motif that tethers the channel to the actin cytoskeleton via NHERF/EBP50 and ERM proteins; dynamic dissociation of NHERF upon PIP2 hydrolysis is a prerequisite for DAG-mediated gating, making the scaffold itself a negative regulator [#8, #14, #47]. Calmodulin binds two C-terminal domains in a Ca2+-dependent manner and, as resolved by cryo-EM, locks the channel closed by ordering the rib helix, while structural studies define the tetrameric six-transmembrane fold, the disulfide-stabilized pore loop, and a conserved S4-S5 linker glycine (G503) that couples to S6 residue S623 during gating [#5, #15, #17, #27]. TRPC4 assembles as homotetramers or heteromultimers exclusively with TRPC1, TRPC3, and TRPC5, with the first ankyrin-like repeat and N-terminal self-association domains driving assembly and a distinct N-terminal domain (aa 23-29) plus the PDZ motif directing plasma-membrane targeting [#6, #9, #18, #30, #31, #46]. In native tissues TRPC4 underlies store-operated Ca2+ entry in vascular endothelium required for vasorelaxation and microvascular permeability [#0, #4], the muscarinic cation current (mICAT) coupling muscarinic receptors to intestinal smooth muscle contraction [#7], and Gq/11–Gi/o coincidence-detector plateau depolarizations in lateral septal and amygdala neurons as well as heteromeric TRPC1/4/5 channels supporting hippocampal synaptic transmission and working memory [#18, #32, #34]. A gain-of-function TRPC4-I957V variant is associated with reduced myocardial infarction risk [#56].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Establishing how TRPC4 is gated answered whether it is a store-operated channel or a receptor-operated one — TRPC4 was shown to be activated by Gq/11 receptors and RTKs independent of store depletion, defining it as a receptor-operated cation channel.\",\n      \"evidence\": \"Heterologous expression in HEK293 with patch clamp, Mn2+ quench, and GTPγS infusion\",\n      \"pmids\": [\"10837492\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify which G-protein subunit directly couples to the channel\", \"Native physiological agonists not defined\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Knockout and antisense studies resolved whether TRPC4 contributes to native store-operated currents, establishing it as an indispensable component of endothelial SOC required for agonist-induced Ca2+ entry and vasorelaxation.\",\n      \"evidence\": \"TRPC4-/- mice and bovine adrenal antisense knockdown with electrophysiology, Ca2+ imaging, and vascular tension assays\",\n      \"pmids\": [\"11175743\", \"10816590\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Apparent conflict with the receptor-operated gating model not reconciled\", \"Molecular composition of the native SOC complex undefined\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Splice-variant analysis explained why TRPC4 activity varies — the TRPC4α C-terminus acts as an autoinhibitory domain whose truncation restores activity, and FRET confirmed homo- and heteromultimer assembly of α and β.\",\n      \"evidence\": \"Heterologous expression, whole-cell patch clamp, FRET, GFP-fusion trafficking in HEK293 cells\",\n      \"pmids\": [\"11713258\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of the autoinhibition (lipid vs. scaffold) not yet identified\", \"Physiological relevance of α/β ratio in native tissue unknown\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Identifying scaffold partners addressed how TRPC4 is organized and localized — NHERF/EBP50 binds the channel and PLCβ, linking it to the ERM-actin cytoskeleton and forming a signaling complex in native brain.\",\n      \"evidence\": \"Reciprocal Co-IP from transfected cells and mouse brain plus GST pull-down\",\n      \"pmids\": [\"10980202\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of the complex for gating not established at this stage\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Mapping calmodulin binding addressed Ca2+-dependent feedback — TRPC4 binds CaM at two C-terminal domains in a Ca2+-dependent manner with defined affinities.\",\n      \"evidence\": \"CaM-Sepharose affinity, GST pull-down, dansyl-CaM peptide fluorimetry\",\n      \"pmids\": [\"11311128\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional effect of CaM binding on channel gating not shown until structural work\", \"Stoichiometry in the assembled channel unknown\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Knockout studies extended TRPC4's endothelial role to barrier function, showing TRPC4-dependent Ca2+ entry drives actin stress-fiber formation, cell retraction, and increased lung microvascular permeability, and to pacemaker ICC where TRPC4 localizes to caveolae.\",\n      \"evidence\": \"TRPC4-/- lung endothelial cells, isolated-perfused lung filtration measurement, caveolae fractionation and Ca2+ imaging in ICC\",\n      \"pmids\": [\"12114324\", \"11897792\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct demonstration that caveolar localization gates ICC pacemaking lacking\", \"Link between Ca2+ entry and cytoskeletal contraction mechanistically incomplete\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Surface-targeting studies defined how TRPC4 reaches the plasma membrane — the C-terminal TRL PDZ motif and its EBP50/ERM interaction control membrane localization, with motif deletion causing perinuclear/Golgi retention.\",\n      \"evidence\": \"Immunofluorescence, surface biotinylation, EBP50 truncation mutants in HEK293; biophysical channel characterization in GI smooth muscle\",\n      \"pmids\": [\"12154080\", \"12388058\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trafficking machinery downstream of the motif not identified\", \"Relationship of TRL-dependent targeting to PIP2 inhibition not yet linked\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Cytoskeletal anchoring partners addressed how the channel's gating machinery is assembled at the membrane — protein 4.1 and ZO-1 bind TRPC4, and protein 4.1 interaction is essential for endothelial store-operated channel gating.\",\n      \"evidence\": \"Co-IP, domain deletion, peptide competition, electrophysiology in endothelial and astrocyte systems\",\n      \"pmids\": [\"16254212\", \"15540229\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How spectrin-actin coupling mechanically gates the pore is unresolved\", \"ZO-1 and protein 4.1 contributions to native gating in other tissues untested\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Heteromultimer studies established TRPC4's partner specificity beyond homotetramers, showing TRPC3-TRPC4 form a redox-sensitive heteromeric channel in native endothelium.\",\n      \"evidence\": \"Co-IP, FRET, dominant-negative electrophysiology in porcine aortic endothelial cells\",\n      \"pmids\": [\"16537542\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological stimulus engaging the redox-sensitive heteromer in vivo undefined\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Assembly-domain mapping answered which structural element drives tetramerization — the first ankyrin-like repeat is the minimal domain for homo/heteromeric assembly and acts dominant-negatively when expressed alone.\",\n      \"evidence\": \"FRET, TIRF, dominant-negative electrophysiology, deletion mutants in HEK293\",\n      \"pmids\": [\"17624425\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; in vivo relevance of N-terminal assembly fragments not tested\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identifying the direct G-protein activator answered the long-standing gating question — only Gαi among tested Gα proteins directly activates TRPC4, with M2-receptor coupling blocked by pertussis toxin, distinguishing it from TRPC5/6/V6.\",\n      \"evidence\": \"Whole-cell patch clamp, pertussis toxin, constitutively active Gα and M2 co-expression in HEK293\",\n      \"pmids\": [\"18854172\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single method, single lab\", \"Direct binding not yet demonstrated, only functional selectivity\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Lipid-regulation studies revealed isoform-specific PIP2 control — PIP2 inhibits TRPC4α (not β) through the PDZ-motif/actin linkage, and PIP2 breakdown is a required activation step alongside Ca2+ and Gi/o.\",\n      \"evidence\": \"Patch clamp with intracellular PIP2, in vitro lipid binding, cytochalasin D, PDZ-deletion mutants\",\n      \"pmids\": [\"18230622\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct PIP2 binding residues not yet mapped\", \"How cytoskeletal tethering transduces lipid signal to the gate unclear\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Mapping the Gαi interface answered how Gi/o coupling occurs structurally — Gαi2 directly binds the C-terminal SESTD domain via K715/R716 and fully activates the channel.\",\n      \"evidence\": \"Co-IP, site-directed mutagenesis, constitutively active Gαi2, patch clamp, Ca2+ imaging\",\n      \"pmids\": [\"22457348\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of Gαi2:channel interaction undefined\", \"Coupling to receptor identity in different native tissues incompletely mapped\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Gating-residue and gain-of-function studies defined the pore-coupling mechanism — S4-S5 linker glycine G503 couples to S6 S623, and a TRPC4-I957V variant associated with reduced MI risk shows enhanced activity.\",\n      \"evidence\": \"Site-directed mutagenesis with gain-of-function electrophysiology and homology modeling; HEK293/CHO functional studies of the SNP\",\n      \"pmids\": [\"23677990\", \"21427121\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"G503-S623 coupling inferred from mutagenesis prior to structure\", \"I957V genetic association is correlative for disease risk\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Coincidence-detection studies integrated the activation logic — TRPC4 requires coincident Gi/o and PLCδ1 signaling with PIP2, and dynamic NHERF1/2 dissociation upon PIP2 hydrolysis is the prerequisite for DAG sensitivity, making the scaffold a direct negative regulator.\",\n      \"evidence\": \"Patch clamp, PLCδ1 siRNA, dominant-negative and constitutively active constructs, FRET conformational analysis, PDZ-motif mutagenesis\",\n      \"pmids\": [\"26755577\", \"27994151\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Exact conformational path from C-terminus to pore not resolved at this stage\", \"Generalizability of the PLCδ1 requirement across native tissues untested\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Native interactome and triple-KO behavior established TRPC4's neuronal partner identity and physiology — TRPC1/4/5 assemble exclusively with one another in brain and support hippocampal synaptic transmission and spatial working memory.\",\n      \"evidence\": \"Quantitative mass spectrometry interactome, triple-KO hippocampal slice electrophysiology, in vivo LFP, behavior\",\n      \"pmids\": [\"28790178\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Subunit stoichiometry of native TRPC1/4/5 channels undefined\", \"Receptor coupling driving synaptic TRPC currents not fully mapped\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Cryo-EM structures answered the architecture question — TRPC4 is a tetrameric six-transmembrane channel with a disulfide-stabilized pore loop, defined selectivity filter and lower gate, and cytoplasmic hubs for protein interactions.\",\n      \"evidence\": \"Cryo-EM of mouse (3.3 Å) and zebrafish (3.6 Å) TRPC4\",\n      \"pmids\": [\"30082700\", \"29717981\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Closed/apo states only; activated-state structure not captured\", \"Direct visualization of lipid and G-protein binding not yet achieved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Ligand-bound cryo-EM revealed the CaM-mediated closing mechanism and a drug-binding pocket — CaM binds the rib helix to fix the channel closed, and pyridazinone inhibitors bind a voltage-sensing-like-domain cavity that propagates to the pore.\",\n      \"evidence\": \"Cryo-EM of TRPC4-CaM and TRPC4-inhibitor complexes\",\n      \"pmids\": [\"33236980\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How physiological Ca2+/CaM dynamics tune gating in vivo not established\", \"Structure of the activating PIP2/Gαi-bound state still absent\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Neuronal recordings refined TRPC4 as a bidirectional coincidence sensor — in lateral septal neurons it integrates Gq/11 (mGluR) and Gi/o (GABAB) inputs into plateau depolarizations, with GIRK encoding the relative input strengths as firing patterns.\",\n      \"evidence\": \"Whole-cell slice recordings, receptor pharmacology, TRPC4-/- mice, computational modeling\",\n      \"pmids\": [\"35544691\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular determinants distinguishing coincidence detection across neuron types unclear\", \"In vivo behavioral correlate of the encoding scheme untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the activating conformation is achieved at the structural level — the simultaneous engagement of Gαi2, PIP2 depletion, PLCδ1, NHERF dissociation, and Ca2+/CaM converging on the G503-S623 gate — remains unresolved, as all available high-resolution structures are closed or inhibitor-bound.\",\n      \"evidence\": \"No activated-state structure or integrated reconstitution in the available corpus\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No open-state structure of TRPC4\", \"No reconstitution combining G-protein, lipid, and scaffold inputs\", \"Subunit stoichiometry of native heteromers undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [0, 1, 7, 48]},\n      {\"term_id\": \"GO:0005262\", \"supporting_discovery_ids\": [1, 48]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [12, 28, 29, 34]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [8, 47]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [11, 12, 21, 23, 46]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [2, 8, 10]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [46]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 12, 13, 34]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [18, 32, 33, 34, 51]},\n      {\"term_id\": \"R-HSA-397014\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [0, 3, 4]}\n    ],\n    \"complexes\": [\n      \"TRPC1/4/5 heteromeric channel\",\n      \"TRPC3/4 redox-sensitive heteromeric channel\",\n      \"TRPC1/4 heteromeric channel\",\n      \"TRPC4-NHERF/EBP50 scaffold complex\"\n    ],\n    \"partners\": [\n      \"TRPC1\",\n      \"TRPC5\",\n      \"TRPC3\",\n      \"NHERF1\",\n      \"GNAI2\",\n      \"CALM1\",\n      \"STIM1\",\n      \"SESTD1\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}