{"gene":"FXYD3","run_date":"2026-04-28T17:46:04","timeline":{"discoveries":[{"year":1995,"finding":"FXYD3 (Mat-8) induces hyperpolarization-activated chloride currents when expressed in Xenopus oocytes, functioning as a chloride channel or chloride channel regulator, similar to phospholemman (PLM) but with a distinct cytoplasmic domain lacking PKA/PKC phosphorylation sites.","method":"Electrophysiology in Xenopus oocyte expression system","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — direct functional reconstitution in oocyte expression system, foundational paper with 138 citations","pmids":["7836447"],"is_preprint":false},{"year":2005,"finding":"FXYD3 associates with Na,K-ATPase and decreases both the apparent affinity for Na+ and K+. Unlike other FXYD proteins (type I membrane proteins), mouse FXYD3 may have a second transmembrane-like domain due to a non-cleavable signal peptide. FXYD3 can also associate with H,K-ATPase in Xenopus oocytes but in stomach tissue is associated only with Na,K-ATPase. FXYD3 modulates glycosylation processing of the beta subunit of X,K-ATPase dependent on the signal peptide.","method":"Xenopus oocyte co-expression, electrophysiology, glycosylation analysis","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 — reconstitution in oocytes with functional transport measurements and biochemical characterization, replicated across related studies","pmids":["15743908"],"is_preprint":false},{"year":2006,"finding":"Two human FXYD3 splice variants (short and long) both associate with Na,K-ATPase but not H,K-ATPase or Ca-ATPase. Human FXYD3 has a cleavable signal peptide and adopts type I topology. Short FXYD3 decreases apparent K+ and Na+ affinity of Na,K-ATPase over a large range of membrane potentials, while long FXYD3 decreases apparent K+ affinity only at slightly negative/positive potentials and increases apparent Na+ affinity. Both isoforms induce hyperpolarization-activated current.","method":"Xenopus oocyte co-expression, electrophysiology, co-immunoprecipitation, topology analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — reconstitution with functional transport assays and structural topology determination, multiple orthogonal methods","pmids":["17077088"],"is_preprint":false},{"year":2007,"finding":"Mat-8/FXYD3 co-immunoprecipitates with the Na+/K+-ATPase alpha subunit in colorectal cancer cells. The conserved Gly41 residue in the transmembrane domain is indispensable for association with Na+/K+-ATPase and for plasma membrane localization; Gly41→Arg mutation abolishes both. Cys44→Ala or Cys49→Ala substitutions do not affect association.","method":"Co-immunoprecipitation, site-directed mutagenesis, fluorescent protein tagging and live-cell imaging","journal":"Biological & pharmaceutical bulletin","confidence":"High","confidence_rationale":"Tier 1-2 — reciprocal Co-IP with mutagenesis identifying critical residue","pmids":["17409496"],"is_preprint":false},{"year":2008,"finding":"FXYD3 silencing in Caco-2 intestinal epithelial cells promotes apoptosis and prevents cell differentiation (reduced alkaline phosphatase and villin expression, decreased transepithelial resistance). FXYD3 deficiency increases apparent Na+ and K+ affinities of Na,K-ATPase and decreases maximal Na,K-ATPase activity by reducing its turnover number, accompanied by changes in Na,K-ATPase isozyme expression characteristic of cancer cells.","method":"siRNA knockdown, transepithelial resistance measurement, Na,K-ATPase activity assays, differentiation marker expression","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — clean KD with defined cellular phenotype and direct measurement of Na,K-ATPase activity, multiple orthogonal readouts","pmids":["19109419"],"is_preprint":false},{"year":2009,"finding":"Forced expression of wild-type FXYD3, but not a D19H point mutant (g55c), restores well-demarcated cortical actin distribution in lung cancer cells that had lost FXYD3 expression, indicating FXYD3 plays a role in maintaining cytoskeletal integrity.","method":"Forced expression of wild-type vs. mutant FXYD3, actin staining/imaging","journal":"The American journal of pathology","confidence":"Medium","confidence_rationale":"Tier 2 — mutagenesis with defined phenotypic readout, single lab single study","pmids":["19893046"],"is_preprint":false},{"year":2010,"finding":"Pseudomonas aeruginosa type III effector ExoS directly binds to the transmembrane domain of FXYD3 (the same domain that interacts with Na,K-ATPase), as shown by bacterial two-hybrid screen and pulldown assay. This interaction is proposed to impair Na,K-ATPase-dependent tight junction barrier function, facilitating bacterial translocation across intestinal epithelium.","method":"Bacterial two-hybrid screen, pulldown assay, silkworm infection model","journal":"Infection and immunity","confidence":"Medium","confidence_rationale":"Tier 2-3 — direct binding confirmed by pulldown and two-hybrid, with domain mapping; functional consequence inferred from Na,K-ATPase biology","pmids":["20805335"],"is_preprint":false},{"year":2011,"finding":"TGF-β signaling represses FXYD3 mRNA expression in MCF-10A mammary epithelial cells via Smad3 and the downstream transcriptional repressor ZEB1/δEF1. Silencing ZEB1 up-regulates FXYD3 expression. Smad2 is not required for TGF-β-mediated repression of FXYD3.","method":"siRNA knockdown, TGF-β/TNF-α treatment, pathway inhibitors (TβRI inhibitor, Smad3 inhibitor), RT-PCR","journal":"Biological & pharmaceutical bulletin","confidence":"Medium","confidence_rationale":"Tier 2 — genetic pathway placement with pharmacological and siRNA validation, single lab","pmids":["21372379"],"is_preprint":false},{"year":2014,"finding":"Fxyd3 expression in pancreatic beta-cells is regulated by epigenetic methylation of CpGs in its proximal promoter: gluco-incretin signaling during perinatal development increases promoter methylation, reducing H3K4me3 at the transcriptional start site and silencing Fxyd3. Overexpression of Fxyd3 in beta-cells reduces glucose-induced insulin secretion by acting downstream of plasma membrane depolarization and Ca2+ influx.","method":"Overexpression and knockdown, insulin secretion assays, promoter methylation analysis, ChIP for H3K4me3, transcription reporter assays","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods including gain/loss-of-function, epigenetic analysis, and functional transport assays","pmids":["25058609"],"is_preprint":false},{"year":2015,"finding":"Estrogen and tamoxifen upregulate FXYD3 expression on ER-alpha-positive MCF-7 breast cancer cells in an ER-alpha-dependent manner. ERα associates with the transcription factor ZEB1, and ZEB1 silencing disrupts estrogen- (but not tamoxifen-) induced FXYD3 upregulation, indicating two ER-alpha-dependent mechanisms for FXYD3 regulation.","method":"Flow cytometry with fluorochrome-tagged antibodies, siRNA knockdown, co-immunoprecipitation of ERα and ZEB1","journal":"SpringerPlus","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP and siRNA with defined phenotype, single lab, limited mechanistic depth","pmids":["26090296"],"is_preprint":false},{"year":2016,"finding":"FXYD3 overexpression in MCF-7 breast cancer cells protects Na+/K+-ATPase from oxidative inhibition by facilitating reversal of glutathionylation of the β1 Na+/K+-ATPase subunit. Reducing FXYD3 expression by ~50% increases β1 subunit glutathionylation and reduces Na+/K+-ATPase activity by ~50%. FXYD3 suppression amplifies doxorubicin- and γ-radiation-induced Na+/K+-ATPase inhibition, cell death, and apoptosis.","method":"siRNA knockdown, Na+/K+-ATPase activity assay (colorimetric), glutathionylation measurement, cell viability, caspase 3/7 activation","journal":"Breast cancer research and treatment","confidence":"Medium","confidence_rationale":"Tier 2 — multiple functional assays in single lab with defined molecular mechanism","pmids":["26740212"],"is_preprint":false},{"year":2018,"finding":"FXYD3 interacts with Src and ERα to form an activated signaling complex, triggering non-genomic estrogen signaling. SOX9 directly promotes FXYD3 transcription, and FXYD3 is required for SOX9 nuclear localization, forming a positive regulatory feedback loop. This SOX9/FXYD3/Src axis is required for ER+ breast cancer stem cell maintenance and tamoxifen resistance.","method":"Co-immunoprecipitation, siRNA knockdown, reporter assays, subcellular fractionation/imaging, tamoxifen resistance assays","journal":"Molecular cancer research : MCR","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP showing complex formation and KD with defined phenotype, single lab","pmids":["30206184"],"is_preprint":false},{"year":2022,"finding":"FXYD3 localizes to the basolateral membrane of all airway epithelial cells. siRNA-mediated reduction of FXYD3 decreases ouabain-sensitive short-circuit currents (Na/K-ATPase transport capacity), amiloride-sensitive short-circuit currents, and liquid absorption across intact airway epithelia, demonstrating that FXYD3 facilitates Na+ and liquid absorption by enhancing Na/K-ATPase transport activity.","method":"Single-cell RNA sequencing, immunohistochemistry, siRNA knockdown, short-circuit current measurements with nystatin permeabilization, liquid absorption assay","journal":"American journal of physiology. Cell physiology","confidence":"High","confidence_rationale":"Tier 2 — clean KD with multiple electrophysiological readouts and direct localization, multiple orthogonal methods","pmids":["35993520"],"is_preprint":false},{"year":2023,"finding":"FXYD3 promotes IL-17A signaling in keratinocytes by competitively binding TRAF3, preventing TRAF3 from interacting with IL-17R, thereby promoting the formation of the IL-17R-ACT1 complex. This activates NF-κB and MAPK signaling pathways and promotes proinflammatory factor expression. FXYD3 deletion in keratinocytes attenuates psoriasis-like phenotype in an imiquimod model.","method":"Co-immunoprecipitation, competitive binding assays, siRNA/genetic KO in keratinocytes, in vivo imiquimod psoriasis model, signaling pathway analysis","journal":"Cellular & molecular immunology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP with competition assay, clean KO with defined in vivo and in vitro phenotype, multiple orthogonal methods","pmids":["36693922"],"is_preprint":false},{"year":2025,"finding":"FXYD3 in intestinal goblet cells maintains mucus barrier integrity by interacting with endoplasmic reticulum Ca2+-ATPase SERCA2 to enhance its pump activity. FXYD3 deficiency impairs ER Ca2+ homeostasis and mucin glycosylation, damaging the mucus layer and increasing susceptibility to colitis. Short-chain fatty acids propionate and butyrate promote FXYD3 expression.","method":"Genetic knockout in mouse intestinal epithelium, Co-immunoprecipitation with SERCA2, ER Ca2+ measurements, mucin glycosylation analysis, in vivo colitis model","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 — in vivo KO with defined phenotype, Co-IP identifying binding partner, biochemical functional measurements, multiple orthogonal methods","pmids":["41187059"],"is_preprint":false},{"year":2025,"finding":"FXYD3 directly interacts with IRF7 via its 60-87 amino acid domain. This interaction initiates a positive feedback loop mediated by the cGAS/STING pathway, which is amplified by type I interferon and causes sustained activation of the JAK2/STAT5 signaling pathway, driving malignant progression of intrahepatic cholangiocarcinoma.","method":"Co-immunoprecipitation, domain mapping, single-cell sequencing, spatial transcriptomics, in vitro and in vivo functional assays","journal":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP with domain mapping and pathway placement, single lab","pmids":["41164952"],"is_preprint":false},{"year":2005,"finding":"When expressed in CHO-K1 cells, Mat-8/FXYD3 tagged with DsRed fluorescent protein localizes to intracellular membranes, particularly the endoplasmic reticulum and nuclear envelope, distinct from lysosomes, endosomes, and Golgi bodies.","method":"Stable fluorescent protein tagging, subcellular fractionation by density gradient centrifugation, co-localization with organelle markers","journal":"Biotechnology letters","confidence":"Low","confidence_rationale":"Tier 3 — localization study in non-native cell line, no functional consequence established","pmids":["16132847"],"is_preprint":false}],"current_model":"FXYD3 is a single-pass transmembrane protein that functions primarily as a regulatory subunit (gamma subunit) of Na,K-ATPase, modulating its apparent Na+ and K+ affinities and transport capacity; it also interacts with SERCA2 to enhance ER Ca2+ homeostasis, binds signaling proteins (Src, ERα, IRF7, TRAF3) to modulate estrogen receptor, innate immune (cGAS/STING/JAK2/STAT5), and IL-17A/NF-κB signaling pathways, and protects Na,K-ATPase from oxidative glutathionylation-mediated inhibition, with its expression regulated epigenetically by promoter CpG methylation downstream of gluco-incretin signaling and transcriptionally by TGF-β/Smad3/ZEB1 and SOX9."},"narrative":{"teleology":[{"year":1995,"claim":"The initial functional identity of FXYD3 was established as a membrane protein capable of inducing hyperpolarization-activated chloride currents, placing it in the same functional family as phospholemman but with a distinct regulatory domain.","evidence":"Electrophysiology in Xenopus oocyte heterologous expression system","pmids":["7836447"],"confidence":"High","gaps":["Whether FXYD3 is itself a chloride channel or a regulator of endogenous channels was not resolved","Native tissue function unknown"]},{"year":2005,"claim":"The chloride-channel model was superseded when FXYD3 was shown to be a bona fide Na,K-ATPase-associated regulatory subunit that decreases apparent Na⁺ and K⁺ affinities, redefining its primary molecular function as an ion pump modulator.","evidence":"Xenopus oocyte co-expression with Na,K-ATPase, electrophysiology, glycosylation analysis, and co-immunoprecipitation","pmids":["15743908","17077088"],"confidence":"High","gaps":["Whether splice-variant-specific modulation occurs in native tissues was not demonstrated","Structural basis of differential affinity effects between short and long isoforms unresolved"]},{"year":2007,"claim":"Structure–function dissection identified Gly41 in the transmembrane domain as indispensable for Na,K-ATPase association and plasma membrane targeting, establishing the molecular determinants of the FXYD3–pump interaction.","evidence":"Site-directed mutagenesis, co-immunoprecipitation, and live-cell imaging in colorectal cancer cells","pmids":["17409496"],"confidence":"High","gaps":["No crystal or cryo-EM structure of the FXYD3–Na,K-ATPase complex exists","Whether Gly41 is also required for interactions with other ATPases was not tested"]},{"year":2008,"claim":"Loss-of-function studies demonstrated that FXYD3 is required for intestinal epithelial differentiation, cell survival, and proper Na,K-ATPase turnover number, linking pump regulation to broader epithelial biology.","evidence":"siRNA knockdown in Caco-2 cells with transepithelial resistance, Na,K-ATPase activity assays, and differentiation marker expression","pmids":["19109419"],"confidence":"High","gaps":["In vivo intestinal phenotype of FXYD3 loss not yet established at this point","Mechanism connecting Na,K-ATPase modulation to apoptosis/differentiation unclear"]},{"year":2010,"claim":"The discovery that the Pseudomonas aeruginosa effector ExoS directly binds FXYD3's transmembrane domain — the same interface used for Na,K-ATPase — revealed that pathogens exploit FXYD3 to compromise epithelial barrier function.","evidence":"Bacterial two-hybrid screen, pulldown assay, domain mapping","pmids":["20805335"],"confidence":"Medium","gaps":["Functional consequence of ExoS–FXYD3 interaction on Na,K-ATPase activity was inferred, not directly measured","Relevance in mammalian infection models not confirmed"]},{"year":2011,"claim":"Transcriptional regulation of FXYD3 was placed downstream of TGF-β/Smad3/ZEB1, explaining how epithelial-to-mesenchymal transition signals silence FXYD3.","evidence":"siRNA knockdown, TGF-β treatment, pathway inhibitors, and RT-PCR in MCF-10A cells","pmids":["21372379"],"confidence":"Medium","gaps":["Direct ZEB1 binding to the FXYD3 promoter not shown by ChIP","In vivo relevance to EMT programs not established"]},{"year":2014,"claim":"Epigenetic regulation of FXYD3 was demonstrated in pancreatic β-cells, where gluco-incretin-driven promoter CpG methylation silences FXYD3, and its overexpression reduces glucose-stimulated insulin secretion downstream of Ca²⁺ influx.","evidence":"Overexpression, knockdown, insulin secretion assays, promoter methylation analysis, ChIP for H3K4me3","pmids":["25058609"],"confidence":"High","gaps":["The specific ion transport target (Na,K-ATPase or other) mediating insulin secretion suppression was not identified","Phenotype of Fxyd3 deletion in β-cells in vivo not shown"]},{"year":2016,"claim":"A cytoprotective mechanism was uncovered: FXYD3 facilitates reversal of oxidative glutathionylation on the Na,K-ATPase β1 subunit, protecting pump activity under oxidative stress and conferring resistance to doxorubicin- and radiation-induced cell death.","evidence":"siRNA knockdown, Na,K-ATPase activity assay, glutathionylation measurement, caspase activation in MCF-7 breast cancer cells","pmids":["26740212"],"confidence":"Medium","gaps":["Whether FXYD3 directly catalyzes deglutathionylation or recruits a thiol reductase is unknown","Relevance to therapy resistance in vivo not tested"]},{"year":2018,"claim":"FXYD3 was shown to function beyond ion transport as a signaling scaffold: it forms a complex with Src and ERα for non-genomic estrogen signaling, and participates in a positive feedback loop with SOX9 that maintains breast cancer stem cells and tamoxifen resistance.","evidence":"Co-immunoprecipitation, siRNA, reporter assays, subcellular fractionation in ER+ breast cancer cells","pmids":["30206184"],"confidence":"Medium","gaps":["Direct binding interface between FXYD3 and Src not mapped","SOX9 binding site on FXYD3 promoter confirmed by reporter but not by endogenous ChIP-seq","Independent replication in other ER+ models lacking"]},{"year":2022,"claim":"Physiological relevance of FXYD3 as a Na,K-ATPase modulator was demonstrated in intact human airway epithelia, where FXYD3 knockdown reduced Na,K-ATPase transport capacity, amiloride-sensitive Na⁺ absorption, and transepithelial liquid clearance.","evidence":"scRNA-seq, immunohistochemistry, siRNA knockdown, short-circuit current measurements with nystatin permeabilization, liquid absorption assay","pmids":["35993520"],"confidence":"High","gaps":["In vivo airway phenotype of FXYD3 deletion not tested","Whether FXYD3 modulates ENaC directly or only via Na,K-ATPase is unclear"]},{"year":2023,"claim":"An entirely new signaling axis was revealed: FXYD3 competitively binds TRAF3 to relieve its inhibition of IL-17R, promoting ACT1 recruitment and NF-κB/MAPK activation in keratinocytes, with genetic deletion attenuating psoriasis-like inflammation in vivo.","evidence":"Co-immunoprecipitation, competitive binding assays, genetic KO in keratinocytes, imiquimod psoriasis model","pmids":["36693922"],"confidence":"High","gaps":["Structural basis of FXYD3–TRAF3 interaction not determined","Whether this mechanism operates in other IL-17-responsive cell types is unknown"]},{"year":2025,"claim":"FXYD3 was shown to interact with SERCA2 in intestinal goblet cells, enhancing ER Ca²⁺ pump activity to support mucin glycosylation and mucus barrier integrity, establishing a second P-type ATPase partnership beyond Na,K-ATPase.","evidence":"Intestinal epithelium-specific genetic KO in mice, Co-IP with SERCA2, ER Ca²⁺ measurements, colitis model","pmids":["41187059"],"confidence":"High","gaps":["Whether FXYD3 modulates SERCA2 affinity parameters analogously to Na,K-ATPase is not resolved","Direct structural evidence for the FXYD3–SERCA2 interface absent"]},{"year":2025,"claim":"FXYD3 was found to bind IRF7 via its 60–87 amino acid domain, initiating a cGAS/STING-mediated positive feedback loop that sustains JAK2/STAT5 signaling and drives intrahepatic cholangiocarcinoma progression.","evidence":"Co-immunoprecipitation with domain mapping, single-cell and spatial transcriptomics, in vivo tumor models","pmids":["41164952"],"confidence":"Medium","gaps":["Independent validation of FXYD3–IRF7 interaction in a second lab awaited","Whether this axis operates in normal cholangiocytes or only in cancer is unclear","Causal chain from FXYD3–IRF7 binding to cGAS/STING activation not fully delineated"]},{"year":null,"claim":"Key unresolved questions include the structural basis of FXYD3 interactions with its multiple ATPase and signaling partners, whether its ion-transport and signaling functions are mechanistically linked or independent, and how tissue-specific expression patterns dictate which of its diverse functions predominate.","evidence":"","pmids":[],"confidence":"High","gaps":["No high-resolution structure of FXYD3 in complex with any partner","Whether FXYD3's signaling roles (TRAF3, Src, IRF7) require or are independent of ATPase association is unknown","Comprehensive in vivo phenotyping across tissues using conditional knockouts is incomplete"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,2,4,10,12,14]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[11,13,15]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[3,5,12]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[14]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[13,15]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[11,13,15]}],"complexes":["Na,K-ATPase"],"partners":["ATP1A1","SERCA2","TRAF3","SRC","ESR1","IRF7","SOX9","ZEB1"],"other_free_text":[]},"mechanistic_narrative":"FXYD3 is a single-pass transmembrane protein that functions as a regulatory subunit of P-type ATPases, modulating ion transport, epithelial homeostasis, and cell signaling across diverse tissues. It associates with Na,K-ATPase to alter apparent Na⁺ and K⁺ affinities and maximal pump activity, thereby controlling transepithelial Na⁺ and liquid absorption in airway and intestinal epithelia, and it interacts with SERCA2 in goblet cells to enhance ER Ca²⁺ homeostasis and mucin glycosylation required for mucus barrier integrity [PMID:17077088, PMID:19109419, PMID:35993520, PMID:41187059]. FXYD3 protects the Na,K-ATPase β1 subunit from oxidative glutathionylation-mediated inhibition, and a conserved Gly41 residue in its transmembrane domain is indispensable for pump association and plasma membrane targeting [PMID:17409496, PMID:26740212]. Beyond ion transport, FXYD3 participates in signaling by competitively sequestering TRAF3 to promote IL-17R/ACT1-dependent NF-κB activation in keratinocytes, by forming a Src/ERα complex that drives non-genomic estrogen signaling via a SOX9-dependent transcriptional feedback loop in breast cancer, and by binding IRF7 to sustain cGAS/STING-JAK2/STAT5 signaling in cholangiocarcinoma [PMID:36693922, PMID:30206184, PMID:41164952]."},"prefetch_data":{"uniprot":{"accession":"Q14802","full_name":"FXYD domain-containing ion transport regulator 3","aliases":["Chloride conductance inducer protein Mat-8","Mammary tumor 8 kDa protein","Phospholemman-like","Sodium/potassium-transporting ATPase subunit FXYD3"],"length_aa":87,"mass_kda":9.3,"function":"Associates with and regulates the activity of the sodium/potassium-transporting ATPase (NKA) which transports Na(+) out of the cell and K(+) into the cell (PubMed:17077088). Reduces glutathionylation of the NKA beta-1 subunit ATP1B1, thus reversing glutathionylation-mediated inhibition of ATP1B1 (PubMed:21454534). Induces a hyperpolarization-activated chloride current when expressed in Xenopus oocytes (PubMed:7836447) Decreases the apparent K+ and Na+ affinity of the sodium/potassium-transporting ATPase over a large range of membrane potentials Decreases the apparent K+ affinity of the sodium/potassium-transporting ATPase only at slightly negative and positive membrane potentials and increases the apparent Na+ affinity over a large range of membrane potentials","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q14802/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/FXYD3","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/FXYD3","total_profiled":1310},"omim":[{"mim_id":"604996","title":"FXYD DOMAIN-CONTAINING ION TRANSPORT REGULATOR 3; FXYD3","url":"https://www.omim.org/entry/604996"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Plasma membrane","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"intestine","ntpm":1697.0}],"url":"https://www.proteinatlas.org/search/FXYD3"},"hgnc":{"alias_symbol":["MAT-8"],"prev_symbol":["PLML"]},"alphafold":{"accession":"Q14802","domains":[{"cath_id":"1.20.58","chopping":"2-63","consensus_level":"medium","plddt":76.9376,"start":2,"end":63}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q14802","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q14802-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q14802-F1-predicted_aligned_error_v6.png","plddt_mean":68.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=FXYD3","jax_strain_url":"https://www.jax.org/strain/search?query=FXYD3"},"sequence":{"accession":"Q14802","fasta_url":"https://rest.uniprot.org/uniprotkb/Q14802.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q14802/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q14802"}},"corpus_meta":[{"pmid":"7836447","id":"PMC_7836447","title":"Mat-8, a novel phospholemman-like protein expressed in human breast tumors, induces a chloride conductance in Xenopus oocytes.","date":"1995","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/7836447","citation_count":138,"is_preprint":false},{"pmid":"14654946","id":"PMC_14654946","title":"Up-regulated expression of the MAT-8 gene in prostate cancer and its siRNA-mediated inhibition of expression induces a decrease in proliferation of human prostate carcinoma cells.","date":"2004","source":"International journal of oncology","url":"https://pubmed.ncbi.nlm.nih.gov/14654946","citation_count":82,"is_preprint":false},{"pmid":"20805335","id":"PMC_20805335","title":"Translocation of Pseudomonas aeruginosa from the intestinal tract is mediated by the binding of ExoS to an Na,K-ATPase regulator, FXYD3.","date":"2010","source":"Infection and immunity","url":"https://pubmed.ncbi.nlm.nih.gov/20805335","citation_count":70,"is_preprint":false},{"pmid":"16003754","id":"PMC_16003754","title":"FXYD3 is overexpressed in pancreatic ductal adenocarcinoma and influences pancreatic cancer cell growth.","date":"2006","source":"International journal of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/16003754","citation_count":63,"is_preprint":false},{"pmid":"15743908","id":"PMC_15743908","title":"FXYD3 (Mat-8), a new regulator of Na,K-ATPase.","date":"2005","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/15743908","citation_count":61,"is_preprint":false},{"pmid":"30206184","id":"PMC_30206184","title":"SOX9/FXYD3/Src Axis Is Critical for ER+ Breast Cancer Stem Cell Function.","date":"2018","source":"Molecular cancer research : MCR","url":"https://pubmed.ncbi.nlm.nih.gov/30206184","citation_count":46,"is_preprint":false},{"pmid":"33350586","id":"PMC_33350586","title":"Extracellular vesicles-encapsulated let-7i shed from bone mesenchymal stem cells suppress lung cancer via KDM3A/DCLK1/FXYD3 axis.","date":"2020","source":"Journal of cellular and molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/33350586","citation_count":46,"is_preprint":false},{"pmid":"19893046","id":"PMC_19893046","title":"Down-regulation of FXYD3 expression in human lung cancers: its mechanism and potential role in carcinogenesis.","date":"2009","source":"The American journal of pathology","url":"https://pubmed.ncbi.nlm.nih.gov/19893046","citation_count":36,"is_preprint":false},{"pmid":"33621431","id":"PMC_33621431","title":"KDM5A silencing transcriptionally suppresses the FXYD3-PI3K/AKT axis to inhibit angiogenesis in hepatocellular cancer via miR-433 up-regulation.","date":"2021","source":"Journal of cellular and molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/33621431","citation_count":29,"is_preprint":false},{"pmid":"36693922","id":"PMC_36693922","title":"FXYD3 enhances IL-17A signaling to promote psoriasis by competitively binding TRAF3 in keratinocytes.","date":"2023","source":"Cellular & molecular immunology","url":"https://pubmed.ncbi.nlm.nih.gov/36693922","citation_count":28,"is_preprint":false},{"pmid":"17077088","id":"PMC_17077088","title":"Structural and functional properties of two human FXYD3 (Mat-8) isoforms.","date":"2006","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17077088","citation_count":26,"is_preprint":false},{"pmid":"19955746","id":"PMC_19955746","title":"Expression of FXYD3 protein in relation to biological and clinicopathological variables in colorectal cancers.","date":"2009","source":"Chemotherapy","url":"https://pubmed.ncbi.nlm.nih.gov/19955746","citation_count":26,"is_preprint":false},{"pmid":"19571376","id":"PMC_19571376","title":"FXYD3 protein involved in tumor cell proliferation is overproduced in human breast cancer tissues.","date":"2009","source":"Biological & pharmaceutical bulletin","url":"https://pubmed.ncbi.nlm.nih.gov/19571376","citation_count":23,"is_preprint":false},{"pmid":"17409496","id":"PMC_17409496","title":"Interaction of Mat-8 (FXYD-3) with Na+/K+-ATPase in colorectal cancer cells.","date":"2007","source":"Biological & pharmaceutical bulletin","url":"https://pubmed.ncbi.nlm.nih.gov/17409496","citation_count":23,"is_preprint":false},{"pmid":"21372379","id":"PMC_21372379","title":"Down-regulation of FXYD3 is induced by transforming growth factor-β signaling via ZEB1/δEF1 in human mammary epithelial cells.","date":"2011","source":"Biological & pharmaceutical bulletin","url":"https://pubmed.ncbi.nlm.nih.gov/21372379","citation_count":22,"is_preprint":false},{"pmid":"19109419","id":"PMC_19109419","title":"A link between FXYD3 (Mat-8)-mediated Na,K-ATPase regulation and differentiation of Caco-2 intestinal epithelial cells.","date":"2008","source":"Molecular biology of the 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cell subpopulation with features of drug-tolerant persisters.","date":"2023","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/37966117","citation_count":16,"is_preprint":false},{"pmid":"25013464","id":"PMC_25013464","title":"Expression and clinical significance of FXYD3 in endometrial cancer.","date":"2014","source":"Oncology letters","url":"https://pubmed.ncbi.nlm.nih.gov/25013464","citation_count":16,"is_preprint":false},{"pmid":"37535601","id":"PMC_37535601","title":"TGM2, HMGA2, FXYD3, and LGALS4 genes as biomarkers in acquired oxaliplatin resistance of human colorectal cancer: A systems biology approach.","date":"2023","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/37535601","citation_count":16,"is_preprint":false},{"pmid":"26740212","id":"PMC_26740212","title":"Silencing overexpression of FXYD3 protein in breast cancer cells amplifies effects of doxorubicin and γ-radiation on Na(+)/K(+)-ATPase and cell survival.","date":"2016","source":"Breast cancer research and treatment","url":"https://pubmed.ncbi.nlm.nih.gov/26740212","citation_count":14,"is_preprint":false},{"pmid":"20112499","id":"PMC_20112499","title":"FXYD3 expression in gliomas and its clinicopathological significance.","date":"2009","source":"Oncology research","url":"https://pubmed.ncbi.nlm.nih.gov/20112499","citation_count":13,"is_preprint":false},{"pmid":"24167366","id":"PMC_24167366","title":"Overexpression of FXYD-3 is involved in the tumorigenesis and development of esophageal squamous cell carcinoma.","date":"2013","source":"Disease markers","url":"https://pubmed.ncbi.nlm.nih.gov/24167366","citation_count":13,"is_preprint":false},{"pmid":"32162907","id":"PMC_32162907","title":"Instrument-Free Detection of FXYD3 Using Vial-Based Immunosensor for Earlier and Faster Urothelial Carcinoma Diagnosis.","date":"2020","source":"ACS sensors","url":"https://pubmed.ncbi.nlm.nih.gov/32162907","citation_count":12,"is_preprint":false},{"pmid":"25058609","id":"PMC_25058609","title":"Gluco-incretins regulate beta-cell glucose competence by epigenetic silencing of Fxyd3 expression.","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/25058609","citation_count":11,"is_preprint":false},{"pmid":"25920992","id":"PMC_25920992","title":"External validation of FXYD3 and KRT20 as predictive biomarkers for the presence of micrometastasis in muscle invasive bladder cancer lymph nodes.","date":"2015","source":"Actas urologicas espanolas","url":"https://pubmed.ncbi.nlm.nih.gov/25920992","citation_count":10,"is_preprint":false},{"pmid":"14694902","id":"PMC_14694902","title":"Fxyd3 and Lgi4 expression in the adult mouse: a case of endogenous antisense expression.","date":"2003","source":"Mammalian genome : official journal of the International Mammalian Genome Society","url":"https://pubmed.ncbi.nlm.nih.gov/14694902","citation_count":10,"is_preprint":false},{"pmid":"16132847","id":"PMC_16132847","title":"Stable expression and visualization of Mat-8 (FXYD-3) tagged with a fluorescent protein in Chinese hamster ovary (CHO)-K1 cells.","date":"2005","source":"Biotechnology letters","url":"https://pubmed.ncbi.nlm.nih.gov/16132847","citation_count":10,"is_preprint":false},{"pmid":"26090296","id":"PMC_26090296","title":"Estrogen and tamoxifen up-regulate FXYD3 on breast cancer cells: assessing the differential roles of ER α and ZEB1.","date":"2015","source":"SpringerPlus","url":"https://pubmed.ncbi.nlm.nih.gov/26090296","citation_count":9,"is_preprint":false},{"pmid":"27104167","id":"PMC_27104167","title":"FXYD-3 expression in relation to local recurrence of rectal cancer.","date":"2016","source":"Radiation oncology 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Cell physiology","url":"https://pubmed.ncbi.nlm.nih.gov/35993520","citation_count":4,"is_preprint":false},{"pmid":"39417182","id":"PMC_39417182","title":"Uncovering the role of FXYD3 as a potential oncogene and early biomarker in pancreatic cancer.","date":"2024","source":"American journal of cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/39417182","citation_count":4,"is_preprint":false},{"pmid":"41187059","id":"PMC_41187059","title":"Goblet cell-expressed microprotein FXYD3 determines gut homeostasis by maintaining mucus barrier integrity.","date":"2025","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/41187059","citation_count":1,"is_preprint":false},{"pmid":"41164952","id":"PMC_41164952","title":"FXYD3 Promotes Tumor Progression by Binding With IRF7 to Regulate JAK2/STAT5 Signaling in Intrahepatic Cholangiocarcinoma.","date":"2025","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/41164952","citation_count":1,"is_preprint":false},{"pmid":"39783776","id":"PMC_39783776","title":"FXYD3 Is Frequently Expressed in Pancreatic Ductal Adenocarcinoma but Does Not Predict Survival.","date":"2025","source":"Cancer medicine","url":"https://pubmed.ncbi.nlm.nih.gov/39783776","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":20259,"output_tokens":4362,"usd":0.063104},"stage2":{"model":"claude-opus-4-6","input_tokens":7874,"output_tokens":3594,"usd":0.19383},"total_usd":0.256934,"stage1_batch_id":"msgbatch_0118RScg3WGaGkaZRtWZbtce","stage2_batch_id":"msgbatch_01Cpfzvtbj42aqp5oYZ5Te2s","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1995,\n      \"finding\": \"FXYD3 (Mat-8) induces hyperpolarization-activated chloride currents when expressed in Xenopus oocytes, functioning as a chloride channel or chloride channel regulator, similar to phospholemman (PLM) but with a distinct cytoplasmic domain lacking PKA/PKC phosphorylation sites.\",\n      \"method\": \"Electrophysiology in Xenopus oocyte expression system\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct functional reconstitution in oocyte expression system, foundational paper with 138 citations\",\n      \"pmids\": [\"7836447\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"FXYD3 associates with Na,K-ATPase and decreases both the apparent affinity for Na+ and K+. Unlike other FXYD proteins (type I membrane proteins), mouse FXYD3 may have a second transmembrane-like domain due to a non-cleavable signal peptide. FXYD3 can also associate with H,K-ATPase in Xenopus oocytes but in stomach tissue is associated only with Na,K-ATPase. FXYD3 modulates glycosylation processing of the beta subunit of X,K-ATPase dependent on the signal peptide.\",\n      \"method\": \"Xenopus oocyte co-expression, electrophysiology, glycosylation analysis\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstitution in oocytes with functional transport measurements and biochemical characterization, replicated across related studies\",\n      \"pmids\": [\"15743908\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Two human FXYD3 splice variants (short and long) both associate with Na,K-ATPase but not H,K-ATPase or Ca-ATPase. Human FXYD3 has a cleavable signal peptide and adopts type I topology. Short FXYD3 decreases apparent K+ and Na+ affinity of Na,K-ATPase over a large range of membrane potentials, while long FXYD3 decreases apparent K+ affinity only at slightly negative/positive potentials and increases apparent Na+ affinity. Both isoforms induce hyperpolarization-activated current.\",\n      \"method\": \"Xenopus oocyte co-expression, electrophysiology, co-immunoprecipitation, topology analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstitution with functional transport assays and structural topology determination, multiple orthogonal methods\",\n      \"pmids\": [\"17077088\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Mat-8/FXYD3 co-immunoprecipitates with the Na+/K+-ATPase alpha subunit in colorectal cancer cells. The conserved Gly41 residue in the transmembrane domain is indispensable for association with Na+/K+-ATPase and for plasma membrane localization; Gly41→Arg mutation abolishes both. Cys44→Ala or Cys49→Ala substitutions do not affect association.\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis, fluorescent protein tagging and live-cell imaging\",\n      \"journal\": \"Biological & pharmaceutical bulletin\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — reciprocal Co-IP with mutagenesis identifying critical residue\",\n      \"pmids\": [\"17409496\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"FXYD3 silencing in Caco-2 intestinal epithelial cells promotes apoptosis and prevents cell differentiation (reduced alkaline phosphatase and villin expression, decreased transepithelial resistance). FXYD3 deficiency increases apparent Na+ and K+ affinities of Na,K-ATPase and decreases maximal Na,K-ATPase activity by reducing its turnover number, accompanied by changes in Na,K-ATPase isozyme expression characteristic of cancer cells.\",\n      \"method\": \"siRNA knockdown, transepithelial resistance measurement, Na,K-ATPase activity assays, differentiation marker expression\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KD with defined cellular phenotype and direct measurement of Na,K-ATPase activity, multiple orthogonal readouts\",\n      \"pmids\": [\"19109419\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Forced expression of wild-type FXYD3, but not a D19H point mutant (g55c), restores well-demarcated cortical actin distribution in lung cancer cells that had lost FXYD3 expression, indicating FXYD3 plays a role in maintaining cytoskeletal integrity.\",\n      \"method\": \"Forced expression of wild-type vs. mutant FXYD3, actin staining/imaging\",\n      \"journal\": \"The American journal of pathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mutagenesis with defined phenotypic readout, single lab single study\",\n      \"pmids\": [\"19893046\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Pseudomonas aeruginosa type III effector ExoS directly binds to the transmembrane domain of FXYD3 (the same domain that interacts with Na,K-ATPase), as shown by bacterial two-hybrid screen and pulldown assay. This interaction is proposed to impair Na,K-ATPase-dependent tight junction barrier function, facilitating bacterial translocation across intestinal epithelium.\",\n      \"method\": \"Bacterial two-hybrid screen, pulldown assay, silkworm infection model\",\n      \"journal\": \"Infection and immunity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — direct binding confirmed by pulldown and two-hybrid, with domain mapping; functional consequence inferred from Na,K-ATPase biology\",\n      \"pmids\": [\"20805335\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"TGF-β signaling represses FXYD3 mRNA expression in MCF-10A mammary epithelial cells via Smad3 and the downstream transcriptional repressor ZEB1/δEF1. Silencing ZEB1 up-regulates FXYD3 expression. Smad2 is not required for TGF-β-mediated repression of FXYD3.\",\n      \"method\": \"siRNA knockdown, TGF-β/TNF-α treatment, pathway inhibitors (TβRI inhibitor, Smad3 inhibitor), RT-PCR\",\n      \"journal\": \"Biological & pharmaceutical bulletin\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic pathway placement with pharmacological and siRNA validation, single lab\",\n      \"pmids\": [\"21372379\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Fxyd3 expression in pancreatic beta-cells is regulated by epigenetic methylation of CpGs in its proximal promoter: gluco-incretin signaling during perinatal development increases promoter methylation, reducing H3K4me3 at the transcriptional start site and silencing Fxyd3. Overexpression of Fxyd3 in beta-cells reduces glucose-induced insulin secretion by acting downstream of plasma membrane depolarization and Ca2+ influx.\",\n      \"method\": \"Overexpression and knockdown, insulin secretion assays, promoter methylation analysis, ChIP for H3K4me3, transcription reporter assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods including gain/loss-of-function, epigenetic analysis, and functional transport assays\",\n      \"pmids\": [\"25058609\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Estrogen and tamoxifen upregulate FXYD3 expression on ER-alpha-positive MCF-7 breast cancer cells in an ER-alpha-dependent manner. ERα associates with the transcription factor ZEB1, and ZEB1 silencing disrupts estrogen- (but not tamoxifen-) induced FXYD3 upregulation, indicating two ER-alpha-dependent mechanisms for FXYD3 regulation.\",\n      \"method\": \"Flow cytometry with fluorochrome-tagged antibodies, siRNA knockdown, co-immunoprecipitation of ERα and ZEB1\",\n      \"journal\": \"SpringerPlus\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP and siRNA with defined phenotype, single lab, limited mechanistic depth\",\n      \"pmids\": [\"26090296\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"FXYD3 overexpression in MCF-7 breast cancer cells protects Na+/K+-ATPase from oxidative inhibition by facilitating reversal of glutathionylation of the β1 Na+/K+-ATPase subunit. Reducing FXYD3 expression by ~50% increases β1 subunit glutathionylation and reduces Na+/K+-ATPase activity by ~50%. FXYD3 suppression amplifies doxorubicin- and γ-radiation-induced Na+/K+-ATPase inhibition, cell death, and apoptosis.\",\n      \"method\": \"siRNA knockdown, Na+/K+-ATPase activity assay (colorimetric), glutathionylation measurement, cell viability, caspase 3/7 activation\",\n      \"journal\": \"Breast cancer research and treatment\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple functional assays in single lab with defined molecular mechanism\",\n      \"pmids\": [\"26740212\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"FXYD3 interacts with Src and ERα to form an activated signaling complex, triggering non-genomic estrogen signaling. SOX9 directly promotes FXYD3 transcription, and FXYD3 is required for SOX9 nuclear localization, forming a positive regulatory feedback loop. This SOX9/FXYD3/Src axis is required for ER+ breast cancer stem cell maintenance and tamoxifen resistance.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, reporter assays, subcellular fractionation/imaging, tamoxifen resistance assays\",\n      \"journal\": \"Molecular cancer research : MCR\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP showing complex formation and KD with defined phenotype, single lab\",\n      \"pmids\": [\"30206184\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"FXYD3 localizes to the basolateral membrane of all airway epithelial cells. siRNA-mediated reduction of FXYD3 decreases ouabain-sensitive short-circuit currents (Na/K-ATPase transport capacity), amiloride-sensitive short-circuit currents, and liquid absorption across intact airway epithelia, demonstrating that FXYD3 facilitates Na+ and liquid absorption by enhancing Na/K-ATPase transport activity.\",\n      \"method\": \"Single-cell RNA sequencing, immunohistochemistry, siRNA knockdown, short-circuit current measurements with nystatin permeabilization, liquid absorption assay\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KD with multiple electrophysiological readouts and direct localization, multiple orthogonal methods\",\n      \"pmids\": [\"35993520\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"FXYD3 promotes IL-17A signaling in keratinocytes by competitively binding TRAF3, preventing TRAF3 from interacting with IL-17R, thereby promoting the formation of the IL-17R-ACT1 complex. This activates NF-κB and MAPK signaling pathways and promotes proinflammatory factor expression. FXYD3 deletion in keratinocytes attenuates psoriasis-like phenotype in an imiquimod model.\",\n      \"method\": \"Co-immunoprecipitation, competitive binding assays, siRNA/genetic KO in keratinocytes, in vivo imiquimod psoriasis model, signaling pathway analysis\",\n      \"journal\": \"Cellular & molecular immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP with competition assay, clean KO with defined in vivo and in vitro phenotype, multiple orthogonal methods\",\n      \"pmids\": [\"36693922\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"FXYD3 in intestinal goblet cells maintains mucus barrier integrity by interacting with endoplasmic reticulum Ca2+-ATPase SERCA2 to enhance its pump activity. FXYD3 deficiency impairs ER Ca2+ homeostasis and mucin glycosylation, damaging the mucus layer and increasing susceptibility to colitis. Short-chain fatty acids propionate and butyrate promote FXYD3 expression.\",\n      \"method\": \"Genetic knockout in mouse intestinal epithelium, Co-immunoprecipitation with SERCA2, ER Ca2+ measurements, mucin glycosylation analysis, in vivo colitis model\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo KO with defined phenotype, Co-IP identifying binding partner, biochemical functional measurements, multiple orthogonal methods\",\n      \"pmids\": [\"41187059\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"FXYD3 directly interacts with IRF7 via its 60-87 amino acid domain. This interaction initiates a positive feedback loop mediated by the cGAS/STING pathway, which is amplified by type I interferon and causes sustained activation of the JAK2/STAT5 signaling pathway, driving malignant progression of intrahepatic cholangiocarcinoma.\",\n      \"method\": \"Co-immunoprecipitation, domain mapping, single-cell sequencing, spatial transcriptomics, in vitro and in vivo functional assays\",\n      \"journal\": \"Advanced science (Weinheim, Baden-Wurttemberg, Germany)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP with domain mapping and pathway placement, single lab\",\n      \"pmids\": [\"41164952\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"When expressed in CHO-K1 cells, Mat-8/FXYD3 tagged with DsRed fluorescent protein localizes to intracellular membranes, particularly the endoplasmic reticulum and nuclear envelope, distinct from lysosomes, endosomes, and Golgi bodies.\",\n      \"method\": \"Stable fluorescent protein tagging, subcellular fractionation by density gradient centrifugation, co-localization with organelle markers\",\n      \"journal\": \"Biotechnology letters\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — localization study in non-native cell line, no functional consequence established\",\n      \"pmids\": [\"16132847\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"FXYD3 is a single-pass transmembrane protein that functions primarily as a regulatory subunit (gamma subunit) of Na,K-ATPase, modulating its apparent Na+ and K+ affinities and transport capacity; it also interacts with SERCA2 to enhance ER Ca2+ homeostasis, binds signaling proteins (Src, ERα, IRF7, TRAF3) to modulate estrogen receptor, innate immune (cGAS/STING/JAK2/STAT5), and IL-17A/NF-κB signaling pathways, and protects Na,K-ATPase from oxidative glutathionylation-mediated inhibition, with its expression regulated epigenetically by promoter CpG methylation downstream of gluco-incretin signaling and transcriptionally by TGF-β/Smad3/ZEB1 and SOX9.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"FXYD3 is a single-pass transmembrane protein that functions as a regulatory subunit of P-type ATPases, modulating ion transport, epithelial homeostasis, and cell signaling across diverse tissues. It associates with Na,K-ATPase to alter apparent Na⁺ and K⁺ affinities and maximal pump activity, thereby controlling transepithelial Na⁺ and liquid absorption in airway and intestinal epithelia, and it interacts with SERCA2 in goblet cells to enhance ER Ca²⁺ homeostasis and mucin glycosylation required for mucus barrier integrity [PMID:17077088, PMID:19109419, PMID:35993520, PMID:41187059]. FXYD3 protects the Na,K-ATPase β1 subunit from oxidative glutathionylation-mediated inhibition, and a conserved Gly41 residue in its transmembrane domain is indispensable for pump association and plasma membrane targeting [PMID:17409496, PMID:26740212]. Beyond ion transport, FXYD3 participates in signaling by competitively sequestering TRAF3 to promote IL-17R/ACT1-dependent NF-κB activation in keratinocytes, by forming a Src/ERα complex that drives non-genomic estrogen signaling via a SOX9-dependent transcriptional feedback loop in breast cancer, and by binding IRF7 to sustain cGAS/STING-JAK2/STAT5 signaling in cholangiocarcinoma [PMID:36693922, PMID:30206184, PMID:41164952].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"The initial functional identity of FXYD3 was established as a membrane protein capable of inducing hyperpolarization-activated chloride currents, placing it in the same functional family as phospholemman but with a distinct regulatory domain.\",\n      \"evidence\": \"Electrophysiology in Xenopus oocyte heterologous expression system\",\n      \"pmids\": [\"7836447\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether FXYD3 is itself a chloride channel or a regulator of endogenous channels was not resolved\",\n        \"Native tissue function unknown\"\n      ]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"The chloride-channel model was superseded when FXYD3 was shown to be a bona fide Na,K-ATPase-associated regulatory subunit that decreases apparent Na⁺ and K⁺ affinities, redefining its primary molecular function as an ion pump modulator.\",\n      \"evidence\": \"Xenopus oocyte co-expression with Na,K-ATPase, electrophysiology, glycosylation analysis, and co-immunoprecipitation\",\n      \"pmids\": [\"15743908\", \"17077088\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether splice-variant-specific modulation occurs in native tissues was not demonstrated\",\n        \"Structural basis of differential affinity effects between short and long isoforms unresolved\"\n      ]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Structure–function dissection identified Gly41 in the transmembrane domain as indispensable for Na,K-ATPase association and plasma membrane targeting, establishing the molecular determinants of the FXYD3–pump interaction.\",\n      \"evidence\": \"Site-directed mutagenesis, co-immunoprecipitation, and live-cell imaging in colorectal cancer cells\",\n      \"pmids\": [\"17409496\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No crystal or cryo-EM structure of the FXYD3–Na,K-ATPase complex exists\",\n        \"Whether Gly41 is also required for interactions with other ATPases was not tested\"\n      ]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Loss-of-function studies demonstrated that FXYD3 is required for intestinal epithelial differentiation, cell survival, and proper Na,K-ATPase turnover number, linking pump regulation to broader epithelial biology.\",\n      \"evidence\": \"siRNA knockdown in Caco-2 cells with transepithelial resistance, Na,K-ATPase activity assays, and differentiation marker expression\",\n      \"pmids\": [\"19109419\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"In vivo intestinal phenotype of FXYD3 loss not yet established at this point\",\n        \"Mechanism connecting Na,K-ATPase modulation to apoptosis/differentiation unclear\"\n      ]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"The discovery that the Pseudomonas aeruginosa effector ExoS directly binds FXYD3's transmembrane domain — the same interface used for Na,K-ATPase — revealed that pathogens exploit FXYD3 to compromise epithelial barrier function.\",\n      \"evidence\": \"Bacterial two-hybrid screen, pulldown assay, domain mapping\",\n      \"pmids\": [\"20805335\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Functional consequence of ExoS–FXYD3 interaction on Na,K-ATPase activity was inferred, not directly measured\",\n        \"Relevance in mammalian infection models not confirmed\"\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Transcriptional regulation of FXYD3 was placed downstream of TGF-β/Smad3/ZEB1, explaining how epithelial-to-mesenchymal transition signals silence FXYD3.\",\n      \"evidence\": \"siRNA knockdown, TGF-β treatment, pathway inhibitors, and RT-PCR in MCF-10A cells\",\n      \"pmids\": [\"21372379\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct ZEB1 binding to the FXYD3 promoter not shown by ChIP\",\n        \"In vivo relevance to EMT programs not established\"\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Epigenetic regulation of FXYD3 was demonstrated in pancreatic β-cells, where gluco-incretin-driven promoter CpG methylation silences FXYD3, and its overexpression reduces glucose-stimulated insulin secretion downstream of Ca²⁺ influx.\",\n      \"evidence\": \"Overexpression, knockdown, insulin secretion assays, promoter methylation analysis, ChIP for H3K4me3\",\n      \"pmids\": [\"25058609\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The specific ion transport target (Na,K-ATPase or other) mediating insulin secretion suppression was not identified\",\n        \"Phenotype of Fxyd3 deletion in β-cells in vivo not shown\"\n      ]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"A cytoprotective mechanism was uncovered: FXYD3 facilitates reversal of oxidative glutathionylation on the Na,K-ATPase β1 subunit, protecting pump activity under oxidative stress and conferring resistance to doxorubicin- and radiation-induced cell death.\",\n      \"evidence\": \"siRNA knockdown, Na,K-ATPase activity assay, glutathionylation measurement, caspase activation in MCF-7 breast cancer cells\",\n      \"pmids\": [\"26740212\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether FXYD3 directly catalyzes deglutathionylation or recruits a thiol reductase is unknown\",\n        \"Relevance to therapy resistance in vivo not tested\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"FXYD3 was shown to function beyond ion transport as a signaling scaffold: it forms a complex with Src and ERα for non-genomic estrogen signaling, and participates in a positive feedback loop with SOX9 that maintains breast cancer stem cells and tamoxifen resistance.\",\n      \"evidence\": \"Co-immunoprecipitation, siRNA, reporter assays, subcellular fractionation in ER+ breast cancer cells\",\n      \"pmids\": [\"30206184\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct binding interface between FXYD3 and Src not mapped\",\n        \"SOX9 binding site on FXYD3 promoter confirmed by reporter but not by endogenous ChIP-seq\",\n        \"Independent replication in other ER+ models lacking\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Physiological relevance of FXYD3 as a Na,K-ATPase modulator was demonstrated in intact human airway epithelia, where FXYD3 knockdown reduced Na,K-ATPase transport capacity, amiloride-sensitive Na⁺ absorption, and transepithelial liquid clearance.\",\n      \"evidence\": \"scRNA-seq, immunohistochemistry, siRNA knockdown, short-circuit current measurements with nystatin permeabilization, liquid absorption assay\",\n      \"pmids\": [\"35993520\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"In vivo airway phenotype of FXYD3 deletion not tested\",\n        \"Whether FXYD3 modulates ENaC directly or only via Na,K-ATPase is unclear\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"An entirely new signaling axis was revealed: FXYD3 competitively binds TRAF3 to relieve its inhibition of IL-17R, promoting ACT1 recruitment and NF-κB/MAPK activation in keratinocytes, with genetic deletion attenuating psoriasis-like inflammation in vivo.\",\n      \"evidence\": \"Co-immunoprecipitation, competitive binding assays, genetic KO in keratinocytes, imiquimod psoriasis model\",\n      \"pmids\": [\"36693922\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis of FXYD3–TRAF3 interaction not determined\",\n        \"Whether this mechanism operates in other IL-17-responsive cell types is unknown\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"FXYD3 was shown to interact with SERCA2 in intestinal goblet cells, enhancing ER Ca²⁺ pump activity to support mucin glycosylation and mucus barrier integrity, establishing a second P-type ATPase partnership beyond Na,K-ATPase.\",\n      \"evidence\": \"Intestinal epithelium-specific genetic KO in mice, Co-IP with SERCA2, ER Ca²⁺ measurements, colitis model\",\n      \"pmids\": [\"41187059\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether FXYD3 modulates SERCA2 affinity parameters analogously to Na,K-ATPase is not resolved\",\n        \"Direct structural evidence for the FXYD3–SERCA2 interface absent\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"FXYD3 was found to bind IRF7 via its 60–87 amino acid domain, initiating a cGAS/STING-mediated positive feedback loop that sustains JAK2/STAT5 signaling and drives intrahepatic cholangiocarcinoma progression.\",\n      \"evidence\": \"Co-immunoprecipitation with domain mapping, single-cell and spatial transcriptomics, in vivo tumor models\",\n      \"pmids\": [\"41164952\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Independent validation of FXYD3–IRF7 interaction in a second lab awaited\",\n        \"Whether this axis operates in normal cholangiocytes or only in cancer is unclear\",\n        \"Causal chain from FXYD3–IRF7 binding to cGAS/STING activation not fully delineated\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis of FXYD3 interactions with its multiple ATPase and signaling partners, whether its ion-transport and signaling functions are mechanistically linked or independent, and how tissue-specific expression patterns dictate which of its diverse functions predominate.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No high-resolution structure of FXYD3 in complex with any partner\",\n        \"Whether FXYD3's signaling roles (TRAF3, Src, IRF7) require or are independent of ATPase association is unknown\",\n        \"Comprehensive in vivo phenotyping across tissues using conditional knockouts is incomplete\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 2, 4, 10, 12, 14]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [11, 13, 15]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [3, 5, 12]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [14]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0382551\", \"supporting_discovery_ids\": [1, 2, 4, 12]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [13, 15]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [11, 13, 15]}\n    ],\n    \"complexes\": [\n      \"Na,K-ATPase\"\n    ],\n    \"partners\": [\n      \"ATP1A1\",\n      \"SERCA2\",\n      \"TRAF3\",\n      \"SRC\",\n      \"ESR1\",\n      \"IRF7\",\n      \"SOX9\",\n      \"ZEB1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}