{"gene":"ATP2B3","run_date":"2026-04-28T17:12:37","timeline":{"discoveries":[{"year":2013,"finding":"Somatic hotspot mutations in ATP2B3 (encoding plasma membrane Ca2+ ATPase) were identified in aldosterone-producing adenomas (APAs). Electrophysiological ex vivo studies on primary adrenal adenoma cells provided evidence for inappropriate membrane depolarization in cells with ATP2B3 alterations, leading to autonomous aldosterone secretion.","method":"Exome sequencing, functional in vitro studies, electrophysiological ex vivo patch-clamp","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (exome sequencing, functional assays, electrophysiology) replicated across large APA cohort","pmids":["23416519"],"is_preprint":false},{"year":2013,"finding":"Somatic ATP2B3 mutations in APAs result in upregulation of CYP11B2 (aldosterone synthase) gene expression and its transcriptional regulator NR4A2 in HAC15 adrenal cells, establishing a mechanistic link between ATP2B3 loss-of-function and dysregulated aldosterone production.","method":"Overexpression in HAC15 adrenal cells, gene expression assays, whole-cell patch-clamp","journal":"Hypertension","confidence":"High","confidence_rationale":"Tier 2 — reciprocal functional assays with multiple readouts (CYP11B2 expression, NR4A2, patch-clamp) in relevant cell line","pmids":["24082052"],"is_preprint":false},{"year":2016,"finding":"The APA-associated ATP2B3 Leu425_Val426del mutation causes: (1) loss of physiological Ca2+ pump function (reduced Ca2+ export capacity); (2) increased Ca2+ influx via depolarization-activated Ca2+ channels; and (3) a Na+-dependent inward current that strongly depolarizes the plasma membrane—collectively promoting CYP11B2 expression and aldosterone production.","method":"Ca2+ measurements in NCI-H295R and HEK-293 cells, patch-clamp electrophysiology, mRNA expression assays, aldosterone production assays","journal":"Endocrinology","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal methods including ion transport assays, electrophysiology, and functional aldosterone readout in two cell lines","pmids":["27035656"],"is_preprint":false},{"year":2007,"finding":"14-3-3epsilon protein interacts with PMCA3 (ATP2B3) in a phosphorylation-independent manner, and this interaction inhibits PMCA3 pump activity, reducing the ability of cells to restore basal Ca2+ concentration following an InsP3-induced Ca2+ transient.","method":"Two-hybrid assay, co-immunoprecipitation in HeLa cells, GST pull-down, aequorin-based Ca2+ measurement in CHO cells","journal":"Cell calcium","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal binding assays plus functional Ca2+ extrusion readout","pmids":["18029012"],"is_preprint":false},{"year":1995,"finding":"PMCA3 protein is expressed in rat brain neurons and localizes primarily to granule cell processes in the cerebellum (granule cell and molecular layers), choroid plexus, and hippocampal dendritic fields, with localization pattern co-distributing with the axonal marker GAP-43 in cerebellar molecular layer.","method":"In situ hybridization, immunoblot with affinity-purified anti-peptide antibodies, immunohistochemistry","journal":"Brain research. Molecular brain research","confidence":"Medium","confidence_rationale":"Tier 2 — direct localization by IHC/ISH with functional context (neuronal processes), single lab","pmids":["7770003"],"is_preprint":false},{"year":1994,"finding":"ATP2B3 (PMCA3) was localized to human chromosome Xq28 by fluorescence in situ hybridization (FISH), somatic cell hybrid analysis, and genetic linkage analysis, identifying it as a candidate gene for X-linked neurological diseases mapping to distal Xq.","method":"FISH, somatic cell hybrid analysis, genetic linkage analysis","journal":"Cytogenetics and cell genetics","confidence":"Medium","confidence_rationale":"Tier 2 — direct chromosomal localization by multiple methods, single lab","pmids":["8187550"],"is_preprint":false},{"year":2017,"finding":"A novel PMCA3 G733R mutation in the catalytic P-domain impairs the pump's ability to control cellular Ca2+ handling under both basal and stimulated conditions, with homology modeling and molecular dynamics indicating the mutation disrupts the 3D configuration of the local P-domain structure.","method":"Biochemical Ca2+ handling assays in cells, homology modeling, molecular dynamics simulation","journal":"Biochimica et biophysica acta. Molecular basis of disease","confidence":"Medium","confidence_rationale":"Tier 2–3 — functional cell-based Ca2+ assay combined with structural modeling; single lab","pmids":["28807751"],"is_preprint":false},{"year":2022,"finding":"The ataxia-linked E1081Q mutation in ATP2B3 immediately upstream of the calmodulin-binding domain has a splicing variant-dependent effect: in the full-length 'b' variant it abolishes sub-plasma membrane Ca2+ reduction capacity, while in the truncated 'a' variant it increases Ca2+ extrusion activity in sub-PM microdomains.","method":"Biochemical Ca2+ pump activity assays, sub-plasma membrane Ca2+ microdomain measurements, molecular studies of splice variants","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 — functional assays with splice-variant distinction; single lab, moderate evidence","pmids":["36207321"],"is_preprint":false},{"year":2012,"finding":"Downregulation of PMCA3 in PC12 cells accelerates differentiation, forms longer neurites, alters expression of voltage-dependent Ca2+ channels (VDCCs) increasing their contribution to Ca2+ influx, and triggers compensatory upregulation of constitutive PMCA1/PMCA4 isoforms and SERCA.","method":"Antisense stable transfection in PC12 cells, dibutyryl-cAMP differentiation assay, Ca2+ influx measurements, immunoblot for PMCA isoforms and SERCA","journal":"Cell calcium","confidence":"Medium","confidence_rationale":"Tier 2–3 — loss-of-function with defined cellular phenotype and compensatory pathway placement; single lab","pmids":["22921123"],"is_preprint":false},{"year":2021,"finding":"A novel somatic ATP2B3 K416_F418delinsN mutation in APA causes increased CYP11B2 expression and aldosterone production when transfected into HAC15 adrenal cells, confirming functional pathogenicity of mutations in the codon 416–418 region.","method":"Somatic mutation sequencing, HAC15 cell transfection, CYP11B2 expression and aldosterone production assay","journal":"Cancers","confidence":"Low","confidence_rationale":"Tier 3 — single functional assay in cell line, single case report","pmids":["34572956"],"is_preprint":false},{"year":2023,"finding":"ATP2B3 inhibition (knockdown) in HT-22 neuronal cells alleviates erastin-induced ferroptosis by modulating the P62-KEAP1-NRF2-HO-1 oxidative stress pathway, reducing ROS production and reversing ferroptosis-associated cell death.","method":"siRNA knockdown in HT-22 cells, ROS measurement, protein expression analysis (P62, NRF2, HO-1, NQO1, KEAP1), cell viability assay, TMT-based proteomics","journal":"International journal of molecular sciences","confidence":"Low","confidence_rationale":"Tier 3 — single lab, pathway placement by KD/OE without direct mechanistic link to pump activity","pmids":["37298147"],"is_preprint":false},{"year":2011,"finding":"PMCA3 knockdown in GH3 pituitary cells results in increased GAD65 expression, indicating that PMCA3 participates in the regulation of GABA synthesis through Ca2+ homeostasis maintenance.","method":"Antisense knockdown in GH3 cells, enzyme activity assays (GAD, GABA-T), immunoblot","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 — single lab, single method, indirect pathway link","pmids":["21798237"],"is_preprint":false}],"current_model":"ATP2B3 (PMCA3) is a plasma membrane Ca2+-ATPase that actively extrudes Ca2+ from the cytoplasm to maintain Ca2+ homeostasis, particularly in neurons and adrenal cells; somatic loss-of-function mutations in its transmembrane domain (near codons 416–426) reduce Ca2+ export, trigger membrane depolarization and Na+-dependent inward currents, increase intracellular Ca2+, and thereby activate CYP11B2 expression and aldosterone production in aldosterone-producing adenomas, while its activity is also inhibited by 14-3-3epsilon binding and mutations in its catalytic P-domain or calmodulin-binding domain cause cerebellar ataxia by impairing sub-plasma membrane Ca2+ microdomain regulation."},"narrative":{"teleology":[{"year":1994,"claim":"Mapping ATP2B3 to Xq28 established it as an X-linked candidate for neurological disease, framing subsequent genetic studies.","evidence":"FISH, somatic cell hybrid, and linkage analysis in human samples","pmids":["8187550"],"confidence":"Medium","gaps":["No disease-causing mutations identified at this stage","Functional relevance of chromosomal position not tested"]},{"year":1995,"claim":"Demonstrating that PMCA3 protein localizes to cerebellar granule cell processes and hippocampal dendritic fields revealed its neuron-specific Ca²⁺ extrusion role, distinguishing it from ubiquitous PMCA isoforms.","evidence":"Immunohistochemistry and in situ hybridization in rat brain","pmids":["7770003"],"confidence":"Medium","gaps":["Localization shown only in rat; human tissue confirmation lacking","No functional consequence of regional expression tested"]},{"year":2007,"claim":"Identification of 14-3-3ε as a phosphorylation-independent inhibitor of PMCA3 pump activity established the first known regulatory partner that attenuates Ca²⁺ clearance.","evidence":"Two-hybrid, co-immunoprecipitation, GST pull-down, aequorin-based Ca²⁺ measurements in CHO cells","pmids":["18029012"],"confidence":"High","gaps":["Physiological context of 14-3-3ε regulation (e.g., neuronal vs. adrenal) not addressed","Binding site on PMCA3 not mapped"]},{"year":2012,"claim":"Showing that PMCA3 knockdown in PC12 cells accelerates neurite outgrowth and triggers compensatory upregulation of PMCA1/4 and SERCA demonstrated that PMCA3 loss remodels Ca²⁺ signaling networks during neuronal differentiation.","evidence":"Antisense stable transfection in PC12 cells with differentiation assays and immunoblot","pmids":["22921123"],"confidence":"Medium","gaps":["Relevance to in vivo neuronal development not tested","Compensatory mechanisms complicate isolation of PMCA3-specific roles"]},{"year":2013,"claim":"Discovery of recurrent somatic ATP2B3 mutations in aldosterone-producing adenomas, coupled with electrophysiological evidence for membrane depolarization and CYP11B2/NR4A2 upregulation, established a direct mechanistic link between PMCA3 loss-of-function and autonomous aldosterone secretion.","evidence":"Exome sequencing of APA cohort; patch-clamp on primary adenoma cells; overexpression in HAC15 cells with gene expression readout","pmids":["23416519","24082052"],"confidence":"High","gaps":["Precise structural basis for mutation-induced ion leak not resolved","Whether mutations act purely through loss of Ca²⁺ extrusion or also gain-of-function Na⁺ conductance remained unclear"]},{"year":2016,"claim":"Mechanistic dissection of the L425_V426del mutation resolved that APA-linked ATP2B3 mutations simultaneously abolish Ca²⁺ pump function and introduce a Na⁺-dependent inward current, explaining both elevated intracellular Ca²⁺ and membrane depolarization.","evidence":"Ca²⁺ imaging in NCI-H295R and HEK-293 cells, patch-clamp electrophysiology, aldosterone production assays","pmids":["27035656"],"confidence":"High","gaps":["Na⁺ conductance pathway through the mutant pump not structurally characterized","In vivo confirmation of dual mechanism lacking"]},{"year":2017,"claim":"Identification of the catalytic P-domain G733R mutation as impairing Ca²⁺ handling, supported by molecular dynamics, linked PMCA3 catalytic domain integrity to cerebellar ataxia pathogenesis.","evidence":"Cellular Ca²⁺ assays combined with homology modeling and molecular dynamics","pmids":["28807751"],"confidence":"Medium","gaps":["No patient genotype–phenotype segregation data provided","Structural model based on homology, not experimental structure"]},{"year":2022,"claim":"Demonstrating that the ataxia-linked E1081Q mutation near the calmodulin-binding domain has opposite effects in the 'a' versus 'b' splice variants revealed that PMCA3 splice context determines whether a mutation causes gain or loss of sub-PM Ca²⁺ extrusion.","evidence":"Sub-plasma membrane Ca²⁺ microdomain measurements and pump activity assays comparing splice variants","pmids":["36207321"],"confidence":"Medium","gaps":["Relative expression of splice variants in cerebellar neurons not quantified","How splice-specific effects translate to selective cerebellar vulnerability unknown"]},{"year":null,"claim":"Key unresolved questions include the high-resolution structural basis for mutation-induced Na⁺ leak through PMCA3, the in vivo contribution of 14-3-3ε regulation to neuronal or adrenal Ca²⁺ homeostasis, and whether PMCA3 splice variant ratios explain cell-type-specific disease manifestations.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No cryo-EM or crystal structure of PMCA3 available","In vivo knockout/knockin mouse models with neuronal or adrenal phenotyping not reported in the timeline","Relative pathogenic contribution of Ca²⁺ extrusion loss versus Na⁺ leak in APA not resolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[0,2,3,6,7]},{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[2,3,6,7]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,2,3,4,7]}],"pathway":[{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[0,2,3,6,7]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,2]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[4,8]}],"complexes":[],"partners":["YWHAE","CYP11B2","NR4A2"],"other_free_text":[]},"mechanistic_narrative":"ATP2B3 (PMCA3) is a plasma membrane Ca²⁺-ATPase that extrudes cytoplasmic Ca²⁺ to maintain intracellular calcium homeostasis, with critical roles in neuronal signaling and adrenal aldosterone regulation. In the brain, PMCA3 localizes to cerebellar granule cell processes and hippocampal dendritic fields, where it controls sub-plasma membrane Ca²⁺ microdomains; mutations in its catalytic P-domain (G733R) or calmodulin-binding region (E1081Q) impair Ca²⁺ extrusion in a splice variant–dependent manner and cause cerebellar ataxia [PMID:28807751, PMID:36207321, PMID:7770003]. In adrenal aldosterone-producing adenomas, somatic loss-of-function mutations near transmembrane codons 416–426 abolish Ca²⁺ export, generate Na⁺-dependent inward currents that depolarize the plasma membrane, elevate intracellular Ca²⁺, and drive CYP11B2 (aldosterone synthase) expression and autonomous aldosterone production [PMID:23416519, PMID:27035656, PMID:24082052]. PMCA3 activity is negatively regulated by phosphorylation-independent binding of 14-3-3ε, which reduces Ca²⁺ clearance following intracellular Ca²⁺ transients [PMID:18029012]."},"prefetch_data":{"uniprot":{"accession":"Q16720","full_name":"Plasma membrane calcium-transporting ATPase 3","aliases":["Plasma membrane calcium ATPase isoform 3","Plasma membrane calcium pump isoform 3"],"length_aa":1220,"mass_kda":134.2,"function":"ATP-driven Ca(2+) ion pump involved in the maintenance of basal intracellular Ca(2+) levels at the presynaptic terminals (PubMed:18029012, PubMed:22912398, PubMed:25953895, PubMed:27035656). Uses ATP as an energy source to transport cytosolic Ca(2+) ions across the plasma membrane to the extracellular compartment (PubMed:25953895, PubMed:27035656). May counter-transport protons, but the mechanism and the stoichiometry of this Ca(2+)/H(+) exchange remains to be established (By similarity)","subcellular_location":"Cell membrane; Presynaptic cell membrane","url":"https://www.uniprot.org/uniprotkb/Q16720/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ATP2B3","classification":"Not Classified","n_dependent_lines":5,"n_total_lines":1208,"dependency_fraction":0.0041390728476821195},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"ATP2B1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/ATP2B3","total_profiled":1310},"omim":[{"mim_id":"302500","title":"SPINOCEREBELLAR ATAXIA, X-LINKED 1; SCAX1","url":"https://www.omim.org/entry/302500"},{"mim_id":"300014","title":"ATPase, Ca(2+)-TRANSPORTING, PLASMA MEMBRANE, 3; ATP2B3","url":"https://www.omim.org/entry/300014"},{"mim_id":"182310","title":"ATPase, Na+/K+ TRANSPORTING, ALPHA-1 POLYPEPTIDE; ATP1A1","url":"https://www.omim.org/entry/182310"},{"mim_id":"108733","title":"ATPase, Ca(2+)-TRANSPORTING, PLASMA MEMBRANE, 2; ATP2B2","url":"https://www.omim.org/entry/108733"},{"mim_id":"108732","title":"ATPase, Ca(2+)-TRANSPORTING, PLASMA MEMBRANE, 4; ATP2B4","url":"https://www.omim.org/entry/108732"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Plasma membrane","reliability":"Approved"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"choroid plexus","ntpm":106.2}],"url":"https://www.proteinatlas.org/search/ATP2B3"},"hgnc":{"alias_symbol":["PMCA3","CFAP39"],"prev_symbol":["SCAX1","CLA2"]},"alphafold":{"accession":"Q16720","domains":[{"cath_id":"2.70.150.10","chopping":"30-88_211-289","consensus_level":"medium","plddt":85.6199,"start":30,"end":289},{"cath_id":"1.20.1110.10","chopping":"358-444_840-1066","consensus_level":"high","plddt":84.5335,"start":358,"end":1066},{"cath_id":"3.40.50.1000","chopping":"453-478_684-830","consensus_level":"high","plddt":86.8229,"start":453,"end":830},{"cath_id":"3.40.1110.10","chopping":"483-679","consensus_level":"high","plddt":86.569,"start":483,"end":679}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q16720","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q16720-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q16720-F1-predicted_aligned_error_v6.png","plddt_mean":74.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ATP2B3","jax_strain_url":"https://www.jax.org/strain/search?query=ATP2B3"},"sequence":{"accession":"Q16720","fasta_url":"https://rest.uniprot.org/uniprotkb/Q16720.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q16720/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q16720"}},"corpus_meta":[{"pmid":"23416519","id":"PMC_23416519","title":"Somatic mutations in ATP1A1 and ATP2B3 lead to aldosterone-producing adenomas and secondary hypertension.","date":"2013","source":"Nature genetics","url":"https://pubmed.ncbi.nlm.nih.gov/23416519","citation_count":443,"is_preprint":false},{"pmid":"24082052","id":"PMC_24082052","title":"Somatic ATP1A1, ATP2B3, and KCNJ5 mutations in aldosterone-producing adenomas.","date":"2013","source":"Hypertension (Dallas, Tex. : 1979)","url":"https://pubmed.ncbi.nlm.nih.gov/24082052","citation_count":135,"is_preprint":false},{"pmid":"17071747","id":"PMC_17071747","title":"CLA-1 and its splicing variant CLA-2 mediate bacterial adhesion and cytosolic bacterial invasion in mammalian cells.","date":"2006","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/17071747","citation_count":57,"is_preprint":false},{"pmid":"27035656","id":"PMC_27035656","title":"Cellular Pathophysiology of an Adrenal Adenoma-Associated Mutant of the Plasma Membrane Ca(2+)-ATPase ATP2B3.","date":"2016","source":"Endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/27035656","citation_count":50,"is_preprint":false},{"pmid":"7770003","id":"PMC_7770003","title":"Localization of the plasma membrane Ca(2+)-ATPase isoform PMCA3 in rat cerebellum, choroid plexus and hippocampus.","date":"1995","source":"Brain research. Molecular brain research","url":"https://pubmed.ncbi.nlm.nih.gov/7770003","citation_count":45,"is_preprint":false},{"pmid":"8187550","id":"PMC_8187550","title":"Localization of two genes encoding plasma membrane Ca2+ ATPases isoforms 2 (ATP2B2) and 3 (ATP2B3) to human chromosomes 3p26-->p25 and Xq28, respectively.","date":"1994","source":"Cytogenetics and cell genetics","url":"https://pubmed.ncbi.nlm.nih.gov/8187550","citation_count":32,"is_preprint":false},{"pmid":"21464475","id":"PMC_21464475","title":"Dynamic alternations in cellular and molecular components during blossom-end rot development in tomatoes expressing sCAX1, a constitutively active Ca2+/H+ antiporter from Arabidopsis.","date":"2011","source":"Plant physiology","url":"https://pubmed.ncbi.nlm.nih.gov/21464475","citation_count":27,"is_preprint":false},{"pmid":"18029012","id":"PMC_18029012","title":"Inhibitory interaction of the 14-3-3 proteins with ubiquitous (PMCA1) and tissue-specific (PMCA3) isoforms of the plasma membrane Ca2+ pump.","date":"2007","source":"Cell calcium","url":"https://pubmed.ncbi.nlm.nih.gov/18029012","citation_count":26,"is_preprint":false},{"pmid":"15277554","id":"PMC_15277554","title":"Characterization and expression of plasma membrane Ca2+ ATPase (PMCA3) in the crayfish Procambarus clarkii antennal gland during molting.","date":"2004","source":"The Journal of experimental biology","url":"https://pubmed.ncbi.nlm.nih.gov/15277554","citation_count":24,"is_preprint":false},{"pmid":"22921123","id":"PMC_22921123","title":"Downregulation of PMCA2 or PMCA3 reorganizes Ca(2+) handling systems in differentiating PC12 cells.","date":"2012","source":"Cell calcium","url":"https://pubmed.ncbi.nlm.nih.gov/22921123","citation_count":24,"is_preprint":false},{"pmid":"23773993","id":"PMC_23773993","title":"Exome sequencing identification of a GJB1 missense mutation in a kindred with X-linked spinocerebellar ataxia (SCA-X1).","date":"2013","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/23773993","citation_count":18,"is_preprint":false},{"pmid":"2584925","id":"PMC_2584925","title":"Analysis of a novel VHS107 haplotype in CLA-2 and WSA mice. Evidence for gene conversion among IgVH genes in outbred populations.","date":"1989","source":"The Journal of experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/2584925","citation_count":14,"is_preprint":false},{"pmid":"28807751","id":"PMC_28807751","title":"A novel PMCA3 mutation in an ataxic patient with hypomorphic phosphomannomutase 2 (PMM2) heterozygote mutations: Biochemical characterization of the pump defect.","date":"2017","source":"Biochimica et biophysica acta. Molecular basis of disease","url":"https://pubmed.ncbi.nlm.nih.gov/28807751","citation_count":12,"is_preprint":false},{"pmid":"37298147","id":"PMC_37298147","title":"ATP2B3 Inhibition Alleviates Erastin-Induced Ferroptosis in HT-22 Cells through the P62-KEAP1-NRF2-HO-1 Pathway.","date":"2023","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/37298147","citation_count":6,"is_preprint":false},{"pmid":"23007728","id":"PMC_23007728","title":"Ectopic expression of a maize calreticulin mitigates calcium deficiency-like disorders in sCAX1-expressing tobacco and tomato.","date":"2012","source":"Plant molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/23007728","citation_count":5,"is_preprint":false},{"pmid":"26481629","id":"PMC_26481629","title":"A Novel Somatic Deletion Mutation of ATP2B3 in Aldosterone-Producing Adenoma.","date":"2015","source":"Endocrine pathology","url":"https://pubmed.ncbi.nlm.nih.gov/26481629","citation_count":5,"is_preprint":false},{"pmid":"21798237","id":"PMC_21798237","title":"GABA-shunt enzymes activity in GH3 cells with reduced level of PMCA2 or PMCA3 isoform.","date":"2011","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/21798237","citation_count":4,"is_preprint":false},{"pmid":"36207321","id":"PMC_36207321","title":"The ataxia-linked E1081Q mutation affects the sub-plasma membrane Ca2+-microdomains by tuning PMCA3 activity.","date":"2022","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/36207321","citation_count":3,"is_preprint":false},{"pmid":"34572956","id":"PMC_34572956","title":"Characteristics of a Novel ATP2B3 K416_F418delinsN Mutation in a Classical Aldosterone-Producing Adenoma.","date":"2021","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/34572956","citation_count":1,"is_preprint":false},{"pmid":"36081280","id":"PMC_36081280","title":"Expression, purification and structure determination of the chlorinase ClA2.","date":"2022","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/36081280","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11694,"output_tokens":2729,"usd":0.038009},"stage2":{"model":"claude-opus-4-6","input_tokens":6087,"output_tokens":2269,"usd":0.13074},"total_usd":0.168749,"stage1_batch_id":"msgbatch_01TqNM6p6FsoMBT2jzQTwKBr","stage2_batch_id":"msgbatch_01Ya473eco8sTHbvRymQaU8o","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2013,\n      \"finding\": \"Somatic hotspot mutations in ATP2B3 (encoding plasma membrane Ca2+ ATPase) were identified in aldosterone-producing adenomas (APAs). Electrophysiological ex vivo studies on primary adrenal adenoma cells provided evidence for inappropriate membrane depolarization in cells with ATP2B3 alterations, leading to autonomous aldosterone secretion.\",\n      \"method\": \"Exome sequencing, functional in vitro studies, electrophysiological ex vivo patch-clamp\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (exome sequencing, functional assays, electrophysiology) replicated across large APA cohort\",\n      \"pmids\": [\"23416519\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Somatic ATP2B3 mutations in APAs result in upregulation of CYP11B2 (aldosterone synthase) gene expression and its transcriptional regulator NR4A2 in HAC15 adrenal cells, establishing a mechanistic link between ATP2B3 loss-of-function and dysregulated aldosterone production.\",\n      \"method\": \"Overexpression in HAC15 adrenal cells, gene expression assays, whole-cell patch-clamp\",\n      \"journal\": \"Hypertension\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal functional assays with multiple readouts (CYP11B2 expression, NR4A2, patch-clamp) in relevant cell line\",\n      \"pmids\": [\"24082052\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The APA-associated ATP2B3 Leu425_Val426del mutation causes: (1) loss of physiological Ca2+ pump function (reduced Ca2+ export capacity); (2) increased Ca2+ influx via depolarization-activated Ca2+ channels; and (3) a Na+-dependent inward current that strongly depolarizes the plasma membrane—collectively promoting CYP11B2 expression and aldosterone production.\",\n      \"method\": \"Ca2+ measurements in NCI-H295R and HEK-293 cells, patch-clamp electrophysiology, mRNA expression assays, aldosterone production assays\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal methods including ion transport assays, electrophysiology, and functional aldosterone readout in two cell lines\",\n      \"pmids\": [\"27035656\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"14-3-3epsilon protein interacts with PMCA3 (ATP2B3) in a phosphorylation-independent manner, and this interaction inhibits PMCA3 pump activity, reducing the ability of cells to restore basal Ca2+ concentration following an InsP3-induced Ca2+ transient.\",\n      \"method\": \"Two-hybrid assay, co-immunoprecipitation in HeLa cells, GST pull-down, aequorin-based Ca2+ measurement in CHO cells\",\n      \"journal\": \"Cell calcium\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal binding assays plus functional Ca2+ extrusion readout\",\n      \"pmids\": [\"18029012\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"PMCA3 protein is expressed in rat brain neurons and localizes primarily to granule cell processes in the cerebellum (granule cell and molecular layers), choroid plexus, and hippocampal dendritic fields, with localization pattern co-distributing with the axonal marker GAP-43 in cerebellar molecular layer.\",\n      \"method\": \"In situ hybridization, immunoblot with affinity-purified anti-peptide antibodies, immunohistochemistry\",\n      \"journal\": \"Brain research. Molecular brain research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct localization by IHC/ISH with functional context (neuronal processes), single lab\",\n      \"pmids\": [\"7770003\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"ATP2B3 (PMCA3) was localized to human chromosome Xq28 by fluorescence in situ hybridization (FISH), somatic cell hybrid analysis, and genetic linkage analysis, identifying it as a candidate gene for X-linked neurological diseases mapping to distal Xq.\",\n      \"method\": \"FISH, somatic cell hybrid analysis, genetic linkage analysis\",\n      \"journal\": \"Cytogenetics and cell genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct chromosomal localization by multiple methods, single lab\",\n      \"pmids\": [\"8187550\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"A novel PMCA3 G733R mutation in the catalytic P-domain impairs the pump's ability to control cellular Ca2+ handling under both basal and stimulated conditions, with homology modeling and molecular dynamics indicating the mutation disrupts the 3D configuration of the local P-domain structure.\",\n      \"method\": \"Biochemical Ca2+ handling assays in cells, homology modeling, molecular dynamics simulation\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular basis of disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — functional cell-based Ca2+ assay combined with structural modeling; single lab\",\n      \"pmids\": [\"28807751\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The ataxia-linked E1081Q mutation in ATP2B3 immediately upstream of the calmodulin-binding domain has a splicing variant-dependent effect: in the full-length 'b' variant it abolishes sub-plasma membrane Ca2+ reduction capacity, while in the truncated 'a' variant it increases Ca2+ extrusion activity in sub-PM microdomains.\",\n      \"method\": \"Biochemical Ca2+ pump activity assays, sub-plasma membrane Ca2+ microdomain measurements, molecular studies of splice variants\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional assays with splice-variant distinction; single lab, moderate evidence\",\n      \"pmids\": [\"36207321\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Downregulation of PMCA3 in PC12 cells accelerates differentiation, forms longer neurites, alters expression of voltage-dependent Ca2+ channels (VDCCs) increasing their contribution to Ca2+ influx, and triggers compensatory upregulation of constitutive PMCA1/PMCA4 isoforms and SERCA.\",\n      \"method\": \"Antisense stable transfection in PC12 cells, dibutyryl-cAMP differentiation assay, Ca2+ influx measurements, immunoblot for PMCA isoforms and SERCA\",\n      \"journal\": \"Cell calcium\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — loss-of-function with defined cellular phenotype and compensatory pathway placement; single lab\",\n      \"pmids\": [\"22921123\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"A novel somatic ATP2B3 K416_F418delinsN mutation in APA causes increased CYP11B2 expression and aldosterone production when transfected into HAC15 adrenal cells, confirming functional pathogenicity of mutations in the codon 416–418 region.\",\n      \"method\": \"Somatic mutation sequencing, HAC15 cell transfection, CYP11B2 expression and aldosterone production assay\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single functional assay in cell line, single case report\",\n      \"pmids\": [\"34572956\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ATP2B3 inhibition (knockdown) in HT-22 neuronal cells alleviates erastin-induced ferroptosis by modulating the P62-KEAP1-NRF2-HO-1 oxidative stress pathway, reducing ROS production and reversing ferroptosis-associated cell death.\",\n      \"method\": \"siRNA knockdown in HT-22 cells, ROS measurement, protein expression analysis (P62, NRF2, HO-1, NQO1, KEAP1), cell viability assay, TMT-based proteomics\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single lab, pathway placement by KD/OE without direct mechanistic link to pump activity\",\n      \"pmids\": [\"37298147\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"PMCA3 knockdown in GH3 pituitary cells results in increased GAD65 expression, indicating that PMCA3 participates in the regulation of GABA synthesis through Ca2+ homeostasis maintenance.\",\n      \"method\": \"Antisense knockdown in GH3 cells, enzyme activity assays (GAD, GABA-T), immunoblot\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single lab, single method, indirect pathway link\",\n      \"pmids\": [\"21798237\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ATP2B3 (PMCA3) is a plasma membrane Ca2+-ATPase that actively extrudes Ca2+ from the cytoplasm to maintain Ca2+ homeostasis, particularly in neurons and adrenal cells; somatic loss-of-function mutations in its transmembrane domain (near codons 416–426) reduce Ca2+ export, trigger membrane depolarization and Na+-dependent inward currents, increase intracellular Ca2+, and thereby activate CYP11B2 expression and aldosterone production in aldosterone-producing adenomas, while its activity is also inhibited by 14-3-3epsilon binding and mutations in its catalytic P-domain or calmodulin-binding domain cause cerebellar ataxia by impairing sub-plasma membrane Ca2+ microdomain regulation.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"ATP2B3 (PMCA3) is a plasma membrane Ca²⁺-ATPase that extrudes cytoplasmic Ca²⁺ to maintain intracellular calcium homeostasis, with critical roles in neuronal signaling and adrenal aldosterone regulation. In the brain, PMCA3 localizes to cerebellar granule cell processes and hippocampal dendritic fields, where it controls sub-plasma membrane Ca²⁺ microdomains; mutations in its catalytic P-domain (G733R) or calmodulin-binding region (E1081Q) impair Ca²⁺ extrusion in a splice variant–dependent manner and cause cerebellar ataxia [PMID:28807751, PMID:36207321, PMID:7770003]. In adrenal aldosterone-producing adenomas, somatic loss-of-function mutations near transmembrane codons 416–426 abolish Ca²⁺ export, generate Na⁺-dependent inward currents that depolarize the plasma membrane, elevate intracellular Ca²⁺, and drive CYP11B2 (aldosterone synthase) expression and autonomous aldosterone production [PMID:23416519, PMID:27035656, PMID:24082052]. PMCA3 activity is negatively regulated by phosphorylation-independent binding of 14-3-3ε, which reduces Ca²⁺ clearance following intracellular Ca²⁺ transients [PMID:18029012].\",\n  \"teleology\": [\n    {\n      \"year\": 1994,\n      \"claim\": \"Mapping ATP2B3 to Xq28 established it as an X-linked candidate for neurological disease, framing subsequent genetic studies.\",\n      \"evidence\": \"FISH, somatic cell hybrid, and linkage analysis in human samples\",\n      \"pmids\": [\"8187550\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No disease-causing mutations identified at this stage\", \"Functional relevance of chromosomal position not tested\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Demonstrating that PMCA3 protein localizes to cerebellar granule cell processes and hippocampal dendritic fields revealed its neuron-specific Ca²⁺ extrusion role, distinguishing it from ubiquitous PMCA isoforms.\",\n      \"evidence\": \"Immunohistochemistry and in situ hybridization in rat brain\",\n      \"pmids\": [\"7770003\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Localization shown only in rat; human tissue confirmation lacking\", \"No functional consequence of regional expression tested\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Identification of 14-3-3ε as a phosphorylation-independent inhibitor of PMCA3 pump activity established the first known regulatory partner that attenuates Ca²⁺ clearance.\",\n      \"evidence\": \"Two-hybrid, co-immunoprecipitation, GST pull-down, aequorin-based Ca²⁺ measurements in CHO cells\",\n      \"pmids\": [\"18029012\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological context of 14-3-3ε regulation (e.g., neuronal vs. adrenal) not addressed\", \"Binding site on PMCA3 not mapped\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Showing that PMCA3 knockdown in PC12 cells accelerates neurite outgrowth and triggers compensatory upregulation of PMCA1/4 and SERCA demonstrated that PMCA3 loss remodels Ca²⁺ signaling networks during neuronal differentiation.\",\n      \"evidence\": \"Antisense stable transfection in PC12 cells with differentiation assays and immunoblot\",\n      \"pmids\": [\"22921123\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relevance to in vivo neuronal development not tested\", \"Compensatory mechanisms complicate isolation of PMCA3-specific roles\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Discovery of recurrent somatic ATP2B3 mutations in aldosterone-producing adenomas, coupled with electrophysiological evidence for membrane depolarization and CYP11B2/NR4A2 upregulation, established a direct mechanistic link between PMCA3 loss-of-function and autonomous aldosterone secretion.\",\n      \"evidence\": \"Exome sequencing of APA cohort; patch-clamp on primary adenoma cells; overexpression in HAC15 cells with gene expression readout\",\n      \"pmids\": [\"23416519\", \"24082052\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise structural basis for mutation-induced ion leak not resolved\", \"Whether mutations act purely through loss of Ca²⁺ extrusion or also gain-of-function Na⁺ conductance remained unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Mechanistic dissection of the L425_V426del mutation resolved that APA-linked ATP2B3 mutations simultaneously abolish Ca²⁺ pump function and introduce a Na⁺-dependent inward current, explaining both elevated intracellular Ca²⁺ and membrane depolarization.\",\n      \"evidence\": \"Ca²⁺ imaging in NCI-H295R and HEK-293 cells, patch-clamp electrophysiology, aldosterone production assays\",\n      \"pmids\": [\"27035656\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Na⁺ conductance pathway through the mutant pump not structurally characterized\", \"In vivo confirmation of dual mechanism lacking\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identification of the catalytic P-domain G733R mutation as impairing Ca²⁺ handling, supported by molecular dynamics, linked PMCA3 catalytic domain integrity to cerebellar ataxia pathogenesis.\",\n      \"evidence\": \"Cellular Ca²⁺ assays combined with homology modeling and molecular dynamics\",\n      \"pmids\": [\"28807751\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No patient genotype–phenotype segregation data provided\", \"Structural model based on homology, not experimental structure\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Demonstrating that the ataxia-linked E1081Q mutation near the calmodulin-binding domain has opposite effects in the 'a' versus 'b' splice variants revealed that PMCA3 splice context determines whether a mutation causes gain or loss of sub-PM Ca²⁺ extrusion.\",\n      \"evidence\": \"Sub-plasma membrane Ca²⁺ microdomain measurements and pump activity assays comparing splice variants\",\n      \"pmids\": [\"36207321\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relative expression of splice variants in cerebellar neurons not quantified\", \"How splice-specific effects translate to selective cerebellar vulnerability unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the high-resolution structural basis for mutation-induced Na⁺ leak through PMCA3, the in vivo contribution of 14-3-3ε regulation to neuronal or adrenal Ca²⁺ homeostasis, and whether PMCA3 splice variant ratios explain cell-type-specific disease manifestations.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No cryo-EM or crystal structure of PMCA3 available\", \"In vivo knockout/knockin mouse models with neuronal or adrenal phenotyping not reported in the timeline\", \"Relative pathogenic contribution of Ca²⁺ extrusion loss versus Na⁺ leak in APA not resolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [0, 2, 3, 6, 7]},\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [2, 3, 6, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 2, 3, 4, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": []},\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [0, 2, 3, 6, 7]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [4, 8]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"YWHAE\",\n      \"CYP11B2\",\n      \"NR4A2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}