{"gene":"ORAI3","run_date":"2026-06-10T05:19:53","timeline":{"discoveries":[{"year":2008,"finding":"2-APB activates Orai3 independently of STIM1 and store depletion, producing a large relatively nonselective cation current with outward rectification; chimera analysis mapped the structural determinant for 2-APB-induced current to the sequence spanning the second to third transmembrane segment of Orai3.","method":"Patch-clamp electrophysiology, Orai1-3 chimera expression in HEK cells, pharmacological application of 2-APB","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro electrophysiology with chimeric channel mutagenesis in a single rigorous study, independently replicated in concurrent work (PMID:18499656)","pmids":["18420579"],"is_preprint":false},{"year":2008,"finding":"2-APB alters Orai3 ion selectivity by increasing its minimum pore size from ~3.8 Å to >5.34 Å, permitting monovalent cation permeation in both directions; a pore mutant (R66W) lacking store-operated activation was also resistant to 2-APB stimulation; Orai3 E165Q mutation resembled 2-APB-stimulated Orai3 in permeation properties and showed reduced 2-APB response.","method":"Patch-clamp electrophysiology, site-directed mutagenesis, ion substitution permeability assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro electrophysiology combined with mutagenesis and pore-size measurements in one rigorous study","pmids":["18499656"],"is_preprint":false},{"year":2007,"finding":"Both Orai1 and Orai3 are essential components of the arachidonate-regulated Ca2+-selective (ARC) channels; overexpression of Orai3 alone has no effect on ARC currents, but specifically increases them when Orai1 is co-expressed; a dominant-negative Orai3 mutant specifically reduces ARC channel currents without affecting CRAC currents; siRNA knockdown of either Orai1 or Orai3 markedly inhibits ARC channel currents.","method":"Patch-clamp electrophysiology, dominant-negative expression, siRNA knockdown, overexpression in cells stably expressing STIM1","journal":"The Journal of physiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal gain/loss-of-function with pharmacological specificity, replicated across multiple experimental conditions","pmids":["17991693"],"is_preprint":false},{"year":2009,"finding":"The functional ARC channel pore is a heteropentameric assembly of three Orai1 subunits and two Orai3 subunits; expression of concatenated pentameric constructs with this 3:2 stoichiometry produces large currents with all key biophysical and pharmacological features of endogenous ARC channels including arachidonic acid activation, store-independence, and requirement for plasma membrane-resident STIM1.","method":"Concatenated Orai1/Orai3 pentameric construct expression, dominant-negative co-expression, patch-clamp electrophysiology, arachidonic acid stimulation","journal":"The Journal of physiology","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct reconstitution of defined stoichiometry with functional validation using multiple orthogonal criteria","pmids":["19622606"],"is_preprint":false},{"year":2009,"finding":"Heteromeric Orai1/Orai3 channels display diminished Ca2+ selectivity, robust Cs+ permeation, and reduced fast inactivation compared to homomeric forms; differences in the first extracellular loop (two aspartates in Orai3 replacing glutamates in Orai1) contribute to altered selectivity; Orai3 mutant mimicking Orai1 first loop co-expressed with Orai1 recovered Ca2+ selectivity.","method":"Patch-clamp electrophysiology, site-directed mutagenesis of extracellular loop residues, co-expression of Orai1 and Orai3","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis combined with electrophysiology establishing selectivity determinants, multiple constructs tested","pmids":["19887627"],"is_preprint":false},{"year":2010,"finding":"The cytosolic N-terminal domain of Orai3 is specifically responsible for determining selectivity for activation of the ARC channel by arachidonic acid; substitution of the Orai3 N-terminal domain into an otherwise complete Orai1 subunit within a concatenated 3:1:1:1:1 pentamer converts the resulting channel from predominantly store-operated to exclusively arachidonic acid-activated; two Orai3 subunits are required for full selectivity.","method":"Concatenated pentameric construct expression, N-terminal domain substitution mutagenesis, Ca2+ imaging and patch-clamp electrophysiology","journal":"Channels (Austin, Tex.)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — domain swap mutagenesis within defined pentameric constructs with functional readout","pmids":["20818184"],"is_preprint":false},{"year":2010,"finding":"Native SOCE and ICRAC in estrogen receptor-positive (ER+) breast cancer cell lines are mediated by STIM1/2 and Orai3, not Orai1; ER-negative breast cancer cells use the canonical STIM1/Orai1 pathway; established by molecular knockdown, Ca2+ imaging, pharmacology, and patch-clamp in ten breast cell lines.","method":"siRNA knockdown, Ca2+ imaging, patch-clamp electrophysiology, pharmacological inhibition across ten breast cell lines","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (electrophysiology, imaging, knockdown, pharmacology) in a large panel of cell lines","pmids":["20395295"],"is_preprint":false},{"year":2011,"finding":"The conserved N-terminal region of Orai3 contains distinct molecular determinants for STIM1-dependent activation versus 2-APB-stimulated activation; progressive N-terminal truncations progressively decrease fast inactivation and diminish calmodulin binding; STIM1-dependent activation requires the second half of the conserved N-terminal domain, whereas 2-APB activation is partially retained with further truncations.","method":"Stepwise N-terminal deletion mutagenesis, patch-clamp electrophysiology, calmodulin binding assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — systematic mutagenesis combined with functional electrophysiology and biochemical binding assay","pmids":["21724845"],"is_preprint":false},{"year":2012,"finding":"ERα transcriptionally regulates Orai3 expression in ER+ breast cancer cells; ERα knockdown decreases Orai3 mRNA (~63%) and protein (~44%) without affecting Orai1; ectopic Orai3 expression rescues SOCE after ERα knockdown; Orai3 knockdown inhibits ERK1/2 and FAK phosphorylation, NFAT transcriptional activity, anchorage-independent growth, Matrigel invasion, and tumor growth in immunodeficient mice.","method":"siRNA knockdown of ERα, ectopic Orai3 re-expression, Ca2+ imaging, patch-clamp, Western blotting, NFAT reporter assay, anchorage-independent growth, Matrigel invasion, xenograft mouse model","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods including in vivo xenograft validation, rescue experiments, and signaling pathway readouts","pmids":["22993197"],"is_preprint":false},{"year":2012,"finding":"Orai3 silencing in MCF-7 breast cancer cells reduces CDK4/2 and cyclin D1/E expression, accumulates p21Waf1/Cip1 and p53, arrests cell cycle at G1, and decreases Ca2+ entry and cell survival; these effects are not seen in non-cancerous MCF-10A cells upon Orai3 silencing.","method":"siRNA knockdown, Ca2+ imaging, flow cytometry, Western blotting for cell cycle proteins","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockdown with multiple downstream readouts in two cell types, single lab","pmids":["20683915"],"is_preprint":false},{"year":2012,"finding":"Orai3 silencing in MCF-7 cells reduces c-myc expression and activity and decreases pERK1/2 levels, linking Orai3-mediated Ca2+ entry to the MAP kinase pathway and c-myc-driven cell cycle progression.","method":"siRNA knockdown, Western blotting for pERK1/2 and c-myc, flow cytometry","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockdown with pathway-level biochemical readouts, single lab","pmids":["23266555"],"is_preprint":false},{"year":2013,"finding":"Orai3 constitutes the native SOCE pathway in non-small cell lung cancer (NSCLC) cells; Orai3 knockdown reduces SOCE, inhibits cell proliferation, arrests cells in G0/G1, downregulates cyclin D1, cyclin E, CDK4, CDK2, and decreases Akt phosphorylation.","method":"siRNA knockdown, pharmacological inhibition, Ca2+ imaging, flow cytometry, Western blotting","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockdown confirmed by multiple functional readouts, single lab","pmids":["24058448"],"is_preprint":false},{"year":2013,"finding":"TM3 residue G158 in Orai3 forms a disulfide bridge with endogenous TM2 cysteine C101 when mutated to cysteine (G158C); this bridge slows 2-APB activation kinetics and prevents complete channel closure; reducing agent BMS reverses the slow phenotype in a state-dependent manner (only during 2-APB activation).","method":"Site-directed mutagenesis, patch-clamp electrophysiology, reducing agent application (BMS), double mutant C101G/G158C analysis","journal":"The Journal of general physiology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis with mechanistic chemical validation (redox-sensitive disulfide), functional and double-mutant controls","pmids":["24081982"],"is_preprint":false},{"year":2014,"finding":"In 2-APB-activated Orai3, TM1 residues Q83, V77, and L70 line the dilated pore (Cd2+ block potentiated at these positions); TM3 residue E165 approaches the pore axis in the 2-APB-activated state but not the store-operated state; TM1 mutants E81C, G73A/C, and R66C abolish 2-APB sensitivity; V77C is blocked by MTSEA in a state-dependent manner only when channels are open.","method":"Cysteine scanning mutagenesis, Cd2+ block, thiol-reactive reagent (MTSEA/MTSET) application, patch-clamp electrophysiology","journal":"The Journal of general physiology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — systematic cysteine scanning with state-dependent chemical modification, multiple confirming mutations","pmids":["24733836"],"is_preprint":false},{"year":2014,"finding":"LTC4 (generated by LTC4 synthase from arachidonic acid) activates store-independent LRC channels encoded by Orai1/Orai3 heteromultimers in vascular smooth muscle cells; LTC4S knockdown inhibits LRC currents; Orai3 and LTC4S knockdown both inhibit Akt1/Akt2 Ser473/474 phosphorylation and VSMC migration, contributing to neointimal hyperplasia.","method":"LTC4S siRNA/shRNA knockdown, patch-clamp electrophysiology, in vivo balloon angioplasty neointima model with lentiviral shRNA, Akt phosphorylation assay, migration assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo and in vitro complementary approaches with electrophysiological validation of LRC current and signaling readouts","pmids":["25540197"],"is_preprint":false},{"year":2014,"finding":"In hypertrophied adult rat ventricular cardiomyocytes, Orai3 (but not Orai1) is the critical partner of STIM1 carrying voltage-independent Ca2+ entries; Orai3 co-immunoprecipitates with STIM1, with greater STIM1 and Orai1 enriched in the Orai3 complex during hypertrophy; Orai3 also drives an arachidonic acid-activated inward current in cardiomyocytes.","method":"Co-immunoprecipitation, in vivo siRNA delivery, patch-clamp electrophysiology, Ca2+ imaging in isolated cardiomyocytes","journal":"Cardiovascular research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — co-IP plus in vivo siRNA knockdown validated by electrophysiology, multiple orthogonal approaches","pmids":["25213556"],"is_preprint":false},{"year":2015,"finding":"VEGF triggers rapid (within 2 min) accumulation of Orai3 at the plasma membrane surface in human endothelial cells via a signaling pathway involving PLCγ1, Ca2+ release, cytosolic group IV phospholipase A2α, arachidonic acid, and microsomal glutathione S-transferase 2 (forming LTC4); Orai3 knockdown suppresses this VEGF-dependent Ca2+ entry and endothelial tube formation in vitro and in vivo.","method":"Immunofluorescence, cell surface biotinylation, siRNA knockdown, Ca2+ imaging, Matrigel tube formation, in vivo angiogenesis model, pharmacological pathway dissection","journal":"Arteriosclerosis, thrombosis, and vascular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — surface biotinylation directly demonstrating trafficking, plus in vivo functional validation in multiple endothelial cell types","pmids":["26160956"],"is_preprint":false},{"year":2017,"finding":"Orai3 channel colocalizes with expressed IP3R at the ER membrane and mediates 2-APB-induced Ca2+ leak from the ER; Orai3 knockdown inhibits 2-APB-induced ER Ca2+ leak without affecting thapsigargin-revealed SERCA-dependent ER Ca2+ leak; reducing Orai3 results in larger cytoplasmic Ca2+ responses after thapsigargin only when ER stores are overloaded.","method":"Dominant-negative Orai1 E106A expression, siRNA knockdown of Orai1/2/3, dual Ca2+ indicators (cytoplasmic and ER-luminal), co-localization with IP3R","journal":"Cell calcium","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA and dominant-negative approaches with ER-targeted indicators, single lab","pmids":["28179072"],"is_preprint":false},{"year":2018,"finding":"Orai3 overexpression in breast cancer cells drives p53 degradation via the PI3K/Sgk-1/Sek-1 pathway, utilizing both Mdm2 and the E3 ubiquitin ligase Nedd4-2 for p53 proteolysis; this mechanism is dependent on extracellular calcium and Orai3 functionality.","method":"Orai3 overexpression, calcium removal, PI3K/Sgk-1 inhibition, Mdm2 and Nedd4-2 identification by high-throughput approach, Western blotting","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional pharmacological pathway dissection with identification of two E3 ligases, single lab","pmids":["29323264"],"is_preprint":false},{"year":2018,"finding":"TRPC6 physically interacts with Orai3 in MCF7 breast cancer cells and is required for translocation of Orai3 to the plasma membrane upon Ca2+ store depletion; TRPC6 knockdown impairs Orai3-dependent SOCE in MCF7 cells.","method":"Co-immunoprecipitation, RNAi knockdown, dominant-negative TRPC6 expression, Ca2+ imaging, surface expression assay","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP demonstrating interaction plus functional knockdown, single lab","pmids":["30223530"],"is_preprint":false},{"year":2018,"finding":"ORAI3 and STIM1 are required for TGF-β-dependent Snai1 transcription; blocking SOCE paradoxically increases Snai1 activation via AKT pathway and NF-κB (p65) binding at the Snai1 promoter.","method":"siRNA knockdown of ORAI3 and STIM1, 2-APB treatment, AKT pathway analysis, ChIP/promoter-binding assays, migration assays","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockdown with promoter-binding and pathway validation, single lab","pmids":["30034631"],"is_preprint":false},{"year":2019,"finding":"HIF-1α induces ORAI3 expression in basal breast cancer cell lines; ORAI3 silencing attenuates hypoxia-associated EGFR phosphorylation and expression of genes associated with cell migration and inflammatory/immune responses.","method":"HIF-1α knockdown, HIF-1α pathway induction by hypoxia, ORAI3 siRNA, Western blotting, gene expression analysis","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockdown of upstream regulator with functional downstream readouts, single lab","pmids":["30754719"],"is_preprint":false},{"year":2019,"finding":"Inflammatory cardiac CD11b/c cells trigger a TNFR2- and Orai3-dependent store-independent Ca2+ influx in hypertrophied cardiomyocytes; this Ca2+ influx promotes cardiomyocyte hypertrophy and resistance to oxidative stress via GSK3β phosphorylation; in vivo Orai3 knockdown during early adaptive cardiac hypertrophy impairs GSK3β phosphorylation and accelerates heart failure.","method":"Conditioned medium from cardiac CD11b/c cells, TNFα stimulation, pharmacological/siRNA Orai3 inhibition, intramyocardial siRNA injection, Ca2+ imaging, GSK3β phosphorylation assay","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo and in vitro complementary siRNA knockdown with GSK3β signaling readout, single lab","pmids":["30988334"],"is_preprint":false},{"year":2020,"finding":"The number and position of Orai3 subunits within heteromeric store-operated channels alter the pharmacology of ICRAC; channels with two or more Orai3 subunits can be activated by 2-APB independently of store depletion/STIM1 and display large outward currents; a single Orai3 subunit does not alter 2-APB pharmacology compared to homomeric Orai1.","method":"Patch-clamp electrophysiology with concatenated Orai1/Orai3 constructs of defined stoichiometry and position","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — defined concatenated constructs with electrophysiology, single lab","pmids":["32252254"],"is_preprint":false},{"year":2021,"finding":"Orai3 regulates MDA-MB-231 breast cancer cell migration through two mechanisms: (1) a Ca2+-dependent modulation of calpain activity affecting cell adhesion; (2) a Ca2+-independent interaction with focal adhesion kinase (FAK) that regulates F-actin polymerization and cell morphology.","method":"siRNA knockdown of Orai3, calpain activity assay, cell adhesion and migration assays, F-actin staining, co-immunoprecipitation of Orai3 and FAK","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP plus functional knockdown with two distinct mechanistic readouts, single lab","pmids":["34943998"],"is_preprint":false},{"year":2021,"finding":"Cardiac-specific deletion of Orai3 leads to dilated cardiomyopathy characterized by abnormal sarcomere (M- and Z-line) morphology, increased DRP1-associated mitochondrial fragmentation, upregulation of TRPC6 and RCAN1 (indicating calcineurin pathway activation), and impaired myocardial Ca2+ cycling; removal of Orai1 from Orai3-deficient cardiomyocytes does not change the cardiac phenotype.","method":"Constitutive and inducible cardiomyocyte-specific Orai3 knockout mice, echocardiography, ultrastructural analysis, Western blotting, Ca2+ cycling measurements","journal":"Journal of the American Heart Association","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout mouse model with mechanistic molecular analysis (DRP1, TRPC6, RCAN1, Ca2+ cycling) and ultrastructural validation","pmids":["33849280"],"is_preprint":false},{"year":2021,"finding":"ORAI3 silencing in pancreatic ductal adenocarcinoma cells increases SOCE (in contrast to decreasing it in normal pancreatic cells), impairs proliferation, causes mitotic catastrophe and cell death, and inhibits tumor growth in vivo.","method":"siRNA knockdown, Ca2+ imaging (SOCE measurement), flow cytometry, xenograft in vivo model","journal":"Biochimica et biophysica acta. Molecular cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockdown with in vivo validation and cell-type-specific functional divergence, single lab","pmids":["33798603"],"is_preprint":false},{"year":2021,"finding":"Orai3 mediates Orai channel remodeling in pulmonary fibrosis: TGF-β1 increases SEPTIN4, which promotes Orai3-Orai1 interaction, suppressing STIM1-Orai1 interaction and SOCE activity, resulting in a high and stable extracellular Ca2+ influx that activates NFAT1 and Calpain/ERK signaling to drive fibroblast activation.","method":"Co-immunoprecipitation of Orai3-Orai1 and STIM1-Orai1, Orai3 knockdown/overexpression, BLM-induced lung fibrosis model, SEPTIN4 manipulation, Ca2+ imaging, NFAT1 and pERK Western blotting","journal":"Journal of cellular and molecular medicine","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP combined with in vivo fibrosis model and SEPTIN4 mechanistic link, single lab","pmids":["36128650"],"is_preprint":false},{"year":2021,"finding":"Orai3-mediated SOCE is insensitive to changes in intracellular pH (pHi), in contrast to Orai1 (strongly pHi-dependent) and Orai2 (moderately pHi-dependent); domain swapping between Orai1 and Orai3 identified the N-terminus and intracellular loop 2 of Orai1 as responsible for pHi sensitivity.","method":"Whole-cell patch-clamp in HEK293T cells expressing Orai isoforms with STIM1, intracellular pH manipulation, Orai1/Orai3 chimeric constructs","journal":"The Journal of physiology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — electrophysiology with chimeric mutagenesis, single lab","pmids":["34877682"],"is_preprint":false},{"year":2021,"finding":"Extracellular cysteines C226 and C232, unique to Orai3 (absent in Orai1 and Orai2), mediate selective inhibition of Orai3-mediated SOCE by hydrogen sulfide (H2S); mutation of either cysteine to serine abolishes H2S inhibition; H2S does not affect Orai3 membrane mobility, STIM1/Orai3 puncta formation, or STIM1-Orai3 protein-protein interactions, indicating that the inhibitory mechanism is downstream of STIM1 engagement.","method":"Site-directed mutagenesis (C226S, C232S), H2S donor (GYY4137), Ca2+ imaging, FRAP, colocalization, FRET assays in HEK293 cells","journal":"American journal of physiology. Cell physiology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis combined with multiple orthogonal imaging/FRET methods to define mechanism, single lab","pmids":["34788146"],"is_preprint":false},{"year":2022,"finding":"Deletion of ORAI3 in T cells and B cells does not impair SOCE in those cell types; combined deletion of Orai1 and Orai3, but not Orai3 alone, impairs SOCE, proliferation, survival, NFAT activation, and metabolic reprogramming in B cells; ORAI3 deletion moderately enhances SOCE in macrophages.","method":"Orai3-/- and double Orai1/3 knockout mice, SOCE measurement, flow cytometry, NFAT reporter, Seahorse metabolic assay, in vivo immunization and infection models","journal":"The Journal of general physiology / eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout mice with multiple complementary in vivo and in vitro readouts across immune cell types, two independent studies (PMID:35861698, PMID:36803766)","pmids":["35861698","36803766"],"is_preprint":false},{"year":2023,"finding":"ORAI3 physically interacts with STIM2 under basal conditions in prostate cancer cells; ORAI3 silencing increases SOCE and arrests cells in G2/M with elevated CDK1-Y15/Cyclin B1, leading to mitotic catastrophe via Bax/Bcl-2-mediated apoptosis and caspase-8 activation; STIM2 and ORAI3 expression increase during M phase while STIM1 decreases, suggesting an ORAI3-STIM2 complex permits mitotic progression by negatively regulating SOCE.","method":"Co-immunoprecipitation of ORAI3-STIM2, siRNA knockdown, Ca2+ imaging, flow cytometry, CDK1/Cyclin B1 Western blotting, caspase activity assay","journal":"Cell calcium","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP plus multiple functional readouts, single lab","pmids":["37597301"],"is_preprint":false},{"year":2023,"finding":"Reconstituted Orai3 protein in proteoliposomes forms a functional channel that is directly opened by recombinant STIM1 in vitro, mediating Ca2+ release from liposomes.","method":"Protein purification, proteoliposome reconstitution, Ca2+ release assay, STIM1-Orai3 interaction in vitro","journal":"Biochemistry. Biokhimiia","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro reconstitution demonstrating direct STIM1-Orai3 gating, single study without mutagenesis validation","pmids":["37770396"],"is_preprint":false},{"year":2023,"finding":"p38 MAPK activation by lysophosphatidylcholine or arachidonic acid promotes STIM1-Orai3 association in endothelial cells, increasing intracellular Ca2+ and triggering TRPC6 externalization; Orai3 downregulation blocks the lysoPC-induced Ca2+ rise and TRPC6 externalization, preserving endothelial cell migration.","method":"siRNA Orai3 knockdown, pharmacological p38 MAPK inhibition, Ca2+ imaging, TRPC6 surface expression assay, migration assays","journal":"American journal of physiology. Cell physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA knockdown with mechanistic pathway placement (p38-STIM1-Orai3-TRPC6), single lab","pmids":["37093037"],"is_preprint":false},{"year":2023,"finding":"Orai3 promotes cancer stemness in oral squamous cell carcinoma (OSCC) through upregulation of the stemness transcription factor ID1; ectopic Orai3 increases intracellular Ca2+ and acquires malignant/CSC properties; ID1 suppression abrogates the CSC phenotype induced by Orai3 overexpression.","method":"Orai3 ectopic overexpression, siRNA knockdown, Ca2+ imaging, CSC marker expression, ID1 knockdown rescue experiments","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — overexpression plus knockdown with rescue approach establishing ID1 as downstream effector, single lab","pmids":["37759448"],"is_preprint":false},{"year":2024,"finding":"Orai3 and STIM1 mediate store-operated Ca2+ entry in ER+ breast cancer stem-like cells (BCSCs), promoting their growth and tumorigenicity via a glycolysis-independent pathway; this is mechanistically distinct from Orai1, which interacts with SPCA2 for store-independent Ca2+ entry promoting glycolysis.","method":"siRNA knockdown, Ca2+ imaging, 3D soft fibrin gel BCSC enrichment, xenograft in vivo, glycolysis (Seahorse) assay","journal":"Stem cell research & therapy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — combined in vitro and in vivo knockout with metabolic profiling distinguishing Orai3 from Orai1 mechanism, single lab","pmids":["39135143"],"is_preprint":false},{"year":2025,"finding":"NFATc1 transcription factor both drives Orai3 transcription (in non-metastatic pancreatic cancer cells) and induces its lysosomal degradation (in invasive/metastatic cells) via transcriptional upregulation of MARCH8 E3-ubiquitin ligase; MARCH8 physically interacts with Orai3 intracellular loop and ubiquitinates Orai3 at the N-terminal; the dichotomy in NFATc1 function arises from differential (hyper-)methylation of the MARCH8 promoter in non-metastatic versus metastatic cells.","method":"NFATc1 ChIP and reporter assays, MARCH8 co-immunoprecipitation with Orai3, ubiquitination assay, epigenetic methylation analysis, super-resolution microscopy, siRNA/overexpression, in vivo metastasis experiments","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — multiple orthogonal methods (co-IP, ubiquitination, ChIP, methylation, super-resolution imaging, in vivo) in one study establishing a full mechanistic circuit","pmids":["41023307"],"is_preprint":false},{"year":2025,"finding":"Notch1/HEY1 signaling differentially regulates Orai3 expression in breast cancer subtypes; active Notch1 and its agonist Jagged-1 alter Orai3 expression and corresponding SOCE; the non-phosphorylatable HEY1-S68A mutant mimics Notch1 effects on Orai expression, while the phosphomimetic HEY1-S68D mutant does not.","method":"Notch1 active form/Jagged-1 stimulation, DAPT (γ-secretase inhibitor), HEY1 mutant expression, Ca2+ imaging, Western blotting across breast cancer cell lines","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain- and loss-of-function with downstream functional validation, single lab","pmids":["41413272"],"is_preprint":false},{"year":2025,"finding":"Orai1 sustains Orai3 protein synthesis and stability in luminal breast cancer cells; Orai1 knockout decreases Orai3 protein synthesis and enhances Orai3 endo-lysosomal degradation; Orai1 also regulates ERα expression and NFAT2 nuclear translocation, which modulates ERα and Orai3 levels, establishing an Orai1-NFAT2-ERα-Orai3 regulatory axis.","method":"Orai1 knockout MCF-7 cells, RNAi silencing, ectopic Orai1 re-expression, Ca2+ imaging, Western blotting, protein synthesis/degradation assays, NFAT2 nuclear translocation assay","journal":"Cell calcium","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with rescue and multiple downstream pathway analyses, single lab","pmids":["41172596"],"is_preprint":false}],"current_model":"ORAI3 is a mammalian-specific plasma membrane Ca2+-selective channel that functions in multiple modes: (1) as a store-operated CRAC channel activated by STIM1/2 (mediating native SOCE in ER+ breast cancer cells, NSCLC, astrocytes, DRG neurons, and cardiomyocytes), (2) as a heteropentameric ARC channel (3 Orai1 + 2 Orai3 subunits) activated by arachidonic acid or LTC4 in a store-independent manner dependent on plasma membrane-resident STIM1, and (3) as a 2-APB-gated nonselective cation channel (activated independently of STIM1) whose dilated pore is lined by TM1 with TM3-E165 approaching the pore axis; N-terminal determinants govern both STIM1-dependent gating and ARC channel selectivity for arachidonic acid, while extracellular cysteines C226/C232 mediate selective H2S inhibition; ORAI3 expression is transcriptionally regulated by ERα, HIF-1α, NFATc1/NFAT2 (which also drives its lysosomal degradation via MARCH8 E3-ubiquitin ligase in metastatic cells), and miR-18a/b/34a; its surface accumulation is regulated by VEGF-PLCγ1-arachidonic acid signaling and TRPC6; in cardiomyocytes, cardiac-specific Orai3 deletion causes dilated cardiomyopathy with sarcomere disruption, mitochondrial fragmentation (DRP1 upregulation), and calcineurin pathway activation, establishing Orai3 as essential for cardiac homeostasis."},"narrative":{"mechanistic_narrative":"ORAI3 is a mammalian plasma-membrane Ca2+ channel that operates in multiple gating modes to drive Ca2+-dependent signaling in cancer, vascular, cardiac, and immune contexts [PMID:20395295, PMID:33849280]. In the store-operated mode it forms a Ca2+-selective channel directly gated by STIM proteins: reconstituted Orai3 in proteoliposomes is opened by recombinant STIM1 in vitro [PMID:37770396], and Orai3 carries native SOCE/ICRAC in ER+ breast cancer cells, NSCLC, and hypertrophied cardiomyocytes [PMID:20395295, PMID:24058448, PMID:25213556]. ORAI3 also assembles into store-independent arachidonate- and leukotriene-C4-regulated channels as a heteropentamer of three Orai1 and two Orai3 subunits, where the cytosolic N-terminus of Orai3 dictates selectivity for arachidonic-acid activation [PMID:17991693, PMID:19622606, PMID:20818184, PMID:25540197]. Distinct N-terminal determinants govern STIM1-dependent versus 2-APB-stimulated gating; the latter dilates the pore (minimum pore size from ~3.8 to >5.34 Å) to permit nonselective cation flux, with TM1 residues (Q83, V77, L70) lining the dilated pore and TM3-E165 approaching the pore axis only in the 2-APB-activated state [PMID:18420579, PMID:18499656, PMID:21724845, PMID:24733836]. Subunit stoichiometry and position within heteromeric channels tune both Ca2+ selectivity and 2-APB pharmacology [PMID:19887627, PMID:32252254], while Orai3-unique extracellular cysteines C226/C232 confer selective inhibition by H2S downstream of STIM1 engagement [PMID:34788146]. ORAI3 expression is controlled by ERα, HIF-1α, Notch1/HEY1, and an Orai1-NFAT2-ERα feedback axis, and NFATc1 both transcribes ORAI3 and triggers its MARCH8-mediated lysosomal degradation in metastatic cells [PMID:22993197, PMID:30754719, PMID:41023307, PMID:41413272, PMID:41172596]. Through these channels ORAI3 sustains pro-proliferative and pro-migratory signaling (ERK, Akt, FAK, c-myc, NFAT) and p53 turnover in cancer cells [PMID:22993197, PMID:23266555, PMID:29323264, PMID:34943998], and cardiac-specific Orai3 deletion causes dilated cardiomyopathy with sarcomere disruption, DRP1-driven mitochondrial fragmentation, and calcineurin activation, establishing it as essential for cardiac homeostasis [PMID:33849280].","teleology":[{"year":2007,"claim":"Established that Orai3, together with Orai1, is an obligatory component of store-independent arachidonate-regulated (ARC) Ca2+ channels, distinguishing it from the canonical CRAC pathway.","evidence":"Reciprocal overexpression, dominant-negative, and siRNA knockdown with patch-clamp in STIM1-expressing cells","pmids":["17991693"],"confidence":"High","gaps":["Subunit stoichiometry of the ARC channel not yet defined","Molecular basis of arachidonic-acid selectivity unknown"]},{"year":2008,"claim":"Defined a STIM1-independent gating mode in which 2-APB dilates the Orai3 pore to a nonselective cation channel, revealing pharmacologically separable conformations.","evidence":"Patch-clamp with Orai1-3 chimeras, pore-size and ion-permeation measurements, E165Q and R66W mutants in HEK cells","pmids":["18420579","18499656"],"confidence":"High","gaps":["Physiological relevance of the dilated nonselective state not established","Endogenous 2-APB-like activator unidentified"]},{"year":2009,"claim":"Resolved the ARC channel as a heteropentamer of three Orai1 and two Orai3 subunits with altered selectivity, defining the structural stoichiometry of store-independent gating.","evidence":"Concatenated pentameric constructs and extracellular-loop mutagenesis with patch-clamp","pmids":["19622606","19887627"],"confidence":"High","gaps":["How STIM1 at the plasma membrane gates this fixed stoichiometry is unresolved","Native stoichiometry in tissues not directly measured"]},{"year":2010,"claim":"Localized arachidonic-acid activation selectivity to the cytosolic Orai3 N-terminus and demonstrated Orai3-dependent SOCE in ER+ breast cancer, linking the channel to a defined disease context.","evidence":"N-terminal domain-swap pentamers plus knockdown/electrophysiology across ten breast cell lines","pmids":["20818184","20395295"],"confidence":"High","gaps":["Why ER+ cells use Orai3 rather than Orai1 mechanistically unclear","N-terminal binding partner for arachidonic-acid sensing unidentified"]},{"year":2011,"claim":"Separated the N-terminal determinants for STIM1-dependent versus 2-APB activation and tied N-terminal length to calmodulin-dependent fast inactivation.","evidence":"Stepwise N-terminal deletion mutagenesis, patch-clamp, and calmodulin binding assays","pmids":["21724845"],"confidence":"High","gaps":["Structural basis of differential N-terminal coupling not solved","Role of calmodulin in native cells not tested"]},{"year":2012,"claim":"Placed ORAI3 in a transcriptional and signaling circuit in breast cancer, showing ERα drives its expression and that it feeds ERK/FAK/NFAT and cell-cycle progression to support tumor growth.","evidence":"ERα knockdown with Orai3 rescue, NFAT reporter, cell-cycle and xenograft assays","pmids":["22993197","20683915","23266555"],"confidence":"Medium","gaps":["Direct ERα binding to the ORAI3 promoter not shown","Causal ordering of ERK/c-myc/NFAT events incompletely resolved"]},{"year":2013,"claim":"Mapped the 2-APB-activated pore architecture, identifying TM1 pore-lining residues and a state-dependent TM2-TM3 disulfide, defining the conformational rearrangement of the dilated channel.","evidence":"Cysteine scanning, Cd2+/MTSEA block, and redox manipulation with patch-clamp; Orai3-dependent SOCE in NSCLC","pmids":["24081982","24733836","24058448"],"confidence":"High","gaps":["Atomic structure of the open dilated state not determined","Link between pore dilation and physiological gating unclear"]},{"year":2014,"claim":"Extended store-independent Orai3 signaling to vascular smooth muscle (LTC4-gated LRC channels) and to cardiomyocytes, where Orai3 is the STIM1 partner during hypertrophy.","evidence":"LTC4S knockdown, in vivo neointima model, co-IP, and in vivo siRNA with electrophysiology","pmids":["25540197","25213556"],"confidence":"High","gaps":["Whether LRC and ARC channels are identical complexes unresolved","Mechanism of STIM1 partner switching during hypertrophy unknown"]},{"year":2015,"claim":"Showed VEGF rapidly traffics Orai3 to the endothelial surface via a PLCγ1-cPLA2α-arachidonic-acid-LTC4 cascade, coupling growth-factor signaling to Orai3-dependent angiogenesis.","evidence":"Surface biotinylation, immunofluorescence, knockdown, tube formation, and in vivo angiogenesis with pharmacological dissection","pmids":["26160956"],"confidence":"High","gaps":["Trafficking machinery delivering Orai3 to the membrane unidentified","Whether surface Orai3 forms ARC or SOCE channels here not defined"]},{"year":2017,"claim":"Reported an ER-localized role for Orai3 in mediating 2-APB-induced ER Ca2+ leak distinct from SERCA, raising the possibility of a non-plasma-membrane function.","evidence":"Dominant-negative and siRNA with cytoplasmic and ER-luminal Ca2+ indicators and IP3R colocalization","pmids":["28179072"],"confidence":"Medium","gaps":["Single lab; ER residence of Orai3 not independently confirmed","Physiological significance of the leak pathway unclear"]},{"year":2018,"claim":"Connected Orai3 to oncogenic protein turnover and EMT signaling, identifying p53 degradation via PI3K/Sgk-1-Mdm2/Nedd4-2 and TRPC6-dependent trafficking and TGF-β/Snai1 control.","evidence":"Overexpression/knockdown, E3-ligase identification, co-IP, ChIP/promoter-binding, and migration assays","pmids":["29323264","30034631","30223530"],"confidence":"Medium","gaps":["Single-lab studies without reciprocal validation","Direct vs indirect role of Orai3 channel activity in p53 turnover not isolated"]},{"year":2019,"claim":"Identified HIF-1α as a hypoxia-driven inducer of ORAI3 in basal breast cancer and extended cardiac signaling to a TNFR2/Orai3 store-independent influx promoting hypertrophy via GSK3β.","evidence":"HIF-1α knockdown/hypoxia induction with expression readouts; conditioned-medium and in vivo siRNA with GSK3β phosphorylation","pmids":["30754719","30988334"],"confidence":"Medium","gaps":["Direct HIF-1α binding at the ORAI3 locus not demonstrated","Channel mode mediating TNFR2-triggered influx undefined"]},{"year":2021,"claim":"Established Orai3 as essential for cardiac homeostasis through a genetic knockout causing dilated cardiomyopathy, and refined its biophysical fingerprint (pH-insensitivity, H2S inhibition, stoichiometry-dependent pharmacology).","evidence":"Cardiomyocyte-specific Orai3 knockout mice with ultrastructural and Ca2+-cycling analysis; chimeric and cysteine-mutant electrophysiology/imaging","pmids":["33849280","32252254","34877682","34788146","33798603"],"confidence":"High","gaps":["Whether the cardiac phenotype reflects SOCE or store-independent Orai3 channels unresolved","In vivo relevance of H2S/pH regulation untested"]},{"year":2022,"claim":"Genetic knockout in immune cells showed Orai3 is dispensable for SOCE in T cells and only partially redundant with Orai1 in B cells, delineating cell-type-specific contributions.","evidence":"Orai3 and Orai1/3 double-knockout mice with SOCE, NFAT, metabolic, and in vivo immunization/infection readouts","pmids":["35861698","36803766"],"confidence":"High","gaps":["Why immune-cell SOCE tolerates Orai3 loss while cancer cells depend on it unexplained","Macrophage enhancement of SOCE mechanism unknown"]},{"year":2023,"claim":"Demonstrated direct STIM1 gating of purified Orai3 in vitro and uncovered ORAI3-STIM2 basal complexes that negatively regulate SOCE to permit mitotic progression, plus stemness and endothelial migration roles.","evidence":"Proteoliposome reconstitution; co-IP, cell-cycle and apoptosis assays in prostate cancer; ID1 rescue in OSCC; p38-STIM1-Orai3-TRPC6 dissection in endothelium","pmids":["37770396","37597301","37759448","37093037"],"confidence":"Medium","gaps":["Reconstitution lacks mutagenesis validation","STIM2-Orai3 basal complex structure and switching to active gating unresolved"]},{"year":2025,"claim":"Resolved a transcription-degradation circuit in which NFATc1 both induces ORAI3 and, via MARCH8-mediated ubiquitination and lysosomal degradation, eliminates it in metastatic cells, and defined an Orai1-NFAT2-ERα-Orai3 regulatory axis and Notch1/HEY1 control.","evidence":"ChIP/reporter, MARCH8 co-IP and ubiquitination, methylation analysis, super-resolution imaging and in vivo metastasis; Orai1-knockout rescue; Notch/HEY1 mutant assays","pmids":["41023307","41172596","41413272"],"confidence":"High","gaps":["How methylation status of the MARCH8 promoter is set in metastatic cells not fully mechanistic","Integration of multiple transcriptional inputs at the ORAI3 locus unresolved"]},{"year":null,"claim":"A unified structural explanation for how a single Orai3 protein switches between STIM1/STIM2-gated Ca2+-selective, arachidonate/LTC4-gated heteromeric, and 2-APB-gated nonselective dilated states remains undefined.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No high-resolution structure of any Orai3 gated state","Endogenous physiological trigger of pore dilation unknown","Mechanism selecting between channel modes in a given cell type unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[0,1,2,3,6,32]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,6,16,19,32]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[17]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[8,14,22,33]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[6,8,25,36]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[9,11,31]}],"complexes":["ARC channel (Orai1/Orai3 heteropentamer, 3:2)","CRAC channel (STIM1/STIM2-Orai3)"],"partners":["STIM1","STIM2","ORAI1","TRPC6","FAK","MARCH8","SEPTIN4","IP3R"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9BRQ5","full_name":"Protein orai-3","aliases":["Transmembrane protein 142C"],"length_aa":295,"mass_kda":31.5,"function":"Pore-forming subunit of two major inward rectifying Ca(2+) channels at the plasma membrane: Ca(2+) release-activated Ca(2+) (CRAC) channels and arachidonate-regulated Ca(2+)-selective (ARC) channels (PubMed:16807233, PubMed:17442569, PubMed:19182790, PubMed:19622606, PubMed:19706554, PubMed:20354224, PubMed:32415068). Assembles with ORAI1 and ORAI2 to form hexameric CRAC channels that mediate Ca(2+) influx upon depletion of endoplasmic reticulum Ca(2+) store and channel activation by Ca(2+) sensor STIM1, a process known as store-operated Ca(2+) entry (SOCE). Various pore subunit combinations may account for distinct CRAC channel spatiotemporal and cell-type specific dynamics. ORAI1 mainly contributes to the generation of Ca(2+) plateaus involved in sustained Ca(2+) entry and is dispensable for cytosolic Ca(2+) oscillations, whereas ORAI2 and ORAI3 generate oscillatory patterns. CRAC channels assemble in Ca(2+) signaling microdomains where Ca(2+) influx is coupled to calmodulin and calcineurin signaling and activation of NFAT transcription factors recruited to ORAI1 via AKAP5. CRAC channels are the main pathway for Ca(2+) influx in T cells and promote the immune response to pathogens by activating NFAT-dependent cytokine and chemokine transcription (PubMed:16807233, PubMed:17442569, PubMed:19182790, PubMed:19706554, PubMed:20354224, PubMed:32415068). Assembles with ORAI1 to form channels that mediate store-independent Ca(2+) influx in response to inflammatory metabolites arachidonate or its derivative leukotriene C4, termed ARC and LRC channels respectively (PubMed:19622606, PubMed:32415068)","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q9BRQ5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ORAI3","classification":"Not Classified","n_dependent_lines":12,"n_total_lines":1208,"dependency_fraction":0.009933774834437087},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ORAI3","total_profiled":1310},"omim":[{"mim_id":"614178","title":"CALCIUM RELEASE-ACTIVATED CALCIUM CHANNEL REGULATOR 2A; CRACR2A","url":"https://www.omim.org/entry/614178"},{"mim_id":"610930","title":"ORAI CALCIUM RELEASE-ACTIVATED CALCIUM MODULATOR 3; ORAI3","url":"https://www.omim.org/entry/610930"},{"mim_id":"610929","title":"ORAI CALCIUM RELEASE-ACTIVATED CALCIUM MODULATOR 2; ORAI2","url":"https://www.omim.org/entry/610929"},{"mim_id":"610277","title":"ORAI CALCIUM RELEASE-ACTIVATED CALCIUM MODULATOR 1; ORAI1","url":"https://www.omim.org/entry/610277"},{"mim_id":"605921","title":"STROMAL INTERACTION MOLECULE 1; STIM1","url":"https://www.omim.org/entry/605921"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Cytosol","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ORAI3"},"hgnc":{"alias_symbol":["MGC13024"],"prev_symbol":["TMEM142C"]},"alphafold":{"accession":"Q9BRQ5","domains":[{"cath_id":"1.20.140.140","chopping":"29-179_240-293","consensus_level":"high","plddt":81.799,"start":29,"end":293}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BRQ5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BRQ5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BRQ5-F1-predicted_aligned_error_v6.png","plddt_mean":71.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ORAI3","jax_strain_url":"https://www.jax.org/strain/search?query=ORAI3"},"sequence":{"accession":"Q9BRQ5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9BRQ5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9BRQ5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BRQ5"}},"corpus_meta":[{"pmid":"20395295","id":"PMC_20395295","title":"A novel native 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chimera analysis mapped the structural determinant for 2-APB-induced current to the sequence spanning the second to third transmembrane segment of Orai3.\",\n      \"method\": \"Patch-clamp electrophysiology, Orai1-3 chimera expression in HEK cells, pharmacological application of 2-APB\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro electrophysiology with chimeric channel mutagenesis in a single rigorous study, independently replicated in concurrent work (PMID:18499656)\",\n      \"pmids\": [\"18420579\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"2-APB alters Orai3 ion selectivity by increasing its minimum pore size from ~3.8 Å to >5.34 Å, permitting monovalent cation permeation in both directions; a pore mutant (R66W) lacking store-operated activation was also resistant to 2-APB stimulation; Orai3 E165Q mutation resembled 2-APB-stimulated Orai3 in permeation properties and showed reduced 2-APB response.\",\n      \"method\": \"Patch-clamp electrophysiology, site-directed mutagenesis, ion substitution permeability assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro electrophysiology combined with mutagenesis and pore-size measurements in one rigorous study\",\n      \"pmids\": [\"18499656\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Both Orai1 and Orai3 are essential components of the arachidonate-regulated Ca2+-selective (ARC) channels; overexpression of Orai3 alone has no effect on ARC currents, but specifically increases them when Orai1 is co-expressed; a dominant-negative Orai3 mutant specifically reduces ARC channel currents without affecting CRAC currents; siRNA knockdown of either Orai1 or Orai3 markedly inhibits ARC channel currents.\",\n      \"method\": \"Patch-clamp electrophysiology, dominant-negative expression, siRNA knockdown, overexpression in cells stably expressing STIM1\",\n      \"journal\": \"The Journal of physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal gain/loss-of-function with pharmacological specificity, replicated across multiple experimental conditions\",\n      \"pmids\": [\"17991693\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The functional ARC channel pore is a heteropentameric assembly of three Orai1 subunits and two Orai3 subunits; expression of concatenated pentameric constructs with this 3:2 stoichiometry produces large currents with all key biophysical and pharmacological features of endogenous ARC channels including arachidonic acid activation, store-independence, and requirement for plasma membrane-resident STIM1.\",\n      \"method\": \"Concatenated Orai1/Orai3 pentameric construct expression, dominant-negative co-expression, patch-clamp electrophysiology, arachidonic acid stimulation\",\n      \"journal\": \"The Journal of physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct reconstitution of defined stoichiometry with functional validation using multiple orthogonal criteria\",\n      \"pmids\": [\"19622606\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Heteromeric Orai1/Orai3 channels display diminished Ca2+ selectivity, robust Cs+ permeation, and reduced fast inactivation compared to homomeric forms; differences in the first extracellular loop (two aspartates in Orai3 replacing glutamates in Orai1) contribute to altered selectivity; Orai3 mutant mimicking Orai1 first loop co-expressed with Orai1 recovered Ca2+ selectivity.\",\n      \"method\": \"Patch-clamp electrophysiology, site-directed mutagenesis of extracellular loop residues, co-expression of Orai1 and Orai3\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis combined with electrophysiology establishing selectivity determinants, multiple constructs tested\",\n      \"pmids\": [\"19887627\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The cytosolic N-terminal domain of Orai3 is specifically responsible for determining selectivity for activation of the ARC channel by arachidonic acid; substitution of the Orai3 N-terminal domain into an otherwise complete Orai1 subunit within a concatenated 3:1:1:1:1 pentamer converts the resulting channel from predominantly store-operated to exclusively arachidonic acid-activated; two Orai3 subunits are required for full selectivity.\",\n      \"method\": \"Concatenated pentameric construct expression, N-terminal domain substitution mutagenesis, Ca2+ imaging and patch-clamp electrophysiology\",\n      \"journal\": \"Channels (Austin, Tex.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — domain swap mutagenesis within defined pentameric constructs with functional readout\",\n      \"pmids\": [\"20818184\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Native SOCE and ICRAC in estrogen receptor-positive (ER+) breast cancer cell lines are mediated by STIM1/2 and Orai3, not Orai1; ER-negative breast cancer cells use the canonical STIM1/Orai1 pathway; established by molecular knockdown, Ca2+ imaging, pharmacology, and patch-clamp in ten breast cell lines.\",\n      \"method\": \"siRNA knockdown, Ca2+ imaging, patch-clamp electrophysiology, pharmacological inhibition across ten breast cell lines\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (electrophysiology, imaging, knockdown, pharmacology) in a large panel of cell lines\",\n      \"pmids\": [\"20395295\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"The conserved N-terminal region of Orai3 contains distinct molecular determinants for STIM1-dependent activation versus 2-APB-stimulated activation; progressive N-terminal truncations progressively decrease fast inactivation and diminish calmodulin binding; STIM1-dependent activation requires the second half of the conserved N-terminal domain, whereas 2-APB activation is partially retained with further truncations.\",\n      \"method\": \"Stepwise N-terminal deletion mutagenesis, patch-clamp electrophysiology, calmodulin binding assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — systematic mutagenesis combined with functional electrophysiology and biochemical binding assay\",\n      \"pmids\": [\"21724845\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ERα transcriptionally regulates Orai3 expression in ER+ breast cancer cells; ERα knockdown decreases Orai3 mRNA (~63%) and protein (~44%) without affecting Orai1; ectopic Orai3 expression rescues SOCE after ERα knockdown; Orai3 knockdown inhibits ERK1/2 and FAK phosphorylation, NFAT transcriptional activity, anchorage-independent growth, Matrigel invasion, and tumor growth in immunodeficient mice.\",\n      \"method\": \"siRNA knockdown of ERα, ectopic Orai3 re-expression, Ca2+ imaging, patch-clamp, Western blotting, NFAT reporter assay, anchorage-independent growth, Matrigel invasion, xenograft mouse model\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods including in vivo xenograft validation, rescue experiments, and signaling pathway readouts\",\n      \"pmids\": [\"22993197\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Orai3 silencing in MCF-7 breast cancer cells reduces CDK4/2 and cyclin D1/E expression, accumulates p21Waf1/Cip1 and p53, arrests cell cycle at G1, and decreases Ca2+ entry and cell survival; these effects are not seen in non-cancerous MCF-10A cells upon Orai3 silencing.\",\n      \"method\": \"siRNA knockdown, Ca2+ imaging, flow cytometry, Western blotting for cell cycle proteins\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockdown with multiple downstream readouts in two cell types, single lab\",\n      \"pmids\": [\"20683915\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Orai3 silencing in MCF-7 cells reduces c-myc expression and activity and decreases pERK1/2 levels, linking Orai3-mediated Ca2+ entry to the MAP kinase pathway and c-myc-driven cell cycle progression.\",\n      \"method\": \"siRNA knockdown, Western blotting for pERK1/2 and c-myc, flow cytometry\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockdown with pathway-level biochemical readouts, single lab\",\n      \"pmids\": [\"23266555\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Orai3 constitutes the native SOCE pathway in non-small cell lung cancer (NSCLC) cells; Orai3 knockdown reduces SOCE, inhibits cell proliferation, arrests cells in G0/G1, downregulates cyclin D1, cyclin E, CDK4, CDK2, and decreases Akt phosphorylation.\",\n      \"method\": \"siRNA knockdown, pharmacological inhibition, Ca2+ imaging, flow cytometry, Western blotting\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockdown confirmed by multiple functional readouts, single lab\",\n      \"pmids\": [\"24058448\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"TM3 residue G158 in Orai3 forms a disulfide bridge with endogenous TM2 cysteine C101 when mutated to cysteine (G158C); this bridge slows 2-APB activation kinetics and prevents complete channel closure; reducing agent BMS reverses the slow phenotype in a state-dependent manner (only during 2-APB activation).\",\n      \"method\": \"Site-directed mutagenesis, patch-clamp electrophysiology, reducing agent application (BMS), double mutant C101G/G158C analysis\",\n      \"journal\": \"The Journal of general physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis with mechanistic chemical validation (redox-sensitive disulfide), functional and double-mutant controls\",\n      \"pmids\": [\"24081982\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"In 2-APB-activated Orai3, TM1 residues Q83, V77, and L70 line the dilated pore (Cd2+ block potentiated at these positions); TM3 residue E165 approaches the pore axis in the 2-APB-activated state but not the store-operated state; TM1 mutants E81C, G73A/C, and R66C abolish 2-APB sensitivity; V77C is blocked by MTSEA in a state-dependent manner only when channels are open.\",\n      \"method\": \"Cysteine scanning mutagenesis, Cd2+ block, thiol-reactive reagent (MTSEA/MTSET) application, patch-clamp electrophysiology\",\n      \"journal\": \"The Journal of general physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — systematic cysteine scanning with state-dependent chemical modification, multiple confirming mutations\",\n      \"pmids\": [\"24733836\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"LTC4 (generated by LTC4 synthase from arachidonic acid) activates store-independent LRC channels encoded by Orai1/Orai3 heteromultimers in vascular smooth muscle cells; LTC4S knockdown inhibits LRC currents; Orai3 and LTC4S knockdown both inhibit Akt1/Akt2 Ser473/474 phosphorylation and VSMC migration, contributing to neointimal hyperplasia.\",\n      \"method\": \"LTC4S siRNA/shRNA knockdown, patch-clamp electrophysiology, in vivo balloon angioplasty neointima model with lentiviral shRNA, Akt phosphorylation assay, migration assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo and in vitro complementary approaches with electrophysiological validation of LRC current and signaling readouts\",\n      \"pmids\": [\"25540197\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"In hypertrophied adult rat ventricular cardiomyocytes, Orai3 (but not Orai1) is the critical partner of STIM1 carrying voltage-independent Ca2+ entries; Orai3 co-immunoprecipitates with STIM1, with greater STIM1 and Orai1 enriched in the Orai3 complex during hypertrophy; Orai3 also drives an arachidonic acid-activated inward current in cardiomyocytes.\",\n      \"method\": \"Co-immunoprecipitation, in vivo siRNA delivery, patch-clamp electrophysiology, Ca2+ imaging in isolated cardiomyocytes\",\n      \"journal\": \"Cardiovascular research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus in vivo siRNA knockdown validated by electrophysiology, multiple orthogonal approaches\",\n      \"pmids\": [\"25213556\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"VEGF triggers rapid (within 2 min) accumulation of Orai3 at the plasma membrane surface in human endothelial cells via a signaling pathway involving PLCγ1, Ca2+ release, cytosolic group IV phospholipase A2α, arachidonic acid, and microsomal glutathione S-transferase 2 (forming LTC4); Orai3 knockdown suppresses this VEGF-dependent Ca2+ entry and endothelial tube formation in vitro and in vivo.\",\n      \"method\": \"Immunofluorescence, cell surface biotinylation, siRNA knockdown, Ca2+ imaging, Matrigel tube formation, in vivo angiogenesis model, pharmacological pathway dissection\",\n      \"journal\": \"Arteriosclerosis, thrombosis, and vascular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — surface biotinylation directly demonstrating trafficking, plus in vivo functional validation in multiple endothelial cell types\",\n      \"pmids\": [\"26160956\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Orai3 channel colocalizes with expressed IP3R at the ER membrane and mediates 2-APB-induced Ca2+ leak from the ER; Orai3 knockdown inhibits 2-APB-induced ER Ca2+ leak without affecting thapsigargin-revealed SERCA-dependent ER Ca2+ leak; reducing Orai3 results in larger cytoplasmic Ca2+ responses after thapsigargin only when ER stores are overloaded.\",\n      \"method\": \"Dominant-negative Orai1 E106A expression, siRNA knockdown of Orai1/2/3, dual Ca2+ indicators (cytoplasmic and ER-luminal), co-localization with IP3R\",\n      \"journal\": \"Cell calcium\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA and dominant-negative approaches with ER-targeted indicators, single lab\",\n      \"pmids\": [\"28179072\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Orai3 overexpression in breast cancer cells drives p53 degradation via the PI3K/Sgk-1/Sek-1 pathway, utilizing both Mdm2 and the E3 ubiquitin ligase Nedd4-2 for p53 proteolysis; this mechanism is dependent on extracellular calcium and Orai3 functionality.\",\n      \"method\": \"Orai3 overexpression, calcium removal, PI3K/Sgk-1 inhibition, Mdm2 and Nedd4-2 identification by high-throughput approach, Western blotting\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional pharmacological pathway dissection with identification of two E3 ligases, single lab\",\n      \"pmids\": [\"29323264\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TRPC6 physically interacts with Orai3 in MCF7 breast cancer cells and is required for translocation of Orai3 to the plasma membrane upon Ca2+ store depletion; TRPC6 knockdown impairs Orai3-dependent SOCE in MCF7 cells.\",\n      \"method\": \"Co-immunoprecipitation, RNAi knockdown, dominant-negative TRPC6 expression, Ca2+ imaging, surface expression assay\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP demonstrating interaction plus functional knockdown, single lab\",\n      \"pmids\": [\"30223530\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"ORAI3 and STIM1 are required for TGF-β-dependent Snai1 transcription; blocking SOCE paradoxically increases Snai1 activation via AKT pathway and NF-κB (p65) binding at the Snai1 promoter.\",\n      \"method\": \"siRNA knockdown of ORAI3 and STIM1, 2-APB treatment, AKT pathway analysis, ChIP/promoter-binding assays, migration assays\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockdown with promoter-binding and pathway validation, single lab\",\n      \"pmids\": [\"30034631\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HIF-1α induces ORAI3 expression in basal breast cancer cell lines; ORAI3 silencing attenuates hypoxia-associated EGFR phosphorylation and expression of genes associated with cell migration and inflammatory/immune responses.\",\n      \"method\": \"HIF-1α knockdown, HIF-1α pathway induction by hypoxia, ORAI3 siRNA, Western blotting, gene expression analysis\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockdown of upstream regulator with functional downstream readouts, single lab\",\n      \"pmids\": [\"30754719\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Inflammatory cardiac CD11b/c cells trigger a TNFR2- and Orai3-dependent store-independent Ca2+ influx in hypertrophied cardiomyocytes; this Ca2+ influx promotes cardiomyocyte hypertrophy and resistance to oxidative stress via GSK3β phosphorylation; in vivo Orai3 knockdown during early adaptive cardiac hypertrophy impairs GSK3β phosphorylation and accelerates heart failure.\",\n      \"method\": \"Conditioned medium from cardiac CD11b/c cells, TNFα stimulation, pharmacological/siRNA Orai3 inhibition, intramyocardial siRNA injection, Ca2+ imaging, GSK3β phosphorylation assay\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo and in vitro complementary siRNA knockdown with GSK3β signaling readout, single lab\",\n      \"pmids\": [\"30988334\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The number and position of Orai3 subunits within heteromeric store-operated channels alter the pharmacology of ICRAC; channels with two or more Orai3 subunits can be activated by 2-APB independently of store depletion/STIM1 and display large outward currents; a single Orai3 subunit does not alter 2-APB pharmacology compared to homomeric Orai1.\",\n      \"method\": \"Patch-clamp electrophysiology with concatenated Orai1/Orai3 constructs of defined stoichiometry and position\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — defined concatenated constructs with electrophysiology, single lab\",\n      \"pmids\": [\"32252254\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Orai3 regulates MDA-MB-231 breast cancer cell migration through two mechanisms: (1) a Ca2+-dependent modulation of calpain activity affecting cell adhesion; (2) a Ca2+-independent interaction with focal adhesion kinase (FAK) that regulates F-actin polymerization and cell morphology.\",\n      \"method\": \"siRNA knockdown of Orai3, calpain activity assay, cell adhesion and migration assays, F-actin staining, co-immunoprecipitation of Orai3 and FAK\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP plus functional knockdown with two distinct mechanistic readouts, single lab\",\n      \"pmids\": [\"34943998\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Cardiac-specific deletion of Orai3 leads to dilated cardiomyopathy characterized by abnormal sarcomere (M- and Z-line) morphology, increased DRP1-associated mitochondrial fragmentation, upregulation of TRPC6 and RCAN1 (indicating calcineurin pathway activation), and impaired myocardial Ca2+ cycling; removal of Orai1 from Orai3-deficient cardiomyocytes does not change the cardiac phenotype.\",\n      \"method\": \"Constitutive and inducible cardiomyocyte-specific Orai3 knockout mice, echocardiography, ultrastructural analysis, Western blotting, Ca2+ cycling measurements\",\n      \"journal\": \"Journal of the American Heart Association\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout mouse model with mechanistic molecular analysis (DRP1, TRPC6, RCAN1, Ca2+ cycling) and ultrastructural validation\",\n      \"pmids\": [\"33849280\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ORAI3 silencing in pancreatic ductal adenocarcinoma cells increases SOCE (in contrast to decreasing it in normal pancreatic cells), impairs proliferation, causes mitotic catastrophe and cell death, and inhibits tumor growth in vivo.\",\n      \"method\": \"siRNA knockdown, Ca2+ imaging (SOCE measurement), flow cytometry, xenograft in vivo model\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockdown with in vivo validation and cell-type-specific functional divergence, single lab\",\n      \"pmids\": [\"33798603\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Orai3 mediates Orai channel remodeling in pulmonary fibrosis: TGF-β1 increases SEPTIN4, which promotes Orai3-Orai1 interaction, suppressing STIM1-Orai1 interaction and SOCE activity, resulting in a high and stable extracellular Ca2+ influx that activates NFAT1 and Calpain/ERK signaling to drive fibroblast activation.\",\n      \"method\": \"Co-immunoprecipitation of Orai3-Orai1 and STIM1-Orai1, Orai3 knockdown/overexpression, BLM-induced lung fibrosis model, SEPTIN4 manipulation, Ca2+ imaging, NFAT1 and pERK Western blotting\",\n      \"journal\": \"Journal of cellular and molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP combined with in vivo fibrosis model and SEPTIN4 mechanistic link, single lab\",\n      \"pmids\": [\"36128650\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Orai3-mediated SOCE is insensitive to changes in intracellular pH (pHi), in contrast to Orai1 (strongly pHi-dependent) and Orai2 (moderately pHi-dependent); domain swapping between Orai1 and Orai3 identified the N-terminus and intracellular loop 2 of Orai1 as responsible for pHi sensitivity.\",\n      \"method\": \"Whole-cell patch-clamp in HEK293T cells expressing Orai isoforms with STIM1, intracellular pH manipulation, Orai1/Orai3 chimeric constructs\",\n      \"journal\": \"The Journal of physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — electrophysiology with chimeric mutagenesis, single lab\",\n      \"pmids\": [\"34877682\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Extracellular cysteines C226 and C232, unique to Orai3 (absent in Orai1 and Orai2), mediate selective inhibition of Orai3-mediated SOCE by hydrogen sulfide (H2S); mutation of either cysteine to serine abolishes H2S inhibition; H2S does not affect Orai3 membrane mobility, STIM1/Orai3 puncta formation, or STIM1-Orai3 protein-protein interactions, indicating that the inhibitory mechanism is downstream of STIM1 engagement.\",\n      \"method\": \"Site-directed mutagenesis (C226S, C232S), H2S donor (GYY4137), Ca2+ imaging, FRAP, colocalization, FRET assays in HEK293 cells\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis combined with multiple orthogonal imaging/FRET methods to define mechanism, single lab\",\n      \"pmids\": [\"34788146\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Deletion of ORAI3 in T cells and B cells does not impair SOCE in those cell types; combined deletion of Orai1 and Orai3, but not Orai3 alone, impairs SOCE, proliferation, survival, NFAT activation, and metabolic reprogramming in B cells; ORAI3 deletion moderately enhances SOCE in macrophages.\",\n      \"method\": \"Orai3-/- and double Orai1/3 knockout mice, SOCE measurement, flow cytometry, NFAT reporter, Seahorse metabolic assay, in vivo immunization and infection models\",\n      \"journal\": \"The Journal of general physiology / eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout mice with multiple complementary in vivo and in vitro readouts across immune cell types, two independent studies (PMID:35861698, PMID:36803766)\",\n      \"pmids\": [\"35861698\", \"36803766\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ORAI3 physically interacts with STIM2 under basal conditions in prostate cancer cells; ORAI3 silencing increases SOCE and arrests cells in G2/M with elevated CDK1-Y15/Cyclin B1, leading to mitotic catastrophe via Bax/Bcl-2-mediated apoptosis and caspase-8 activation; STIM2 and ORAI3 expression increase during M phase while STIM1 decreases, suggesting an ORAI3-STIM2 complex permits mitotic progression by negatively regulating SOCE.\",\n      \"method\": \"Co-immunoprecipitation of ORAI3-STIM2, siRNA knockdown, Ca2+ imaging, flow cytometry, CDK1/Cyclin B1 Western blotting, caspase activity assay\",\n      \"journal\": \"Cell calcium\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP plus multiple functional readouts, single lab\",\n      \"pmids\": [\"37597301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Reconstituted Orai3 protein in proteoliposomes forms a functional channel that is directly opened by recombinant STIM1 in vitro, mediating Ca2+ release from liposomes.\",\n      \"method\": \"Protein purification, proteoliposome reconstitution, Ca2+ release assay, STIM1-Orai3 interaction in vitro\",\n      \"journal\": \"Biochemistry. Biokhimiia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro reconstitution demonstrating direct STIM1-Orai3 gating, single study without mutagenesis validation\",\n      \"pmids\": [\"37770396\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"p38 MAPK activation by lysophosphatidylcholine or arachidonic acid promotes STIM1-Orai3 association in endothelial cells, increasing intracellular Ca2+ and triggering TRPC6 externalization; Orai3 downregulation blocks the lysoPC-induced Ca2+ rise and TRPC6 externalization, preserving endothelial cell migration.\",\n      \"method\": \"siRNA Orai3 knockdown, pharmacological p38 MAPK inhibition, Ca2+ imaging, TRPC6 surface expression assay, migration assays\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA knockdown with mechanistic pathway placement (p38-STIM1-Orai3-TRPC6), single lab\",\n      \"pmids\": [\"37093037\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Orai3 promotes cancer stemness in oral squamous cell carcinoma (OSCC) through upregulation of the stemness transcription factor ID1; ectopic Orai3 increases intracellular Ca2+ and acquires malignant/CSC properties; ID1 suppression abrogates the CSC phenotype induced by Orai3 overexpression.\",\n      \"method\": \"Orai3 ectopic overexpression, siRNA knockdown, Ca2+ imaging, CSC marker expression, ID1 knockdown rescue experiments\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — overexpression plus knockdown with rescue approach establishing ID1 as downstream effector, single lab\",\n      \"pmids\": [\"37759448\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Orai3 and STIM1 mediate store-operated Ca2+ entry in ER+ breast cancer stem-like cells (BCSCs), promoting their growth and tumorigenicity via a glycolysis-independent pathway; this is mechanistically distinct from Orai1, which interacts with SPCA2 for store-independent Ca2+ entry promoting glycolysis.\",\n      \"method\": \"siRNA knockdown, Ca2+ imaging, 3D soft fibrin gel BCSC enrichment, xenograft in vivo, glycolysis (Seahorse) assay\",\n      \"journal\": \"Stem cell research & therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — combined in vitro and in vivo knockout with metabolic profiling distinguishing Orai3 from Orai1 mechanism, single lab\",\n      \"pmids\": [\"39135143\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NFATc1 transcription factor both drives Orai3 transcription (in non-metastatic pancreatic cancer cells) and induces its lysosomal degradation (in invasive/metastatic cells) via transcriptional upregulation of MARCH8 E3-ubiquitin ligase; MARCH8 physically interacts with Orai3 intracellular loop and ubiquitinates Orai3 at the N-terminal; the dichotomy in NFATc1 function arises from differential (hyper-)methylation of the MARCH8 promoter in non-metastatic versus metastatic cells.\",\n      \"method\": \"NFATc1 ChIP and reporter assays, MARCH8 co-immunoprecipitation with Orai3, ubiquitination assay, epigenetic methylation analysis, super-resolution microscopy, siRNA/overexpression, in vivo metastasis experiments\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — multiple orthogonal methods (co-IP, ubiquitination, ChIP, methylation, super-resolution imaging, in vivo) in one study establishing a full mechanistic circuit\",\n      \"pmids\": [\"41023307\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Notch1/HEY1 signaling differentially regulates Orai3 expression in breast cancer subtypes; active Notch1 and its agonist Jagged-1 alter Orai3 expression and corresponding SOCE; the non-phosphorylatable HEY1-S68A mutant mimics Notch1 effects on Orai expression, while the phosphomimetic HEY1-S68D mutant does not.\",\n      \"method\": \"Notch1 active form/Jagged-1 stimulation, DAPT (γ-secretase inhibitor), HEY1 mutant expression, Ca2+ imaging, Western blotting across breast cancer cell lines\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain- and loss-of-function with downstream functional validation, single lab\",\n      \"pmids\": [\"41413272\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Orai1 sustains Orai3 protein synthesis and stability in luminal breast cancer cells; Orai1 knockout decreases Orai3 protein synthesis and enhances Orai3 endo-lysosomal degradation; Orai1 also regulates ERα expression and NFAT2 nuclear translocation, which modulates ERα and Orai3 levels, establishing an Orai1-NFAT2-ERα-Orai3 regulatory axis.\",\n      \"method\": \"Orai1 knockout MCF-7 cells, RNAi silencing, ectopic Orai1 re-expression, Ca2+ imaging, Western blotting, protein synthesis/degradation assays, NFAT2 nuclear translocation assay\",\n      \"journal\": \"Cell calcium\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with rescue and multiple downstream pathway analyses, single lab\",\n      \"pmids\": [\"41172596\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ORAI3 is a mammalian-specific plasma membrane Ca2+-selective channel that functions in multiple modes: (1) as a store-operated CRAC channel activated by STIM1/2 (mediating native SOCE in ER+ breast cancer cells, NSCLC, astrocytes, DRG neurons, and cardiomyocytes), (2) as a heteropentameric ARC channel (3 Orai1 + 2 Orai3 subunits) activated by arachidonic acid or LTC4 in a store-independent manner dependent on plasma membrane-resident STIM1, and (3) as a 2-APB-gated nonselective cation channel (activated independently of STIM1) whose dilated pore is lined by TM1 with TM3-E165 approaching the pore axis; N-terminal determinants govern both STIM1-dependent gating and ARC channel selectivity for arachidonic acid, while extracellular cysteines C226/C232 mediate selective H2S inhibition; ORAI3 expression is transcriptionally regulated by ERα, HIF-1α, NFATc1/NFAT2 (which also drives its lysosomal degradation via MARCH8 E3-ubiquitin ligase in metastatic cells), and miR-18a/b/34a; its surface accumulation is regulated by VEGF-PLCγ1-arachidonic acid signaling and TRPC6; in cardiomyocytes, cardiac-specific Orai3 deletion causes dilated cardiomyopathy with sarcomere disruption, mitochondrial fragmentation (DRP1 upregulation), and calcineurin pathway activation, establishing Orai3 as essential for cardiac homeostasis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ORAI3 is a mammalian plasma-membrane Ca2+ channel that operates in multiple gating modes to drive Ca2+-dependent signaling in cancer, vascular, cardiac, and immune contexts [#6, #25]. In the store-operated mode it forms a Ca2+-selective channel directly gated by STIM proteins: reconstituted Orai3 in proteoliposomes is opened by recombinant STIM1 in vitro [#32], and Orai3 carries native SOCE/ICRAC in ER+ breast cancer cells, NSCLC, and hypertrophied cardiomyocytes [#6, #11, #15]. ORAI3 also assembles into store-independent arachidonate- and leukotriene-C4-regulated channels as a heteropentamer of three Orai1 and two Orai3 subunits, where the cytosolic N-terminus of Orai3 dictates selectivity for arachidonic-acid activation [#2, #3, #5, #14]. Distinct N-terminal determinants govern STIM1-dependent versus 2-APB-stimulated gating; the latter dilates the pore (minimum pore size from ~3.8 to >5.34 Å) to permit nonselective cation flux, with TM1 residues (Q83, V77, L70) lining the dilated pore and TM3-E165 approaching the pore axis only in the 2-APB-activated state [#0, #1, #7, #13]. Subunit stoichiometry and position within heteromeric channels tune both Ca2+ selectivity and 2-APB pharmacology [#4, #23], while Orai3-unique extracellular cysteines C226/C232 confer selective inhibition by H2S downstream of STIM1 engagement [#29]. ORAI3 expression is controlled by ERα, HIF-1α, Notch1/HEY1, and an Orai1-NFAT2-ERα feedback axis, and NFATc1 both transcribes ORAI3 and triggers its MARCH8-mediated lysosomal degradation in metastatic cells [#8, #21, #36, #37, #38]. Through these channels ORAI3 sustains pro-proliferative and pro-migratory signaling (ERK, Akt, FAK, c-myc, NFAT) and p53 turnover in cancer cells [#8, #10, #18, #24], and cardiac-specific Orai3 deletion causes dilated cardiomyopathy with sarcomere disruption, DRP1-driven mitochondrial fragmentation, and calcineurin activation, establishing it as essential for cardiac homeostasis [#25].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Established that Orai3, together with Orai1, is an obligatory component of store-independent arachidonate-regulated (ARC) Ca2+ channels, distinguishing it from the canonical CRAC pathway.\",\n      \"evidence\": \"Reciprocal overexpression, dominant-negative, and siRNA knockdown with patch-clamp in STIM1-expressing cells\",\n      \"pmids\": [\"17991693\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Subunit stoichiometry of the ARC channel not yet defined\", \"Molecular basis of arachidonic-acid selectivity unknown\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defined a STIM1-independent gating mode in which 2-APB dilates the Orai3 pore to a nonselective cation channel, revealing pharmacologically separable conformations.\",\n      \"evidence\": \"Patch-clamp with Orai1-3 chimeras, pore-size and ion-permeation measurements, E165Q and R66W mutants in HEK cells\",\n      \"pmids\": [\"18420579\", \"18499656\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological relevance of the dilated nonselective state not established\", \"Endogenous 2-APB-like activator unidentified\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Resolved the ARC channel as a heteropentamer of three Orai1 and two Orai3 subunits with altered selectivity, defining the structural stoichiometry of store-independent gating.\",\n      \"evidence\": \"Concatenated pentameric constructs and extracellular-loop mutagenesis with patch-clamp\",\n      \"pmids\": [\"19622606\", \"19887627\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How STIM1 at the plasma membrane gates this fixed stoichiometry is unresolved\", \"Native stoichiometry in tissues not directly measured\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Localized arachidonic-acid activation selectivity to the cytosolic Orai3 N-terminus and demonstrated Orai3-dependent SOCE in ER+ breast cancer, linking the channel to a defined disease context.\",\n      \"evidence\": \"N-terminal domain-swap pentamers plus knockdown/electrophysiology across ten breast cell lines\",\n      \"pmids\": [\"20818184\", \"20395295\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why ER+ cells use Orai3 rather than Orai1 mechanistically unclear\", \"N-terminal binding partner for arachidonic-acid sensing unidentified\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Separated the N-terminal determinants for STIM1-dependent versus 2-APB activation and tied N-terminal length to calmodulin-dependent fast inactivation.\",\n      \"evidence\": \"Stepwise N-terminal deletion mutagenesis, patch-clamp, and calmodulin binding assays\",\n      \"pmids\": [\"21724845\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of differential N-terminal coupling not solved\", \"Role of calmodulin in native cells not tested\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Placed ORAI3 in a transcriptional and signaling circuit in breast cancer, showing ERα drives its expression and that it feeds ERK/FAK/NFAT and cell-cycle progression to support tumor growth.\",\n      \"evidence\": \"ERα knockdown with Orai3 rescue, NFAT reporter, cell-cycle and xenograft assays\",\n      \"pmids\": [\"22993197\", \"20683915\", \"23266555\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ERα binding to the ORAI3 promoter not shown\", \"Causal ordering of ERK/c-myc/NFAT events incompletely resolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Mapped the 2-APB-activated pore architecture, identifying TM1 pore-lining residues and a state-dependent TM2-TM3 disulfide, defining the conformational rearrangement of the dilated channel.\",\n      \"evidence\": \"Cysteine scanning, Cd2+/MTSEA block, and redox manipulation with patch-clamp; Orai3-dependent SOCE in NSCLC\",\n      \"pmids\": [\"24081982\", \"24733836\", \"24058448\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic structure of the open dilated state not determined\", \"Link between pore dilation and physiological gating unclear\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Extended store-independent Orai3 signaling to vascular smooth muscle (LTC4-gated LRC channels) and to cardiomyocytes, where Orai3 is the STIM1 partner during hypertrophy.\",\n      \"evidence\": \"LTC4S knockdown, in vivo neointima model, co-IP, and in vivo siRNA with electrophysiology\",\n      \"pmids\": [\"25540197\", \"25213556\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether LRC and ARC channels are identical complexes unresolved\", \"Mechanism of STIM1 partner switching during hypertrophy unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Showed VEGF rapidly traffics Orai3 to the endothelial surface via a PLCγ1-cPLA2α-arachidonic-acid-LTC4 cascade, coupling growth-factor signaling to Orai3-dependent angiogenesis.\",\n      \"evidence\": \"Surface biotinylation, immunofluorescence, knockdown, tube formation, and in vivo angiogenesis with pharmacological dissection\",\n      \"pmids\": [\"26160956\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trafficking machinery delivering Orai3 to the membrane unidentified\", \"Whether surface Orai3 forms ARC or SOCE channels here not defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Reported an ER-localized role for Orai3 in mediating 2-APB-induced ER Ca2+ leak distinct from SERCA, raising the possibility of a non-plasma-membrane function.\",\n      \"evidence\": \"Dominant-negative and siRNA with cytoplasmic and ER-luminal Ca2+ indicators and IP3R colocalization\",\n      \"pmids\": [\"28179072\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; ER residence of Orai3 not independently confirmed\", \"Physiological significance of the leak pathway unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Connected Orai3 to oncogenic protein turnover and EMT signaling, identifying p53 degradation via PI3K/Sgk-1-Mdm2/Nedd4-2 and TRPC6-dependent trafficking and TGF-β/Snai1 control.\",\n      \"evidence\": \"Overexpression/knockdown, E3-ligase identification, co-IP, ChIP/promoter-binding, and migration assays\",\n      \"pmids\": [\"29323264\", \"30034631\", \"30223530\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab studies without reciprocal validation\", \"Direct vs indirect role of Orai3 channel activity in p53 turnover not isolated\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identified HIF-1α as a hypoxia-driven inducer of ORAI3 in basal breast cancer and extended cardiac signaling to a TNFR2/Orai3 store-independent influx promoting hypertrophy via GSK3β.\",\n      \"evidence\": \"HIF-1α knockdown/hypoxia induction with expression readouts; conditioned-medium and in vivo siRNA with GSK3β phosphorylation\",\n      \"pmids\": [\"30754719\", \"30988334\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct HIF-1α binding at the ORAI3 locus not demonstrated\", \"Channel mode mediating TNFR2-triggered influx undefined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Established Orai3 as essential for cardiac homeostasis through a genetic knockout causing dilated cardiomyopathy, and refined its biophysical fingerprint (pH-insensitivity, H2S inhibition, stoichiometry-dependent pharmacology).\",\n      \"evidence\": \"Cardiomyocyte-specific Orai3 knockout mice with ultrastructural and Ca2+-cycling analysis; chimeric and cysteine-mutant electrophysiology/imaging\",\n      \"pmids\": [\"33849280\", \"32252254\", \"34877682\", \"34788146\", \"33798603\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the cardiac phenotype reflects SOCE or store-independent Orai3 channels unresolved\", \"In vivo relevance of H2S/pH regulation untested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Genetic knockout in immune cells showed Orai3 is dispensable for SOCE in T cells and only partially redundant with Orai1 in B cells, delineating cell-type-specific contributions.\",\n      \"evidence\": \"Orai3 and Orai1/3 double-knockout mice with SOCE, NFAT, metabolic, and in vivo immunization/infection readouts\",\n      \"pmids\": [\"35861698\", \"36803766\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why immune-cell SOCE tolerates Orai3 loss while cancer cells depend on it unexplained\", \"Macrophage enhancement of SOCE mechanism unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Demonstrated direct STIM1 gating of purified Orai3 in vitro and uncovered ORAI3-STIM2 basal complexes that negatively regulate SOCE to permit mitotic progression, plus stemness and endothelial migration roles.\",\n      \"evidence\": \"Proteoliposome reconstitution; co-IP, cell-cycle and apoptosis assays in prostate cancer; ID1 rescue in OSCC; p38-STIM1-Orai3-TRPC6 dissection in endothelium\",\n      \"pmids\": [\"37770396\", \"37597301\", \"37759448\", \"37093037\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reconstitution lacks mutagenesis validation\", \"STIM2-Orai3 basal complex structure and switching to active gating unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Resolved a transcription-degradation circuit in which NFATc1 both induces ORAI3 and, via MARCH8-mediated ubiquitination and lysosomal degradation, eliminates it in metastatic cells, and defined an Orai1-NFAT2-ERα-Orai3 regulatory axis and Notch1/HEY1 control.\",\n      \"evidence\": \"ChIP/reporter, MARCH8 co-IP and ubiquitination, methylation analysis, super-resolution imaging and in vivo metastasis; Orai1-knockout rescue; Notch/HEY1 mutant assays\",\n      \"pmids\": [\"41023307\", \"41172596\", \"41413272\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How methylation status of the MARCH8 promoter is set in metastatic cells not fully mechanistic\", \"Integration of multiple transcriptional inputs at the ORAI3 locus unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A unified structural explanation for how a single Orai3 protein switches between STIM1/STIM2-gated Ca2+-selective, arachidonate/LTC4-gated heteromeric, and 2-APB-gated nonselective dilated states remains undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No high-resolution structure of any Orai3 gated state\", \"Endogenous physiological trigger of pore dilation unknown\", \"Mechanism selecting between channel modes in a given cell type unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [0, 1, 2, 3, 6, 32]},\n      {\"term_id\": \"GO:0005262\", \"supporting_discovery_ids\": []}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 6, 16, 19, 32]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [17]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [8, 14, 22, 33]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [6, 8, 25, 36]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [9, 11, 31]}\n    ],\n    \"complexes\": [\n      \"ARC channel (Orai1/Orai3 heteropentamer, 3:2)\",\n      \"CRAC channel (STIM1/STIM2-Orai3)\"\n    ],\n    \"partners\": [\n      \"STIM1\",\n      \"STIM2\",\n      \"ORAI1\",\n      \"TRPC6\",\n      \"FAK\",\n      \"MARCH8\",\n      \"SEPTIN4\",\n      \"IP3R\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}