{"gene":"ATP1A1","run_date":"2026-06-09T22:02:44","timeline":{"discoveries":[{"year":2013,"finding":"Somatic hotspot mutations in ATP1A1 (encoding the Na+/K+-ATPase α1 subunit) cause loss of pump activity and strongly reduced affinity for potassium, leading to inappropriate membrane depolarization in adrenal cells and autonomous aldosterone secretion.","method":"In vitro functional pump activity assays, electrophysiological ex vivo studies on primary adrenal adenoma cells, exome sequencing","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro functional assays of pump activity and electrophysiology on primary cells, replicated across multiple mutation carriers and independently confirmed in follow-up studies","pmids":["23416519"],"is_preprint":false},{"year":2013,"finding":"The ATP1A1 Gly99Arg mutation (somatic, found in APA) severely impairs ATPase activity, reduces apparent Na+ affinity for phosphorylation and K+ affinity for dephosphorylation (indicating decreased Na+ and K+ binding), and causes membrane depolarization when overexpressed in adrenal cells; structural modeling indicates the substitution interferes with the gateway to the ion binding pocket.","method":"In vitro ATPase activity assays, whole-cell patch-clamp electrophysiology in HAC15 adrenal cells, structural homology modeling, CYP11B2 expression assays","journal":"Hypertension (Dallas, Tex. : 1979)","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal in vitro methods (enzymatic assay, electrophysiology, structural modeling) in a single study, consistent with findings from another independent lab (PMID:23416519)","pmids":["24082052"],"is_preprint":false},{"year":2015,"finding":"ATP1A1 is required for coronavirus (MHV, FIPV, MERS-CoV) and VSV entry into host cells; cardiotonic steroid binding to ATP1A1 (at nanomolar concentrations that do not inhibit ion transport) activates Src signaling that inhibits virus entry at an early stage, causing virion accumulation near the cell surface and reduced membrane fusion.","method":"Gene silencing (siRNA), cardiotonic steroid (ouabain, bufalin) treatment, Src kinase inhibitor rescue experiments, viral infection assays, electron microscopy of virion localization","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal genetic (knockdown) and pharmacological approach with Src inhibitor rescue, multiple viral models tested, mechanistic pathway (ATP1A1→Src signaling→viral entry inhibition) established with multiple orthogonal methods","pmids":["25653449"],"is_preprint":false},{"year":2014,"finding":"ATP1A1 (α1-chain of Na/K-ATPase), but not the β1- or β3-chains (ATP1B1/ATP1B3), is required for efficient unconventional secretion of FGF2; in the absence of β-chains, a direct protein-protein interaction between the cytoplasmic domain of ATP1A1 and FGF2 occurs with submicromolar affinity, suggesting ATP1A1 acts as a recruitment factor for FGF2 at the inner leaflet of the plasma membrane.","method":"Unbiased RNAi screen, siRNA knockdown of individual Na/K-ATPase subunits, direct binding assay (cytoplasmic domain of ATP1A1 with FGF2, affinity measurement)","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — genome-wide RNAi screen plus direct in vitro protein interaction assay with affinity measurement; single lab but multiple orthogonal approaches","pmids":["25533462"],"is_preprint":false},{"year":2018,"finding":"De novo mutations in ATP1A1 (catalytic α1 subunit of Na+/K+-ATPase) cause not only loss of pump function but also abnormal cation permeabilities leading to membrane depolarization, underlying renal hypomagnesemia and refractory seizures.","method":"Heterologous expression systems (functional characterization of mutant Na+/K+-ATPase α1 subunits), electrophysiology, whole-exome sequencing","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — functional characterization in heterologous expression systems with electrophysiology, multiple patient mutations characterized","pmids":["30388404"],"is_preprint":false},{"year":2018,"finding":"Missense mutations in ATP1A1 cause autosomal-dominant CMT2 (axonal peripheral neuropathy); two-electrode voltage clamp in Xenopus oocytes demonstrated significant reduction in Na+ current activity in some ouabain-insensitive ATP1A1 mutants, indicating loss-of-function of the Na+/K+ pump. ATP1A1 localizes to the axolemma of myelinated sensory and motor axons and to Schmidt-Lanterman incisures.","method":"Two-electrode voltage clamp in Xenopus oocytes, immunostaining of peripheral nerve axons, whole-exome/genome sequencing across seven countries","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — electrophysiology in Xenopus oocytes (Tier 1 functional assay) plus direct localization by immunostaining, replicated across multiple families and independent labs","pmids":["29499166"],"is_preprint":false},{"year":2019,"finding":"ATP1A1 is required for macropinocytic entry of RSV into respiratory epithelial cells; RSV triggers clustering of ATP1A1 in the plasma membrane (dependent on viral attachment glycoprotein G), activating c-Src kinase signaling that transactivates EGFR at Tyr845, culminating in macropinosome formation and RSV uptake.","method":"Genome-wide siRNA screen, knockdown validation, live-cell imaging, phosphorylation assays (c-Src, EGFR Tyr845), pharmacological inhibitors (ouabain, PST2238), primary human airway epithelial cell validation","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — genome-wide screen with mechanistic follow-up (signaling assays, imaging, inhibitor rescue), validated in primary cells, multiple orthogonal methods in single study","pmids":["31381610"],"is_preprint":false},{"year":2012,"finding":"In zebrafish brain ventricle development, Atp1a1 (Na,K-ATPase α subunit) regulates neuroepithelial permeability and CSF production downstream; RhoA acts downstream of Atp1a1 (and its regulatory subunit Fxyd1) in neuroepithelium formation and permeability modulation, while CSF production requires Atp1a1 but not Fxyd1 or RhoA.","method":"Morpholino knockdown in zebrafish, overexpression, RhoA epistasis experiments, ouabain pharmacological inhibition, intracellular Na+ measurements","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with RhoA established by double loss-of-function, multiple phenotypic readouts; zebrafish model (ortholog), single lab","pmids":["22683378"],"is_preprint":false},{"year":2017,"finding":"ATP1A1 in Sertoli cells regulates tight junctions (claudin 11) and gap junctions (connexin 43) through the Src-EGFR-ERK1/2-CREB signaling pathway; 50 nM ouabain (signaling dose) activates this pathway and increases transepithelial electrical resistance, while 1 mM ouabain (pump-inhibitory dose) has opposite effects; Src and MAPK pathway inhibitors abolish ouabain-induced junctional regulation.","method":"Primary Sertoli cell culture, ouabain treatment, signaling inhibitors, immunoblotting, transepithelial electrical resistance measurements, immunofluorescence, mass spectrometry","journal":"Biology of reproduction","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — pharmacological dissection with inhibitor rescue and multiple signaling readouts; single lab, pathway placement established by inhibitor epistasis","pmids":["28203706"],"is_preprint":false},{"year":2022,"finding":"ATP1A1 overexpressed in tumor cells binds homophilically to ATP1A1 on fibroblasts (heterocellular contact), inducing calcium oscillations, NF-κB activation, and activin A secretion in fibroblasts, which promotes EMT in tumor cells and myofibroblast activation; silencing ATP1A1 or neutralizing activin A suppresses tumor invasion.","method":"Co-immunoprecipitation/pulldown, siRNA silencing, calcium imaging, NF-κB reporter assays, activin A neutralization, invasion assays, primary tumor analysis","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — direct binding demonstrated plus downstream signaling (calcium, NF-κB) and functional rescue; single lab but multiple orthogonal methods","pmids":["35618735"],"is_preprint":false},{"year":2019,"finding":"ATP1A1 knockdown in glioma stem cells inhibits ERK1/2 and AKT pathway activation through suppression of Src phosphorylation, reducing cell proliferation, survival, and causing G1 arrest; rescue of ATP1A1 expression restores proliferation, placing ATP1A1 upstream of Src in this signaling axis.","method":"siRNA knockdown, ATP1A1 re-expression rescue, Western blotting for p-Src/p-ERK1/2/p-AKT, cell cycle analysis, apoptosis assays in primary human GSCs","journal":"Frontiers in oncology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — loss-of-function with rescue, signaling pathway readouts; single lab, pathway placement by epistasis (ATP1A1→Src→ERK/AKT)","pmids":["31114755"],"is_preprint":false},{"year":2021,"finding":"ATP1A1 L104R mutation (APA-associated) stimulates cell proliferation and increases intracellular Ca2+ (measured by Fluo-4), enhances Src phosphorylation, and increases NKA expression in adrenal cells; low-concentration ouabain further potentiates Src phosphorylation in mutant cells.","method":"HAC15 cell transfection with mutant ATP1A1, cell proliferation assays, DNA content analysis, S-phase measurement, Ca2+ fluorescence (Fluo-4), Src phosphorylation (Western blot), transcriptome analysis, immunohistochemistry","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — functional cell assays with multiple readouts (proliferation, Ca2+, Src); single lab, mechanism partially characterized","pmids":["34681640"],"is_preprint":false},{"year":2019,"finding":"ATP1A1 mutations causing intermediate CMT disease lead to proteasomal degradation of the ATP1A1 protein (loss-of-function), without affecting mRNA levels; the mutations (p.S207F and p.G877S) downregulate protein levels by promoting proteasomal turnover.","method":"Whole-exome sequencing, Western blot of protein levels, RT-PCR for mRNA, proteasome inhibitor experiments in patient-derived cells","journal":"Human mutation","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — protein degradation mechanism established by proteasome inhibitor rescue; single lab","pmids":["31373411"],"is_preprint":false},{"year":2023,"finding":"GPR37 interacts with ATP1A1 and promotes its ubiquitination-induced proteasomal degradation, thereby limiting activation of the AKT/mTOR signaling pathway; GPR37 knockdown stabilizes ATP1A1 and increases AKT/mTOR activity.","method":"Co-immunoprecipitation, ubiquitination assays, siRNA knockdown, Western blotting for AKT/mTOR pathway components, ESCC cell functional assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — direct protein interaction (Co-IP) plus functional ubiquitination assay; single lab","pmids":["39730361"],"is_preprint":false},{"year":2025,"finding":"LncDARS-AS1 directly binds ATP1A1 protein, preventing its interaction with UBQLN4 and subsequent proteasomal degradation, thereby enhancing Na+/K+-ATPase activity and promoting osteosarcoma metastasis; digoxin targeting NKA inhibits tumor growth.","method":"ChIRP, mass spectrometry, molecular docking, molecular dynamics simulations, ion-sensitive fluorescent indicators, enzymatic NKA assays, siRNA silencing, in vivo xenograft models","journal":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — direct RNA-protein interaction validated by ChIRP and MS, functional NKA activity measured enzymatically; single lab but multiple orthogonal methods","pmids":["40665639"],"is_preprint":false},{"year":2024,"finding":"Periplogenin (PPG) directly binds ATP1A1 at residue T804 (forming a hydrogen bond); T804A substitution abolishes PPG binding and confers resistance to PPG's cytotoxic activity both in vitro and in vivo in xenograft models.","method":"Forward genetic chemical mutagenesis screen, next-generation sequencing, in vitro hydrogen bond modeling, T804A mutagenesis, in vivo xenograft comparison of wild-type vs. T804A DU145 cells","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — forward genetic screen identifying single amino acid resistance mutation, validated in vivo; direct binding site identified at atomic resolution with in vivo confirmation","pmids":["39227746"],"is_preprint":false},{"year":2023,"finding":"ATP1A1 serves as a receptor/attachment factor for PEDV; the CT structural domain of ATP1A1 interacts with PEDV S1 protein; knockdown or inhibition of ATP1A1 significantly reduces PEDV attachment; ATP1A1 co-localizes with PEDV S1 at the cell surface in early infection.","method":"Virus overlay protein binding assay (VOPBA), mass spectrometry, siRNA knockdown, overexpression, co-localization (immunofluorescence), antibody blocking assay, ouabain/PST2238 inhibitor treatment","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — direct binding identified by VOPBA/MS, functional knockdown and antibody blocking confirm role in attachment; single lab","pmids":["36835408"],"is_preprint":false},{"year":2023,"finding":"ATP1A1-linked CMT disease requires a malfunctioning (dominant-negative or gain-of-abnormal-function) protein product from one allele; haploinsufficiency (protein-null heterozygosity) alone is not sufficient to cause disease in Atp1a1+/- mice up to 18 months, and a human carrier of a protein-null truncation variant (p.Y148*) was phenotypically normal.","method":"Atp1a1+/- heterozygous knockout mouse neuromuscular phenotyping (up to 18 months, with exercise), clinical phenotyping of human protein-null variant carrier","journal":"Biochimica et biophysica acta. Molecular cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean in vivo knockout with longitudinal phenotyping plus human carrier clinical data; single lab, haploinsufficiency model refuted","pmids":["37659504"],"is_preprint":false},{"year":2019,"finding":"The ATP1A1 Pro600Ala mutation (CMT2-causing) is associated with defective in vitro neuronal differentiation of patient-derived iPSCs; ATP1A1 protein is undetectable in the few neurons derived from CMT iPSCs, and electrophysiological properties of derived neurons are less mature compared to controls.","method":"Patient-specific iPSC generation, neural differentiation assays, immunostaining for ATP1A1, electrophysiology of derived neurons","journal":"Journal of the peripheral nervous system : JPNS","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — patient-derived iPSC model with functional readouts; single lab, mechanism of neuronal loss partially established","pmids":["31707753"],"is_preprint":false},{"year":2022,"finding":"In a pancreatic ductal adenocarcinoma context, resibufogenin (RB) binds ATP1A1, activating Na+-K+-ATPase as a signaling receptor that triggers the intracellular MAPK/ERK pathway and Ca2+-mediated Src/FAK/Paxillin focal adhesion pathway, leading to G2/M arrest and inhibition of invasion.","method":"Drug-target binding assays, MAPK/ERK pathway analysis, Ca2+ measurement, Src/FAK/Paxillin phosphorylation assays, cell cycle analysis, invasion assays in GBM cell lines","journal":"Frontiers in pharmacology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — multiple signaling pathway readouts after drug-target engagement; single lab, mechanism inferred from pharmacological binding + downstream effects","pmids":["35656311"],"is_preprint":false},{"year":2017,"finding":"shRNA screen identified ATP1A1 knockdown as conferring sensitivity to aurilide B (a mitochondria-mediated apoptosis inducer); combined ATP1A1 inhibition with ouabain potentiated aurilide B sensitivity, suggesting ATP1A1 regulates mitochondria-mediated apoptosis.","method":"Genome-wide pooled shRNA screen with deep sequencing, ouabain combination treatment, cell viability assays","journal":"Scientific reports","confidence":"Low","confidence_rationale":"Tier 3 / Weak — shRNA screen identification plus pharmacological combination, no direct mechanistic dissection of how ATP1A1 regulates apoptosis; single lab","pmids":["28515454"],"is_preprint":false},{"year":2023,"finding":"ATP1A1 regulates tumor progression in colon cancer through the ERK5 signaling pathway; ATP1A1 knockdown suppresses cell proliferation, migration, and invasion and induces apoptosis, with microarray revealing altered expression of EGFR, MAP2K5, MAPK7, FOS, MYC, and BAD.","method":"siRNA knockdown in HT29 and Caco2 cells, microarray gene expression profiling, cell proliferation/migration/invasion/apoptosis assays","journal":"Annals of surgical oncology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — loss-of-function with microarray, ERK5 pathway placement is inferential from expression data without direct pathway rescue; single lab","pmids":["37407874"],"is_preprint":false},{"year":2022,"finding":"In ATP1A1 mutant APA, the VDR gene promoter is hypomethylated compared to non-functioning adenoma, leading to high VDR expression; VDR suppression abrogates ATP1A1 mutation-mediated cell proliferation in HAC15 cells, placing VDR downstream of ATP1A1 mutation in the proliferative signaling cascade.","method":"DNA methylation analysis, qPCR, siRNA VDR knockdown in HAC15 cells transfected with mutant ATP1A1, cell proliferation assays","journal":"Molecular and cellular endocrinology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — epigenetic and knockdown data; single lab, indirect pathway connection (mutation→methylation→VDR→proliferation)","pmids":["35257799"],"is_preprint":false},{"year":2017,"finding":"ATP1A1 overexpression in RCC cells inhibits proliferation and migration by increasing ROS production and suppressing the Raf/MEK/ERK signaling pathway, while downregulation of ATP1A1 promotes cancer development.","method":"Exogenous overexpression, CCK-8 proliferation assay, Boyden chamber migration assay, flow cytometry for apoptosis and ROS, Western blotting for Raf/MEK/ERK pathway","journal":"Clinical proteomics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — overexpression with signaling readouts; single lab, ROS mechanism inferred without mechanistic dissection","pmids":["28484360"],"is_preprint":false},{"year":2023,"finding":"LAPTM4B interacts with ATP1A1 and prevents its TRIM8-mediated K63-linked ubiquitination and proteasomal degradation, stabilizing ATP1A1 to enhance lysosomal acidification and promote EGFR-TKI resistance in NSCLC.","method":"Co-immunoprecipitation, ubiquitination assays (K63-linkage-specific), siRNA knockdown, Western blotting, lysosomal pH assays, clinical sample analysis","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — direct protein interaction (Co-IP) plus specific ubiquitination assay (K63-linked); single lab but multiple orthogonal methods","pmids":["41362742"],"is_preprint":false},{"year":2025,"finding":"ATP1A1 is required for PRRSV-2 attachment and internalization; PRRSV glycoprotein GP4 interacts with the fourth extracellular region (ER4) of ATP1A1 (dependent on GP4 C-terminus); ATP1A1-Src signaling activates EGFR and caveolin-1 to enable viral uptake via macropinocytosis and caveolae/raft-mediated endocytosis; internalized virions traffic to ATP1A1/CD163-positive early endosomes for uncoating.","method":"siRNA knockdown, overexpression, co-localization imaging, signaling inhibitor assays, GP4-ER4 interaction mapping, synthetic peptide competition, nanobody targeting, multiple PRRSV lineage testing","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (genetic, pharmacological, peptide competition, nanobody), direct domain interaction mapped; single lab","pmids":["41773865"],"is_preprint":false},{"year":2025,"finding":"ATP1A1 directly binds odoamide (marine natural product); mutations at Gly98 and Gly99 of ATP1A1 confer resistance to odoamide cytotoxicity; ATP1A1 was identified as the specific binding protein by affinity chromatography with an odoamide probe, and odoamide-induced apoptotic cell death is critically dependent on interaction with ATP1A1.","method":"Affinity chromatography with odoamide probe, mass spectrometry, resistance mutation mapping (Gly98/Gly99), cell viability assays, JFCR39 cancer panel fingerprinting, gene expression profiling","journal":"Chembiochem : a European journal of chemical biology","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — affinity-based target identification plus resistance mutation mapping identifies binding site; single lab","pmids":["39754293"],"is_preprint":false},{"year":2004,"finding":"Mouse embryos homozygous for a null mutation in the Na/K-ATPase α1-subunit (Atp1a1) are able to undergo compaction and cavitation (blastocyst formation), demonstrating that the α1-isozyme is not strictly required for trophectoderm fluid transport during preimplantation development; other Na/K-ATPase isoforms provide functional redundancy.","method":"Targeted gene disruption (Atp1a1 knockout mice), embryo development analysis from heterozygous matings, morphological assessment of compaction and cavitation","journal":"Mechanisms of development","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean knockout with defined developmental phenotype (negative result: no cavitation defect); single lab","pmids":["15147760"],"is_preprint":false},{"year":2023,"finding":"Calcium oxalate crystal deposition decreases ATP1A1 expression in renal cells and activates the ATP1A1/Src/ROS/p38/JNK/NF-κB signaling pathway; overexpression of ATP1A1 or treatment with pNaKtide (specific inhibitor of ATP1A1/Src complex) inhibits this signaling cascade and reduces crystal-cell adhesion and stone formation.","method":"In vitro crystal deposition experiments, in vivo mouse models, ATP1A1 overexpression, pNaKtide treatment, Western blotting for Src/ROS/p38/JNK/NF-κB","journal":"Redox biology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — in vitro and in vivo experiments with both gain-of-function and pharmacological inhibition; single lab, signaling pathway placed downstream of ATP1A1","pmids":["36871182"],"is_preprint":false},{"year":2023,"finding":"ATP1A1 loss-of-function variant Gly903Arg (p.G903R) causes significantly reduced HEK cell viability in an ouabain resistance assay, demonstrating loss of ATPase function; variant is associated with developmental delay, intellectual disability, and autism.","method":"Whole-exome/genome sequencing, ouabain resistance/cell viability assay in transfected HEK cells with ouabain-insensitive ATP1A1 constructs","journal":"Annals of clinical and translational neurology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — ouabain resistance functional assay in HEK cells; single study, one mutation","pmids":["38504481"],"is_preprint":false},{"year":2019,"finding":"Hereditary spastic paraplegia (HSP) is caused by a de novo ATP1A1 variant (p.L337P); functional studies in heterologous expression systems and homology modeling provide evidence for pathogenicity of this variant affecting Na+/K+-ATPase function.","method":"Whole-exome sequencing, functional characterization in heterologous expression systems, homology modeling","journal":"Clinical genetics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — functional characterization mentioned but abstract provides limited mechanistic detail; single case with heterologous expression","pmids":["31705535"],"is_preprint":false},{"year":2026,"finding":"Combined knockdown of ATP1A1 (Na/K-ATPase) and GJA5 (connexin 40) in iPSC-derived atrial myocardium induces significant beat irregularity and increased conduction heterogeneity, establishing these two factors as cooperative drivers of arrhythmogenesis; this is consistent with an inflammation-driven AF mechanism where both intercellular connectivity and ion flux are disrupted.","method":"siRNA knockdown in 3D iPSC-derived atrial myocardium tissue model, sharp electrode recordings, calcium imaging, multi-electrode assays, lentiviral overexpression, pharmacological modulation, in silico modeling","journal":"Frontiers in immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic (siRNA) and pharmacological manipulation in 3D tissue model with multiple electrophysiological readouts, validated by in silico model; single lab","pmids":["41868610"],"is_preprint":false}],"current_model":"ATP1A1 encodes the ubiquitously expressed α1 catalytic subunit of the Na+/K+-ATPase heterodimer, which hydrolyzes ATP to extrude Na+ and import K+ across the plasma membrane; beyond ion pumping, ATP1A1 acts as a signaling receptor—cardiotonic steroid or ligand binding activates Src kinase, which transactivates EGFR and downstream ERK/AKT pathways to regulate cell proliferation, junctional integrity, unconventional FGF2 secretion, viral entry (via macropinocytosis), and EMT; pathogenic missense mutations cause loss of pump function with abnormal cation permeabilities and membrane depolarization, driving diseases including primary aldosteronism, Charcot-Marie-Tooth neuropathy, hereditary spastic paraplegia, and hypomagnesemia with seizures, while haploinsufficiency alone is insufficient to cause disease, requiring a dominant malfunctioning protein product."},"narrative":{"mechanistic_narrative":"ATP1A1 encodes the catalytic α1 subunit of the Na+/K+-ATPase, an ATP-driven plasma-membrane ion pump whose activity governs cation gradients, membrane potential, and—in mutant or ligand-bound states—intracellular signaling [PMID:23416519, PMID:24082052, PMID:30388404]. Somatic and germline missense mutations impair pump function and reduce Na+ and K+ binding affinities, producing abnormal cation permeabilities and membrane depolarization; structural modeling localizes representative substitutions (e.g. Gly99Arg) to the gateway of the ion-binding pocket [PMID:24082052, PMID:30388404]. This pump dysfunction underlies aldosterone-producing adrenal adenoma through inappropriate depolarization and autonomous aldosterone secretion [PMID:23416519, PMID:24082052], and additional pathogenic variants cause renal hypomagnesemia with seizures [PMID:30388404], axonal Charcot-Marie-Tooth neuropathy where ATP1A1 localizes to the axolemma and Schmidt-Lanterman incisures [PMID:29499166], hereditary spastic paraplegia [PMID:31705535], and a neurodevelopmental phenotype with intellectual disability [PMID:38504481]. CMT-causing variants act not through haploinsufficiency—protein-null heterozygosity is tolerated in mice and humans—but require a malfunctioning dominant protein product, in some cases destabilized by proteasomal degradation [PMID:31373411, PMID:37659504]. Beyond ion transport, ATP1A1 functions as a signaling receptor: cardiotonic steroids or natural-product ligands bind it (with a defined binding site at residue T804 and resistance residues Gly98/Gly99) to trigger Src kinase activation, EGFR transactivation, and downstream ERK/AKT and Ca2+ signaling that regulates cell proliferation, junctional integrity, and EMT [PMID:28203706, PMID:31114755, PMID:34681640, PMID:39227746, PMID:39754293]. This Src-coupled receptor function is exploited by multiple viruses—coronaviruses, RSV, PEDV, and PRRSV—which engage ATP1A1 as an attachment/entry factor driving macropinocytic and caveolae-mediated uptake [PMID:25653449, PMID:31381610, PMID:36835408, PMID:41773865]. ATP1A1 also acts as an unconventional secretion factor, recruiting FGF2 at the inner plasma-membrane leaflet via a direct cytoplasmic-domain interaction [PMID:25533462], and mediates heterocellular tumor-fibroblast contact through homophilic ATP1A1 binding that promotes invasion [PMID:35618735]. ATP1A1 protein abundance is controlled by ubiquitin-dependent turnover involving GPR37, TRIM8/LAPTM4B, and UBQLN4, linking its stability to AKT/mTOR and lysosomal signaling [PMID:39730361, PMID:40665639, PMID:41362742].","teleology":[{"year":2004,"claim":"Tested whether the α1 isoform is individually essential, establishing functional redundancy among Na/K-ATPase isoforms during early development.","evidence":"Targeted Atp1a1 knockout mouse embryos assessed for compaction and cavitation","pmids":["15147760"],"confidence":"Medium","gaps":["Does not address tissue-specific requirements later in development","No assessment of adult phenotype from this null allele"]},{"year":2012,"claim":"Placed Atp1a1 upstream of RhoA in regulating neuroepithelial permeability, separating its ion-transport role in CSF production from its developmental signaling role.","evidence":"Morpholino knockdown and RhoA epistasis in zebrafish brain ventricle development","pmids":["22683378"],"confidence":"Medium","gaps":["Mechanism linking pump activity to RhoA is undefined","Ortholog model; human relevance not tested"]},{"year":2013,"claim":"Established the disease mechanism for aldosterone-producing adenoma: hotspot mutations cause loss of pump activity and reduced K+ affinity, depolarizing adrenal cells to drive autonomous aldosterone secretion.","evidence":"Pump activity and ATPase assays, patch-clamp electrophysiology in adrenal cells, structural modeling, exome sequencing","pmids":["23416519","24082052"],"confidence":"High","gaps":["Does not explain how depolarization couples to CYP11B2/aldosterone transcription mechanistically","Tumor proliferation link not addressed"]},{"year":2014,"claim":"Defined a non-pump function: ATP1A1 directly recruits FGF2 to the inner membrane leaflet for unconventional secretion, independent of β-subunits.","evidence":"Genome-wide RNAi screen plus direct in vitro binding affinity measurement","pmids":["25533462"],"confidence":"High","gaps":["Structural basis of the FGF2 interaction not resolved","How recruitment couples to membrane translocation unknown"]},{"year":2015,"claim":"Identified ATP1A1 as a host entry factor for enveloped viruses and showed cardiotonic-steroid-triggered Src signaling can block entry, separating signaling from ion-transport function.","evidence":"siRNA silencing, cardiotonic steroid treatment, Src inhibitor rescue, viral infection and EM across multiple viruses","pmids":["25653449"],"confidence":"High","gaps":["Direct viral-ATP1A1 binding not mapped here","How Src signaling blocks fusion mechanistically unclear"]},{"year":2017,"claim":"Demonstrated dose-dependent receptor signaling: low ouabain activates Src-EGFR-ERK-CREB to strengthen junctions, while pump-inhibitory doses act oppositely.","evidence":"Primary Sertoli cell culture, ouabain dosing, signaling inhibitors, TER measurements","pmids":["28203706"],"confidence":"Medium","gaps":["Endogenous ligand for signaling-dose activation unknown","Single cell type"]},{"year":2018,"claim":"Extended the pathogenic mechanism to abnormal cation permeabilities and to axonal CMT neuropathy, with direct evidence of axolemmal localization.","evidence":"Heterologous expression and Xenopus oocyte electrophysiology of patient mutants, peripheral nerve immunostaining, exome sequencing","pmids":["30388404","29499166"],"confidence":"High","gaps":["How abnormal cation leak versus pump loss differentially drives renal vs neuronal phenotypes unresolved","Cell-type vulnerability basis unknown"]},{"year":2019,"claim":"Showed ATP1A1 drives macropinocytic viral entry via clustering-dependent Src/EGFR-Tyr845 signaling, and that some CMT variants act via proteasomal protein loss rather than mRNA loss.","evidence":"Genome-wide siRNA screen, phosphorylation assays, imaging, inhibitors in airway cells; proteasome inhibitor and mRNA analysis in patient cells; iPSC neuronal differentiation","pmids":["31381610","31373411","31707753","31114755","31705535"],"confidence":"Medium","gaps":["Whether degradation-prone variants act dominant-negatively or by simple loss not fully separated","HSP variant mechanism only partially characterized"]},{"year":2021,"claim":"Linked APA mutations to proliferative signaling by showing the L104R mutant raises intracellular Ca2+ and Src phosphorylation, connecting pump dysfunction to tumor growth.","evidence":"Mutant transfection in HAC15 cells, proliferation, Ca2+ fluorescence, Src phosphorylation, transcriptomics","pmids":["34681640"],"confidence":"Medium","gaps":["Direct causal chain from Ca2+/Src to proliferation not isolated","Single cell line"]},{"year":2022,"claim":"Revealed ATP1A1 as a homophilic heterocellular adhesion/signaling molecule driving tumor EMT through fibroblast NF-κB and activin A secretion.","evidence":"Co-IP/pulldown, calcium imaging, NF-κB reporters, activin A neutralization, invasion assays","pmids":["35618735"],"confidence":"Medium","gaps":["Structural basis of homophilic trans-binding unresolved","Single tumor model context"]},{"year":2023,"claim":"Established ubiquitin-dependent control of ATP1A1 abundance (GPR37, TRIM8/LAPTM4B) coupling its stability to AKT/mTOR and lysosomal signaling, and confirmed that disease requires a dominant malfunctioning product, not haploinsufficiency.","evidence":"Co-IP, ubiquitination assays, knockdowns in cancer cells; Atp1a1+/- mouse longitudinal phenotyping and human protein-null carrier","pmids":["39730361","41362742","37659504","36835408","37407874","36871182"],"confidence":"Medium","gaps":["Hierarchy among competing E3/stabilizing factors unknown","Physiological contexts where each turnover pathway dominates undefined"]},{"year":2024,"claim":"Mapped a defined small-molecule binding site (T804) on ATP1A1 through a resistance mutation, establishing it as a druggable target whose engagement drives cytotoxicity.","evidence":"Forward genetic chemical mutagenesis screen, hydrogen-bond modeling, T804A mutagenesis, in vivo xenografts","pmids":["39227746"],"confidence":"High","gaps":["Downstream pathway from T804 ligand binding not detailed here","Generality across other cardiotonic ligands not tested"]},{"year":2025,"claim":"Identified additional ligand binding determinants (Gly98/Gly99) and a lncRNA (DARS-AS1) that competes with UBQLN4 to stabilize ATP1A1 and enhance pump activity, linking abundance control to metastasis.","evidence":"Affinity chromatography and resistance mapping for odoamide; 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sciences","url":"https://pubmed.ncbi.nlm.nih.gov/36002076","citation_count":3,"is_preprint":false},{"pmid":"38767491","id":"PMC_38767491","title":"Drosophila models used to simulate human ATP1A1 gene mutations that cause Charcot-Marie-Tooth type 2 disease and refractory seizures.","date":"2023","source":"Neural regeneration research","url":"https://pubmed.ncbi.nlm.nih.gov/38767491","citation_count":3,"is_preprint":false},{"pmid":"40665639","id":"PMC_40665639","title":"LncDARS-AS1 Regulates ATP1A1 Stability and Enhances Na+/K+ ATPase Activity to Promote Osteosarcoma Metastasis.","date":"2025","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/40665639","citation_count":2,"is_preprint":false},{"pmid":"37659504","id":"PMC_37659504","title":"ATP1A1-linked diseases require a malfunctioning protein product from one allele.","date":"2023","source":"Biochimica et biophysica acta. Molecular cell research","url":"https://pubmed.ncbi.nlm.nih.gov/37659504","citation_count":2,"is_preprint":false},{"pmid":"37566922","id":"PMC_37566922","title":"Silencing of ATP1A1 attenuates cell membrane disruption by nanosecond electric pulses.","date":"2023","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/37566922","citation_count":2,"is_preprint":false},{"pmid":"19427512","id":"PMC_19427512","title":"RAD51D- and FANCG-dependent base substitution mutagenesis at the ATP1A1 locus in mammalian cells.","date":"2009","source":"Mutation research","url":"https://pubmed.ncbi.nlm.nih.gov/19427512","citation_count":2,"is_preprint":false},{"pmid":"39754293","id":"PMC_39754293","title":"Target Identification of Marine Natural Product Odoamide:Odoamide Induces Apoptotic Cell Death by Targeting ATPase Na+/K+ Transporting Subunit Alpha 1 (ATP1A1).","date":"2025","source":"Chembiochem : a European journal of chemical biology","url":"https://pubmed.ncbi.nlm.nih.gov/39754293","citation_count":2,"is_preprint":false},{"pmid":"41057666","id":"PMC_41057666","title":"pNaktide reverses metabolic reprogramming and disease progression of ATP1A1-deficiency clear cell renal cell carcinoma.","date":"2025","source":"Cancer gene therapy","url":"https://pubmed.ncbi.nlm.nih.gov/41057666","citation_count":1,"is_preprint":false},{"pmid":"38504481","id":"PMC_38504481","title":"Recurrent ATP1A1 variant Gly903Arg causes developmental delay, intellectual disability, and autism.","date":"2024","source":"Annals of clinical and translational neurology","url":"https://pubmed.ncbi.nlm.nih.gov/38504481","citation_count":1,"is_preprint":false},{"pmid":"12537020","id":"PMC_12537020","title":"Targeting of A701G nucleotide at the human ATP1A1 locus using a RNA/DNA chimera.","date":"2002","source":"Nucleosides, nucleotides & nucleic acids","url":"https://pubmed.ncbi.nlm.nih.gov/12537020","citation_count":1,"is_preprint":false},{"pmid":"41773865","id":"PMC_41773865","title":"ATP1A1 enhances porcine reproductive and respiratory syndrome virus type 2 attachment and internalization.","date":"2026","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/41773865","citation_count":0,"is_preprint":false},{"pmid":"37090550","id":"PMC_37090550","title":"ATP1A1 -linked diseases require a malfunctioning protein product from one allele.","date":"2023","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/37090550","citation_count":0,"is_preprint":false},{"pmid":"41362742","id":"PMC_41362742","title":"LAPTM4B Confers Resistance to EGFR-TKIs by Suppressing the Proteasomal Degradation of ATP1A1 in Non-small Cell Lung Cancer.","date":"2026","source":"International journal of biological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/41362742","citation_count":0,"is_preprint":false},{"pmid":"41868610","id":"PMC_41868610","title":"GJA5 and ATP1A1 perturbations recapitulate inflammation-related beat irregularities in iPSC-based atrial myocardium tissue model.","date":"2026","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/41868610","citation_count":0,"is_preprint":false},{"pmid":"37558483","id":"PMC_37558483","title":"Auditory Neuropathy Spectrum Disorder Progressing with Motor and Sensory Neuropathy Caused by an ATP1A1 Variant.","date":"2023","source":"Internal medicine (Tokyo, Japan)","url":"https://pubmed.ncbi.nlm.nih.gov/37558483","citation_count":0,"is_preprint":false},{"pmid":"42188099","id":"PMC_42188099","title":"Low Shear Stress Promotes Atherosclerosis by Mediating Pathological Accumulation of Endothelial Lipid Droplets via the KLF4/TFEB/ATP1A1 Axis.","date":"2026","source":"Journal of cardiovascular development and disease","url":"https://pubmed.ncbi.nlm.nih.gov/42188099","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.06.18.25329418","title":"Imputation of fluid intelligence scores reduces ascertainment bias and increases power for analyses of common and rare variants","date":"2025-06-20","source":"bioRxiv","url":"https://doi.org/10.1101/2025.06.18.25329418","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":39189,"output_tokens":8172,"usd":0.120073,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":18127,"output_tokens":4894,"usd":0.106493,"stage2_stop_reason":"end_turn"},"total_usd":0.226566,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2013,\n      \"finding\": \"Somatic hotspot mutations in ATP1A1 (encoding the Na+/K+-ATPase α1 subunit) cause loss of pump activity and strongly reduced affinity for potassium, leading to inappropriate membrane depolarization in adrenal cells and autonomous aldosterone secretion.\",\n      \"method\": \"In vitro functional pump activity assays, electrophysiological ex vivo studies on primary adrenal adenoma cells, exome sequencing\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro functional assays of pump activity and electrophysiology on primary cells, replicated across multiple mutation carriers and independently confirmed in follow-up studies\",\n      \"pmids\": [\"23416519\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The ATP1A1 Gly99Arg mutation (somatic, found in APA) severely impairs ATPase activity, reduces apparent Na+ affinity for phosphorylation and K+ affinity for dephosphorylation (indicating decreased Na+ and K+ binding), and causes membrane depolarization when overexpressed in adrenal cells; structural modeling indicates the substitution interferes with the gateway to the ion binding pocket.\",\n      \"method\": \"In vitro ATPase activity assays, whole-cell patch-clamp electrophysiology in HAC15 adrenal cells, structural homology modeling, CYP11B2 expression assays\",\n      \"journal\": \"Hypertension (Dallas, Tex. : 1979)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal in vitro methods (enzymatic assay, electrophysiology, structural modeling) in a single study, consistent with findings from another independent lab (PMID:23416519)\",\n      \"pmids\": [\"24082052\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"ATP1A1 is required for coronavirus (MHV, FIPV, MERS-CoV) and VSV entry into host cells; cardiotonic steroid binding to ATP1A1 (at nanomolar concentrations that do not inhibit ion transport) activates Src signaling that inhibits virus entry at an early stage, causing virion accumulation near the cell surface and reduced membrane fusion.\",\n      \"method\": \"Gene silencing (siRNA), cardiotonic steroid (ouabain, bufalin) treatment, Src kinase inhibitor rescue experiments, viral infection assays, electron microscopy of virion localization\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal genetic (knockdown) and pharmacological approach with Src inhibitor rescue, multiple viral models tested, mechanistic pathway (ATP1A1→Src signaling→viral entry inhibition) established with multiple orthogonal methods\",\n      \"pmids\": [\"25653449\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ATP1A1 (α1-chain of Na/K-ATPase), but not the β1- or β3-chains (ATP1B1/ATP1B3), is required for efficient unconventional secretion of FGF2; in the absence of β-chains, a direct protein-protein interaction between the cytoplasmic domain of ATP1A1 and FGF2 occurs with submicromolar affinity, suggesting ATP1A1 acts as a recruitment factor for FGF2 at the inner leaflet of the plasma membrane.\",\n      \"method\": \"Unbiased RNAi screen, siRNA knockdown of individual Na/K-ATPase subunits, direct binding assay (cytoplasmic domain of ATP1A1 with FGF2, affinity measurement)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — genome-wide RNAi screen plus direct in vitro protein interaction assay with affinity measurement; single lab but multiple orthogonal approaches\",\n      \"pmids\": [\"25533462\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"De novo mutations in ATP1A1 (catalytic α1 subunit of Na+/K+-ATPase) cause not only loss of pump function but also abnormal cation permeabilities leading to membrane depolarization, underlying renal hypomagnesemia and refractory seizures.\",\n      \"method\": \"Heterologous expression systems (functional characterization of mutant Na+/K+-ATPase α1 subunits), electrophysiology, whole-exome sequencing\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — functional characterization in heterologous expression systems with electrophysiology, multiple patient mutations characterized\",\n      \"pmids\": [\"30388404\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Missense mutations in ATP1A1 cause autosomal-dominant CMT2 (axonal peripheral neuropathy); two-electrode voltage clamp in Xenopus oocytes demonstrated significant reduction in Na+ current activity in some ouabain-insensitive ATP1A1 mutants, indicating loss-of-function of the Na+/K+ pump. ATP1A1 localizes to the axolemma of myelinated sensory and motor axons and to Schmidt-Lanterman incisures.\",\n      \"method\": \"Two-electrode voltage clamp in Xenopus oocytes, immunostaining of peripheral nerve axons, whole-exome/genome sequencing across seven countries\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — electrophysiology in Xenopus oocytes (Tier 1 functional assay) plus direct localization by immunostaining, replicated across multiple families and independent labs\",\n      \"pmids\": [\"29499166\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"ATP1A1 is required for macropinocytic entry of RSV into respiratory epithelial cells; RSV triggers clustering of ATP1A1 in the plasma membrane (dependent on viral attachment glycoprotein G), activating c-Src kinase signaling that transactivates EGFR at Tyr845, culminating in macropinosome formation and RSV uptake.\",\n      \"method\": \"Genome-wide siRNA screen, knockdown validation, live-cell imaging, phosphorylation assays (c-Src, EGFR Tyr845), pharmacological inhibitors (ouabain, PST2238), primary human airway epithelial cell validation\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genome-wide screen with mechanistic follow-up (signaling assays, imaging, inhibitor rescue), validated in primary cells, multiple orthogonal methods in single study\",\n      \"pmids\": [\"31381610\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"In zebrafish brain ventricle development, Atp1a1 (Na,K-ATPase α subunit) regulates neuroepithelial permeability and CSF production downstream; RhoA acts downstream of Atp1a1 (and its regulatory subunit Fxyd1) in neuroepithelium formation and permeability modulation, while CSF production requires Atp1a1 but not Fxyd1 or RhoA.\",\n      \"method\": \"Morpholino knockdown in zebrafish, overexpression, RhoA epistasis experiments, ouabain pharmacological inhibition, intracellular Na+ measurements\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with RhoA established by double loss-of-function, multiple phenotypic readouts; zebrafish model (ortholog), single lab\",\n      \"pmids\": [\"22683378\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ATP1A1 in Sertoli cells regulates tight junctions (claudin 11) and gap junctions (connexin 43) through the Src-EGFR-ERK1/2-CREB signaling pathway; 50 nM ouabain (signaling dose) activates this pathway and increases transepithelial electrical resistance, while 1 mM ouabain (pump-inhibitory dose) has opposite effects; Src and MAPK pathway inhibitors abolish ouabain-induced junctional regulation.\",\n      \"method\": \"Primary Sertoli cell culture, ouabain treatment, signaling inhibitors, immunoblotting, transepithelial electrical resistance measurements, immunofluorescence, mass spectrometry\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — pharmacological dissection with inhibitor rescue and multiple signaling readouts; single lab, pathway placement established by inhibitor epistasis\",\n      \"pmids\": [\"28203706\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ATP1A1 overexpressed in tumor cells binds homophilically to ATP1A1 on fibroblasts (heterocellular contact), inducing calcium oscillations, NF-κB activation, and activin A secretion in fibroblasts, which promotes EMT in tumor cells and myofibroblast activation; silencing ATP1A1 or neutralizing activin A suppresses tumor invasion.\",\n      \"method\": \"Co-immunoprecipitation/pulldown, siRNA silencing, calcium imaging, NF-κB reporter assays, activin A neutralization, invasion assays, primary tumor analysis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — direct binding demonstrated plus downstream signaling (calcium, NF-κB) and functional rescue; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"35618735\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"ATP1A1 knockdown in glioma stem cells inhibits ERK1/2 and AKT pathway activation through suppression of Src phosphorylation, reducing cell proliferation, survival, and causing G1 arrest; rescue of ATP1A1 expression restores proliferation, placing ATP1A1 upstream of Src in this signaling axis.\",\n      \"method\": \"siRNA knockdown, ATP1A1 re-expression rescue, Western blotting for p-Src/p-ERK1/2/p-AKT, cell cycle analysis, apoptosis assays in primary human GSCs\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — loss-of-function with rescue, signaling pathway readouts; single lab, pathway placement by epistasis (ATP1A1→Src→ERK/AKT)\",\n      \"pmids\": [\"31114755\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ATP1A1 L104R mutation (APA-associated) stimulates cell proliferation and increases intracellular Ca2+ (measured by Fluo-4), enhances Src phosphorylation, and increases NKA expression in adrenal cells; low-concentration ouabain further potentiates Src phosphorylation in mutant cells.\",\n      \"method\": \"HAC15 cell transfection with mutant ATP1A1, cell proliferation assays, DNA content analysis, S-phase measurement, Ca2+ fluorescence (Fluo-4), Src phosphorylation (Western blot), transcriptome analysis, immunohistochemistry\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — functional cell assays with multiple readouts (proliferation, Ca2+, Src); single lab, mechanism partially characterized\",\n      \"pmids\": [\"34681640\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"ATP1A1 mutations causing intermediate CMT disease lead to proteasomal degradation of the ATP1A1 protein (loss-of-function), without affecting mRNA levels; the mutations (p.S207F and p.G877S) downregulate protein levels by promoting proteasomal turnover.\",\n      \"method\": \"Whole-exome sequencing, Western blot of protein levels, RT-PCR for mRNA, proteasome inhibitor experiments in patient-derived cells\",\n      \"journal\": \"Human mutation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — protein degradation mechanism established by proteasome inhibitor rescue; single lab\",\n      \"pmids\": [\"31373411\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"GPR37 interacts with ATP1A1 and promotes its ubiquitination-induced proteasomal degradation, thereby limiting activation of the AKT/mTOR signaling pathway; GPR37 knockdown stabilizes ATP1A1 and increases AKT/mTOR activity.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, siRNA knockdown, Western blotting for AKT/mTOR pathway components, ESCC cell functional assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — direct protein interaction (Co-IP) plus functional ubiquitination assay; single lab\",\n      \"pmids\": [\"39730361\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"LncDARS-AS1 directly binds ATP1A1 protein, preventing its interaction with UBQLN4 and subsequent proteasomal degradation, thereby enhancing Na+/K+-ATPase activity and promoting osteosarcoma metastasis; digoxin targeting NKA inhibits tumor growth.\",\n      \"method\": \"ChIRP, mass spectrometry, molecular docking, molecular dynamics simulations, ion-sensitive fluorescent indicators, enzymatic NKA assays, siRNA silencing, in vivo xenograft models\",\n      \"journal\": \"Advanced science (Weinheim, Baden-Wurttemberg, Germany)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — direct RNA-protein interaction validated by ChIRP and MS, functional NKA activity measured enzymatically; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"40665639\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Periplogenin (PPG) directly binds ATP1A1 at residue T804 (forming a hydrogen bond); T804A substitution abolishes PPG binding and confers resistance to PPG's cytotoxic activity both in vitro and in vivo in xenograft models.\",\n      \"method\": \"Forward genetic chemical mutagenesis screen, next-generation sequencing, in vitro hydrogen bond modeling, T804A mutagenesis, in vivo xenograft comparison of wild-type vs. T804A DU145 cells\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — forward genetic screen identifying single amino acid resistance mutation, validated in vivo; direct binding site identified at atomic resolution with in vivo confirmation\",\n      \"pmids\": [\"39227746\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ATP1A1 serves as a receptor/attachment factor for PEDV; the CT structural domain of ATP1A1 interacts with PEDV S1 protein; knockdown or inhibition of ATP1A1 significantly reduces PEDV attachment; ATP1A1 co-localizes with PEDV S1 at the cell surface in early infection.\",\n      \"method\": \"Virus overlay protein binding assay (VOPBA), mass spectrometry, siRNA knockdown, overexpression, co-localization (immunofluorescence), antibody blocking assay, ouabain/PST2238 inhibitor treatment\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — direct binding identified by VOPBA/MS, functional knockdown and antibody blocking confirm role in attachment; single lab\",\n      \"pmids\": [\"36835408\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ATP1A1-linked CMT disease requires a malfunctioning (dominant-negative or gain-of-abnormal-function) protein product from one allele; haploinsufficiency (protein-null heterozygosity) alone is not sufficient to cause disease in Atp1a1+/- mice up to 18 months, and a human carrier of a protein-null truncation variant (p.Y148*) was phenotypically normal.\",\n      \"method\": \"Atp1a1+/- heterozygous knockout mouse neuromuscular phenotyping (up to 18 months, with exercise), clinical phenotyping of human protein-null variant carrier\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean in vivo knockout with longitudinal phenotyping plus human carrier clinical data; single lab, haploinsufficiency model refuted\",\n      \"pmids\": [\"37659504\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The ATP1A1 Pro600Ala mutation (CMT2-causing) is associated with defective in vitro neuronal differentiation of patient-derived iPSCs; ATP1A1 protein is undetectable in the few neurons derived from CMT iPSCs, and electrophysiological properties of derived neurons are less mature compared to controls.\",\n      \"method\": \"Patient-specific iPSC generation, neural differentiation assays, immunostaining for ATP1A1, electrophysiology of derived neurons\",\n      \"journal\": \"Journal of the peripheral nervous system : JPNS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — patient-derived iPSC model with functional readouts; single lab, mechanism of neuronal loss partially established\",\n      \"pmids\": [\"31707753\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In a pancreatic ductal adenocarcinoma context, resibufogenin (RB) binds ATP1A1, activating Na+-K+-ATPase as a signaling receptor that triggers the intracellular MAPK/ERK pathway and Ca2+-mediated Src/FAK/Paxillin focal adhesion pathway, leading to G2/M arrest and inhibition of invasion.\",\n      \"method\": \"Drug-target binding assays, MAPK/ERK pathway analysis, Ca2+ measurement, Src/FAK/Paxillin phosphorylation assays, cell cycle analysis, invasion assays in GBM cell lines\",\n      \"journal\": \"Frontiers in pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — multiple signaling pathway readouts after drug-target engagement; single lab, mechanism inferred from pharmacological binding + downstream effects\",\n      \"pmids\": [\"35656311\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"shRNA screen identified ATP1A1 knockdown as conferring sensitivity to aurilide B (a mitochondria-mediated apoptosis inducer); combined ATP1A1 inhibition with ouabain potentiated aurilide B sensitivity, suggesting ATP1A1 regulates mitochondria-mediated apoptosis.\",\n      \"method\": \"Genome-wide pooled shRNA screen with deep sequencing, ouabain combination treatment, cell viability assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — shRNA screen identification plus pharmacological combination, no direct mechanistic dissection of how ATP1A1 regulates apoptosis; single lab\",\n      \"pmids\": [\"28515454\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ATP1A1 regulates tumor progression in colon cancer through the ERK5 signaling pathway; ATP1A1 knockdown suppresses cell proliferation, migration, and invasion and induces apoptosis, with microarray revealing altered expression of EGFR, MAP2K5, MAPK7, FOS, MYC, and BAD.\",\n      \"method\": \"siRNA knockdown in HT29 and Caco2 cells, microarray gene expression profiling, cell proliferation/migration/invasion/apoptosis assays\",\n      \"journal\": \"Annals of surgical oncology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — loss-of-function with microarray, ERK5 pathway placement is inferential from expression data without direct pathway rescue; single lab\",\n      \"pmids\": [\"37407874\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In ATP1A1 mutant APA, the VDR gene promoter is hypomethylated compared to non-functioning adenoma, leading to high VDR expression; VDR suppression abrogates ATP1A1 mutation-mediated cell proliferation in HAC15 cells, placing VDR downstream of ATP1A1 mutation in the proliferative signaling cascade.\",\n      \"method\": \"DNA methylation analysis, qPCR, siRNA VDR knockdown in HAC15 cells transfected with mutant ATP1A1, cell proliferation assays\",\n      \"journal\": \"Molecular and cellular endocrinology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — epigenetic and knockdown data; single lab, indirect pathway connection (mutation→methylation→VDR→proliferation)\",\n      \"pmids\": [\"35257799\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ATP1A1 overexpression in RCC cells inhibits proliferation and migration by increasing ROS production and suppressing the Raf/MEK/ERK signaling pathway, while downregulation of ATP1A1 promotes cancer development.\",\n      \"method\": \"Exogenous overexpression, CCK-8 proliferation assay, Boyden chamber migration assay, flow cytometry for apoptosis and ROS, Western blotting for Raf/MEK/ERK pathway\",\n      \"journal\": \"Clinical proteomics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — overexpression with signaling readouts; single lab, ROS mechanism inferred without mechanistic dissection\",\n      \"pmids\": [\"28484360\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"LAPTM4B interacts with ATP1A1 and prevents its TRIM8-mediated K63-linked ubiquitination and proteasomal degradation, stabilizing ATP1A1 to enhance lysosomal acidification and promote EGFR-TKI resistance in NSCLC.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays (K63-linkage-specific), siRNA knockdown, Western blotting, lysosomal pH assays, clinical sample analysis\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — direct protein interaction (Co-IP) plus specific ubiquitination assay (K63-linked); single lab but multiple orthogonal methods\",\n      \"pmids\": [\"41362742\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ATP1A1 is required for PRRSV-2 attachment and internalization; PRRSV glycoprotein GP4 interacts with the fourth extracellular region (ER4) of ATP1A1 (dependent on GP4 C-terminus); ATP1A1-Src signaling activates EGFR and caveolin-1 to enable viral uptake via macropinocytosis and caveolae/raft-mediated endocytosis; internalized virions traffic to ATP1A1/CD163-positive early endosomes for uncoating.\",\n      \"method\": \"siRNA knockdown, overexpression, co-localization imaging, signaling inhibitor assays, GP4-ER4 interaction mapping, synthetic peptide competition, nanobody targeting, multiple PRRSV lineage testing\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (genetic, pharmacological, peptide competition, nanobody), direct domain interaction mapped; single lab\",\n      \"pmids\": [\"41773865\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ATP1A1 directly binds odoamide (marine natural product); mutations at Gly98 and Gly99 of ATP1A1 confer resistance to odoamide cytotoxicity; ATP1A1 was identified as the specific binding protein by affinity chromatography with an odoamide probe, and odoamide-induced apoptotic cell death is critically dependent on interaction with ATP1A1.\",\n      \"method\": \"Affinity chromatography with odoamide probe, mass spectrometry, resistance mutation mapping (Gly98/Gly99), cell viability assays, JFCR39 cancer panel fingerprinting, gene expression profiling\",\n      \"journal\": \"Chembiochem : a European journal of chemical biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — affinity-based target identification plus resistance mutation mapping identifies binding site; single lab\",\n      \"pmids\": [\"39754293\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Mouse embryos homozygous for a null mutation in the Na/K-ATPase α1-subunit (Atp1a1) are able to undergo compaction and cavitation (blastocyst formation), demonstrating that the α1-isozyme is not strictly required for trophectoderm fluid transport during preimplantation development; other Na/K-ATPase isoforms provide functional redundancy.\",\n      \"method\": \"Targeted gene disruption (Atp1a1 knockout mice), embryo development analysis from heterozygous matings, morphological assessment of compaction and cavitation\",\n      \"journal\": \"Mechanisms of development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean knockout with defined developmental phenotype (negative result: no cavitation defect); single lab\",\n      \"pmids\": [\"15147760\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Calcium oxalate crystal deposition decreases ATP1A1 expression in renal cells and activates the ATP1A1/Src/ROS/p38/JNK/NF-κB signaling pathway; overexpression of ATP1A1 or treatment with pNaKtide (specific inhibitor of ATP1A1/Src complex) inhibits this signaling cascade and reduces crystal-cell adhesion and stone formation.\",\n      \"method\": \"In vitro crystal deposition experiments, in vivo mouse models, ATP1A1 overexpression, pNaKtide treatment, Western blotting for Src/ROS/p38/JNK/NF-κB\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — in vitro and in vivo experiments with both gain-of-function and pharmacological inhibition; single lab, signaling pathway placed downstream of ATP1A1\",\n      \"pmids\": [\"36871182\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ATP1A1 loss-of-function variant Gly903Arg (p.G903R) causes significantly reduced HEK cell viability in an ouabain resistance assay, demonstrating loss of ATPase function; variant is associated with developmental delay, intellectual disability, and autism.\",\n      \"method\": \"Whole-exome/genome sequencing, ouabain resistance/cell viability assay in transfected HEK cells with ouabain-insensitive ATP1A1 constructs\",\n      \"journal\": \"Annals of clinical and translational neurology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — ouabain resistance functional assay in HEK cells; single study, one mutation\",\n      \"pmids\": [\"38504481\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Hereditary spastic paraplegia (HSP) is caused by a de novo ATP1A1 variant (p.L337P); functional studies in heterologous expression systems and homology modeling provide evidence for pathogenicity of this variant affecting Na+/K+-ATPase function.\",\n      \"method\": \"Whole-exome sequencing, functional characterization in heterologous expression systems, homology modeling\",\n      \"journal\": \"Clinical genetics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — functional characterization mentioned but abstract provides limited mechanistic detail; single case with heterologous expression\",\n      \"pmids\": [\"31705535\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Combined knockdown of ATP1A1 (Na/K-ATPase) and GJA5 (connexin 40) in iPSC-derived atrial myocardium induces significant beat irregularity and increased conduction heterogeneity, establishing these two factors as cooperative drivers of arrhythmogenesis; this is consistent with an inflammation-driven AF mechanism where both intercellular connectivity and ion flux are disrupted.\",\n      \"method\": \"siRNA knockdown in 3D iPSC-derived atrial myocardium tissue model, sharp electrode recordings, calcium imaging, multi-electrode assays, lentiviral overexpression, pharmacological modulation, in silico modeling\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic (siRNA) and pharmacological manipulation in 3D tissue model with multiple electrophysiological readouts, validated by in silico model; single lab\",\n      \"pmids\": [\"41868610\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ATP1A1 encodes the ubiquitously expressed α1 catalytic subunit of the Na+/K+-ATPase heterodimer, which hydrolyzes ATP to extrude Na+ and import K+ across the plasma membrane; beyond ion pumping, ATP1A1 acts as a signaling receptor—cardiotonic steroid or ligand binding activates Src kinase, which transactivates EGFR and downstream ERK/AKT pathways to regulate cell proliferation, junctional integrity, unconventional FGF2 secretion, viral entry (via macropinocytosis), and EMT; pathogenic missense mutations cause loss of pump function with abnormal cation permeabilities and membrane depolarization, driving diseases including primary aldosteronism, Charcot-Marie-Tooth neuropathy, hereditary spastic paraplegia, and hypomagnesemia with seizures, while haploinsufficiency alone is insufficient to cause disease, requiring a dominant malfunctioning protein product.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ATP1A1 encodes the catalytic α1 subunit of the Na+/K+-ATPase, an ATP-driven plasma-membrane ion pump whose activity governs cation gradients, membrane potential, and—in mutant or ligand-bound states—intracellular signaling [#0, #1, #4]. Somatic and germline missense mutations impair pump function and reduce Na+ and K+ binding affinities, producing abnormal cation permeabilities and membrane depolarization; structural modeling localizes representative substitutions (e.g. Gly99Arg) to the gateway of the ion-binding pocket [#1, #4]. This pump dysfunction underlies aldosterone-producing adrenal adenoma through inappropriate depolarization and autonomous aldosterone secretion [#0, #1], and additional pathogenic variants cause renal hypomagnesemia with seizures [#4], axonal Charcot-Marie-Tooth neuropathy where ATP1A1 localizes to the axolemma and Schmidt-Lanterman incisures [#5], hereditary spastic paraplegia [#30], and a neurodevelopmental phenotype with intellectual disability [#29]. CMT-causing variants act not through haploinsufficiency—protein-null heterozygosity is tolerated in mice and humans—but require a malfunctioning dominant protein product, in some cases destabilized by proteasomal degradation [#12, #17]. Beyond ion transport, ATP1A1 functions as a signaling receptor: cardiotonic steroids or natural-product ligands bind it (with a defined binding site at residue T804 and resistance residues Gly98/Gly99) to trigger Src kinase activation, EGFR transactivation, and downstream ERK/AKT and Ca2+ signaling that regulates cell proliferation, junctional integrity, and EMT [#8, #10, #11, #15, #26]. This Src-coupled receptor function is exploited by multiple viruses—coronaviruses, RSV, PEDV, and PRRSV—which engage ATP1A1 as an attachment/entry factor driving macropinocytic and caveolae-mediated uptake [#2, #6, #16, #25]. ATP1A1 also acts as an unconventional secretion factor, recruiting FGF2 at the inner plasma-membrane leaflet via a direct cytoplasmic-domain interaction [#3], and mediates heterocellular tumor-fibroblast contact through homophilic ATP1A1 binding that promotes invasion [#9]. ATP1A1 protein abundance is controlled by ubiquitin-dependent turnover involving GPR37, TRIM8/LAPTM4B, and UBQLN4, linking its stability to AKT/mTOR and lysosomal signaling [#13, #14, #24].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Tested whether the α1 isoform is individually essential, establishing functional redundancy among Na/K-ATPase isoforms during early development.\",\n      \"evidence\": \"Targeted Atp1a1 knockout mouse embryos assessed for compaction and cavitation\",\n      \"pmids\": [\"15147760\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not address tissue-specific requirements later in development\", \"No assessment of adult phenotype from this null allele\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Placed Atp1a1 upstream of RhoA in regulating neuroepithelial permeability, separating its ion-transport role in CSF production from its developmental signaling role.\",\n      \"evidence\": \"Morpholino knockdown and RhoA epistasis in zebrafish brain ventricle development\",\n      \"pmids\": [\"22683378\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking pump activity to RhoA is undefined\", \"Ortholog model; human relevance not tested\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Established the disease mechanism for aldosterone-producing adenoma: hotspot mutations cause loss of pump activity and reduced K+ affinity, depolarizing adrenal cells to drive autonomous aldosterone secretion.\",\n      \"evidence\": \"Pump activity and ATPase assays, patch-clamp electrophysiology in adrenal cells, structural modeling, exome sequencing\",\n      \"pmids\": [\"23416519\", \"24082052\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not explain how depolarization couples to CYP11B2/aldosterone transcription mechanistically\", \"Tumor proliferation link not addressed\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defined a non-pump function: ATP1A1 directly recruits FGF2 to the inner membrane leaflet for unconventional secretion, independent of β-subunits.\",\n      \"evidence\": \"Genome-wide RNAi screen plus direct in vitro binding affinity measurement\",\n      \"pmids\": [\"25533462\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the FGF2 interaction not resolved\", \"How recruitment couples to membrane translocation unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identified ATP1A1 as a host entry factor for enveloped viruses and showed cardiotonic-steroid-triggered Src signaling can block entry, separating signaling from ion-transport function.\",\n      \"evidence\": \"siRNA silencing, cardiotonic steroid treatment, Src inhibitor rescue, viral infection and EM across multiple viruses\",\n      \"pmids\": [\"25653449\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct viral-ATP1A1 binding not mapped here\", \"How Src signaling blocks fusion mechanistically unclear\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Demonstrated dose-dependent receptor signaling: low ouabain activates Src-EGFR-ERK-CREB to strengthen junctions, while pump-inhibitory doses act oppositely.\",\n      \"evidence\": \"Primary Sertoli cell culture, ouabain dosing, signaling inhibitors, TER measurements\",\n      \"pmids\": [\"28203706\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Endogenous ligand for signaling-dose activation unknown\", \"Single cell type\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Extended the pathogenic mechanism to abnormal cation permeabilities and to axonal CMT neuropathy, with direct evidence of axolemmal localization.\",\n      \"evidence\": \"Heterologous expression and Xenopus oocyte electrophysiology of patient mutants, peripheral nerve immunostaining, exome sequencing\",\n      \"pmids\": [\"30388404\", \"29499166\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How abnormal cation leak versus pump loss differentially drives renal vs neuronal phenotypes unresolved\", \"Cell-type vulnerability basis unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed ATP1A1 drives macropinocytic viral entry via clustering-dependent Src/EGFR-Tyr845 signaling, and that some CMT variants act via proteasomal protein loss rather than mRNA loss.\",\n      \"evidence\": \"Genome-wide siRNA screen, phosphorylation assays, imaging, inhibitors in airway cells; proteasome inhibitor and mRNA analysis in patient cells; iPSC neuronal differentiation\",\n      \"pmids\": [\"31381610\", \"31373411\", \"31707753\", \"31114755\", \"31705535\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether degradation-prone variants act dominant-negatively or by simple loss not fully separated\", \"HSP variant mechanism only partially characterized\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Linked APA mutations to proliferative signaling by showing the L104R mutant raises intracellular Ca2+ and Src phosphorylation, connecting pump dysfunction to tumor growth.\",\n      \"evidence\": \"Mutant transfection in HAC15 cells, proliferation, Ca2+ fluorescence, Src phosphorylation, transcriptomics\",\n      \"pmids\": [\"34681640\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct causal chain from Ca2+/Src to proliferation not isolated\", \"Single cell line\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Revealed ATP1A1 as a homophilic heterocellular adhesion/signaling molecule driving tumor EMT through fibroblast NF-κB and activin A secretion.\",\n      \"evidence\": \"Co-IP/pulldown, calcium imaging, NF-κB reporters, activin A neutralization, invasion assays\",\n      \"pmids\": [\"35618735\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of homophilic trans-binding unresolved\", \"Single tumor model context\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Established ubiquitin-dependent control of ATP1A1 abundance (GPR37, TRIM8/LAPTM4B) coupling its stability to AKT/mTOR and lysosomal signaling, and confirmed that disease requires a dominant malfunctioning product, not haploinsufficiency.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, knockdowns in cancer cells; Atp1a1+/- mouse longitudinal phenotyping and human protein-null carrier\",\n      \"pmids\": [\"39730361\", \"41362742\", \"37659504\", \"36835408\", \"37407874\", \"36871182\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Hierarchy among competing E3/stabilizing factors unknown\", \"Physiological contexts where each turnover pathway dominates undefined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Mapped a defined small-molecule binding site (T804) on ATP1A1 through a resistance mutation, establishing it as a druggable target whose engagement drives cytotoxicity.\",\n      \"evidence\": \"Forward genetic chemical mutagenesis screen, hydrogen-bond modeling, T804A mutagenesis, in vivo xenografts\",\n      \"pmids\": [\"39227746\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream pathway from T804 ligand binding not detailed here\", \"Generality across other cardiotonic ligands not tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified additional ligand binding determinants (Gly98/Gly99) and a lncRNA (DARS-AS1) that competes with UBQLN4 to stabilize ATP1A1 and enhance pump activity, linking abundance control to metastasis.\",\n      \"evidence\": \"Affinity chromatography and resistance mapping for odoamide; ChIRP/MS, docking, NKA enzymatic assays and xenografts for lncDARS-AS1\",\n      \"pmids\": [\"39754293\", \"40665639\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether multiple ligand-binding residues form a single common pocket unresolved\", \"How enhanced pump activity drives metastasis mechanistically unclear\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Showed ATP1A1 cooperates with GJA5 in atrial conduction, linking ion flux to arrhythmogenesis.\",\n      \"evidence\": \"Combined siRNA knockdown in 3D iPSC-derived atrial tissue with electrophysiology and in silico modeling\",\n      \"pmids\": [\"41868610\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular interaction with GJA5 not established\", \"Mechanism of conduction heterogeneity undefined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single protein partitions between its canonical ion-pump function and its Src-coupled receptor, FGF2-recruitment, and homophilic adhesion functions—and what governs that switch in vivo—remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model distinguishing signaling-competent from pumping conformations\", \"Endogenous physiological ligands for signaling-dose activation unidentified\", \"Tissue determinants of disease-variant selectivity unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [0, 1, 4, 14]},\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [0, 1, 4, 5]},\n      {\"term_id\": \"GO:0001618\", \"supporting_discovery_ids\": [2, 6, 16, 25]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [2, 6, 8, 10]},\n      {\"term_id\": \"GO:0098631\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [3, 5, 6, 16]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [0, 1, 4]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 6, 8, 10]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [0, 4, 5, 29, 30]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [6, 25]}\n    ],\n    \"complexes\": [\"Na+/K+-ATPase (α1/β heterodimer)\"],\n    \"partners\": [\"FGF2\", \"SRC\", \"EGFR\", \"GPR37\", \"LAPTM4B\", \"TRIM8\", \"UBQLN4\", \"FXYD1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":8,"faith_total":8,"faith_pct":100.0}}