{"gene":"ANO5","run_date":"2026-06-09T22:02:43","timeline":{"discoveries":[{"year":2007,"finding":"GDD1/TMEM16E (ANO5) protein is an integral membrane glycoprotein that resides predominantly in intracellular vesicles, not at the plasma membrane, as determined by biochemical fractionation and immunohistochemistry using a GDD1-specific antibody.","method":"Biochemical fractionation, immunohistochemistry, subcellular localization with specific antibody","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization experiment with specific antibody, single lab, two orthogonal methods (biochemical + IHC)","pmids":["17418107"],"is_preprint":false},{"year":2010,"finding":"Loss-of-function recessive mutations in ANO5 (splice site, frameshift causing NMD, and missense mutations) cause proximal LGMD2L and distal MMD3 muscular dystrophies, with defective membrane repair documented in patient fibroblasts by membrane-resealing assays.","method":"Genetic mutation analysis, nonsense-mediated mRNA decay assessment, membrane-resealing ability assays, electron microscopy of sarcolemmal lesions","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (genetics, NMD assay, membrane repair assay, EM), replicated across multiple families and populations","pmids":["20096397"],"is_preprint":false},{"year":2014,"finding":"TMEM16E/ANO5 protein is rapidly degraded via the proteasome pathway; degradation is rescued by PI3K pathway inhibition and the chemical chaperone sodium butyrate. The GDD-causing mutant TMEM16E(gdd) exhibits lower stability than wild-type. TMEM16E does not exhibit cell-surface Ca2+-activated Cl- channel (CaCC) activity due to its intracellular vesicle distribution. A putative pore-forming domain of TMEM16E disrupts CaCC activity of TMEM16A via domain swapping.","method":"Proteasome inhibitor assays, PI3K inhibitor rescue, domain-swap experiments, electrophysiology (CaCC assay), site-directed mutagenesis","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro functional assays with mutagenesis and domain swapping, single lab","pmids":["23843187"],"is_preprint":false},{"year":2015,"finding":"TMEM16E carries a 35-amino-acid segment homologous to the scrambling domain in TMEM16F. When this segment replaced the corresponding region of TMEM16A, the chimeric molecule localized to the plasma membrane and supported Ca2+-dependent phospholipid scrambling, demonstrating that TMEM16E harbors a functional scrambling domain.","method":"Chimeric protein construction, plasma membrane localization assay, Ca2+-dependent phospholipid scrambling assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstitution of scrambling activity with chimeric protein and direct functional assay, multiple orthogonal readouts in one study","pmids":["26667038"],"is_preprint":false},{"year":2015,"finding":"TMEM16E is expressed in germ cells during spermatogenesis and localizes to the sperm tail. TMEM16E-deficient male mice show reduced sperm motility and inefficient fertilization of zona-intact eggs in vitro, while morphology, beating, mitochondrial function, capacitation, and zona pellucida binding are unaffected.","method":"TMEM16E-deficient mouse generation, immunolocalization, in vitro fertilization assay, sperm motility analysis","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — knockout mouse model with defined cellular phenotype and direct localization, multiple readouts, single lab","pmids":["26667038"],"is_preprint":false},{"year":2015,"finding":"Genetic disruption of Ano5 in C57BL/6J mice does not cause overt skeletal or cardiac muscle pathology up to 18 months, no significant differences in force production or eccentric contraction force deficit, and cardiac hypertrophy after isoproterenol was indistinguishable from wild type. Microarray identified altered lipid metabolism and complement pathway genes in KO skeletal muscle.","method":"Ano5 knockout mouse generation, histology, force production assays, eccentric contraction assay, echocardiography, microarray","journal":"Skeletal muscle","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with multiple functional readouts, single lab; negative result for muscle pathology is mechanistically informative","pmids":["26693275"],"is_preprint":false},{"year":2017,"finding":"Human TMEM16E/ANO5 displays Ca2+-dependent phospholipid scrambling (PLS) and non-selective ionic currents when overexpressed in mammalian cells, with partial plasma membrane localization. The GDD-causing T513I mutation confers gain-of-function PLS and large ion currents even at low cytosolic Ca2+, whereas the equivalent mutation in TMEM16B paralog had little effect.","method":"Overexpression in mammalian cell lines, phospholipid scrambling assay, patch-clamp electrophysiology, site-directed mutagenesis","journal":"Cellular and molecular life sciences : CMLS","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct biochemical reconstitution of PLS and electrophysiology with mutagenesis, multiple orthogonal methods, single lab","pmids":["29124309"],"is_preprint":false},{"year":2017,"finding":"ANO5 protein is expressed as a single 107 kDa polypeptide in human skeletal muscle. Truncating mutations (c.191dupA, c.1261C>T) abolish ANO5 expression entirely; non-truncating pathogenic point mutations reduce ANO5 expression in the membrane fraction, consistent with protein destabilization and degradation.","method":"Western blot with validated monoclonal antibody, tissue fractionation of patient muscle biopsies","journal":"Neuropathology and applied neurobiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — validated antibody, fractionation, multiple patient samples with different mutations; single lab","pmids":["28489263"],"is_preprint":false},{"year":2018,"finding":"ANO5 overexpression confers Ca2+-dependent phospholipid scrambling and a nonselective ionic current to HEK-293 cells. Ano5-/- mouse muscle precursor cells (MPCs) exhibit defective cell fusion in culture (fewer nuclei per myotube), decreased Ca2+-dependent phosphatidylserine exposure, and reduced Ca2+-dependent outwardly rectifying ionic currents. Viral re-introduction of ANO5 rescues fusion, scrambling, and ionic currents.","method":"HEK-293 overexpression, phospholipid scrambling assay, patch-clamp electrophysiology, Ano5-/- MPC isolation, myotube fusion assay, viral rescue experiment","journal":"The Journal of general physiology","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstitution in HEK cells, KO cell functional assay, viral rescue, multiple orthogonal methods; directly establishes ANO5-mediated scrambling and its role in muscle cell fusion","pmids":["30257928"],"is_preprint":false},{"year":2018,"finding":"Immunofluorescence staining of healthy muscle cryosections with a validated monoclonal antibody localizes ANO5 to the sarcoplasmic reticulum. A LGMD2L patient with the c.191dupA homozygous mutation showed no ANO5 signal. A truncated ANO5 peptide (first 121 aa) resulting from a novel splice variant forms aggregates when expressed in C2C12 cells.","method":"Immunofluorescence with validated monoclonal antibody (N421A/85), transfection of truncated ANO5 constructs into C2C12 cells, observation of aggregate formation","journal":"The journal of pathology. Clinical research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization with validated antibody, functional consequence (aggregate formation) shown in cell line; single lab","pmids":["29665321"],"is_preprint":false},{"year":2018,"finding":"CRISPR-mediated frame-disrupting ANO5 mutations in rabbits (exon 12/13 indels) lead to typical muscular dystrophy signs: increased serum CK, muscle necrosis, regeneration, fatty replacement, and fibrosis, establishing a rabbit animal model that recapitulates human disease.","method":"CRISPR/Cas9 knock-in in rabbits, serum CK measurement, histology, muscle imaging","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — defined loss-of-function model with multiple histological readouts, single lab","pmids":["29789544"],"is_preprint":false},{"year":2019,"finding":"ANO5-deficient patient myoblasts (carrying c.2272C>T mutation causing protein degradation) show normal myogenesis but defective plasma membrane repair (PMR). The repair deficit is linked to impaired ER-mediated cytosolic Ca2+ clearance after focal injury. Wild-type ANO5 expression or pharmacological prevention of cytosolic Ca2+ overload rescues PMR in patient cells.","method":"Patient-derived myoblast lines, plasma membrane repair assay (focal laser injury), Ca2+ imaging, WT ANO5 rescue by transfection, pharmacological Ca2+ modulation","journal":"Cell death discovery","confidence":"High","confidence_rationale":"Tier 2 / Strong — patient cells, multiple orthogonal methods (PMR assay, Ca2+ imaging, genetic rescue, pharmacological rescue), mechanistic link to ER Ca2+ clearance","pmids":["31341644"],"is_preprint":false},{"year":2019,"finding":"ANO5 knockdown in C2C12 myotubes causes clustered/aggregated nuclei (nuclear positioning defect) associated with reduced Kif5b protein expression. ANO5 knockdown also impairs depolarization-induced Ca2+ transients and reduces SR Ca2+ storage, associated with reduced DHPR and SERCA1 protein expression and disrupted DHPR–RyR1 co-localization.","method":"shRNA knockdown in C2C12 myoblasts, immunofluorescence for nuclear positioning, western blot for Kif5b/DHPR/SERCA1, Ca2+ imaging, co-localization analysis","journal":"The Korean journal of physiology & pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — shRNA KD with defined cellular phenotypes and multiple protein readouts; single lab","pmids":["31680776"],"is_preprint":false},{"year":2020,"finding":"Using HEK293-based functional assays, MD-associated ANO5 mutations are associated with loss-of-function phospholipid scrambling, while GDD-associated mutations are associated with gain-of-function scrambling, demonstrating opposite effects on ANO5 activity depending on mutation class.","method":"HEK293 overexpression, phospholipid scrambling assay, site-directed mutagenesis of multiple patient-associated variants","journal":"Human mutation","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct reconstitution of scrambling activity with multiple disease mutations, systematic functional characterization; single lab with multiple orthogonal variants tested","pmids":["32112655"],"is_preprint":false},{"year":2021,"finding":"BVES is a novel interacting protein of ANO5 identified by proximity labeling (BioID2) and confirmed by co-immunoprecipitation. The N-terminus of ANO5 mediates interaction with the C-terminus of BVES. ANO5 and BVES co-localize at the endoplasmic reticulum membrane in muscle cells. Genome-editing disruption of either ANO5 or BVES significantly suppresses C2C12 myoblast differentiation.","method":"BioID2 proximity labeling, mass spectrometry, co-immunoprecipitation, co-localization by immunofluorescence, CRISPR/Cas9 disruption, myoblast differentiation assay","journal":"Cell & bioscience","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP confirming interaction, BioID2 identification, domain mapping, localization, and functional knockout phenotype; multiple orthogonal methods, single lab","pmids":["34963485"],"is_preprint":false},{"year":2021,"finding":"A targeted out-of-frame deletion Ano5 mouse model shows progressive muscle loss, increased muscle weakness, and persistent myofiber regeneration without chronic inflammation, recapitulating mild-to-moderate LGMD2L. These features are not associated with changes in calcium-activated sarcolemmal chloride channel activity or impaired in vivo regenerative myogenesis.","method":"Knockout mouse generation, histology, grip/force measurements, chloride channel electrophysiology, regeneration assays","journal":"Journal of neuromuscular diseases","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse with multiple functional readouts, negative result for Cl- channel at sarcolemma is mechanistically informative; single lab","pmids":["34633328"],"is_preprint":false},{"year":2022,"finding":"Ablation of Ano5 in mice reduces intracellular calcium transients in osteoblasts and osteoclasts, leading to defective osteoblast and osteoclast differentiation. Ano5 KO mice exhibit low bone volume, abnormal calcium deposits, and reduced WNT/β-Catenin signaling in osteoblasts and RANKL-NFATc1 signaling in osteoclasts. Parathyroid hormone treatment enhances bone strength in KO mice.","method":"Ano5 knockout mouse model, calcium imaging, osteoblast/osteoclast differentiation assays, western blot for signaling pathways, PTH treatment in vivo","journal":"NPJ genomic medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse with Ca2+ imaging and pathway analysis, multiple orthogonal readouts; single lab","pmids":["35982081"],"is_preprint":false},{"year":2022,"finding":"Male Ano5-/- mice show elevated serum CK after exercise and defective plasma membrane repair after laser injury, while female Ano5-/- mice are indistinguishable from wild type by these measures. Female Ano5-/- mice exhibit features of cardiomyopathy by echocardiography despite normal skeletal muscle repair, demonstrating sex-dependent tissue involvement.","method":"Ano5-/- mouse model, serum CK measurement after exercise, laser injury membrane repair assay, echocardiography","journal":"American journal of physiology. Cell physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse with multiple functional readouts, sex-stratified analysis; single lab","pmids":["35020501"],"is_preprint":false},{"year":2023,"finding":"TMEM16E (ANO5) is required for endothelial cell procoagulant activity via phosphatidylserine externalization. In an intravital laser-injury thrombosis model, PS externalization is concentrated at the vessel wall. TMEM16E-null mice show reduced vessel-wall-dependent fibrin formation. The TMEM16 inhibitor benzbromarone prevents PS externalization, EC procoagulant activity, and protects mice from thrombosis.","method":"Focused genetic screen, in vitro PS externalization assay, intravital laser-injury mouse thrombosis model, TMEM16E-null mice, pharmacological inhibition with benzbromarone","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO mouse + pharmacological inhibition + in vivo model + in vitro assays, multiple orthogonal methods establishing procoagulant PS externalization function","pmids":["36951953"],"is_preprint":false},{"year":2023,"finding":"Ano5 deficiency impairs osteoclastogenesis: Ano5-/- mice exhibit inhibited formation of multinucleated osteoclasts, reduced TRAP activity, decreased expression of Nfatc1, c-Fos, and osteoclast genes, disrupted actin ring formation, and less mineral resorption. RANKL-induced NF-κB signaling is suppressed in Ano5-/- osteoclasts, and this can be partially rescued by NF-κB activator.","method":"Ano5-/- mouse model, TRAP staining, phalloidin staining for actin rings, bone resorption assay, qRT-PCR, western blot for NF-κB signaling, pharmacological NF-κB activation","journal":"Oral diseases","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse with multiple functional assays and pathway rescue; single lab","pmids":["36989132"],"is_preprint":false},{"year":2025,"finding":"TMEM16E activation promotes macropinocytosis essential for plasma membrane integrity. TMEM16E externalizes phosphatidylserine associated with resting growth factor receptors, interacts with and signals through EGFR in a ligand-independent manner, stimulates PI3K, and facilitates macropinocytosis and internalization of membrane-bound annexin V. TMEM16E is internalized during this process but returns to the plasma membrane.","method":"Co-immunoprecipitation (TMEM16E–EGFR interaction), PI3K activity assay, macropinocytosis assay, annexin V internalization assay, amiloride inhibition, live imaging","journal":"Communications biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, functional assays with pharmacological inhibitors, multiple cellular readouts; single lab","pmids":["39794444"],"is_preprint":false},{"year":2021,"finding":"Introduction of the GDD-causing p.Cys360Tyr mutation in Ano5 knock-in mice recapitulates GDD skeletal features (massive jawbones, bowing tibia, bone fragility, sclerosis). Knock-in osteoblasts show increased osteoblastogenesis and hypermineralized bone matrix. Knock-in osteoclasts show decreased osteoclastogenesis and disrupted actin ring formation.","method":"Knock-in mouse model (Cys360Tyr), skeletal phenotyping, calvaria-derived osteoblast cultures, bone marrow-derived macrophage osteoclast cultures, ALP/TRAP staining, phalloidin staining","journal":"Journal of bone and mineral research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knock-in mouse with multiple cellular assays; single lab","pmids":["34841576"],"is_preprint":false}],"current_model":"ANO5/TMEM16E is an intracellular (sarcoplasmic reticulum/endoplasmic reticulum-resident) Ca2+-activated phospholipid scramblase that also generates nonselective ionic currents; its scrambling activity is required for muscle precursor cell fusion and plasma membrane repair (via regulation of ER-mediated cytosolic Ca2+ clearance), and loss-of-function mutations cause LGMD2L/MMD3 while gain-of-function GDD mutations constitutively activate scrambling—ANO5 also interacts with BVES at the ER membrane to regulate myoblast differentiation, localizes to the sperm tail to support motility, and at the endothelial cell surface drives procoagulant phosphatidylserine externalization and ligand-independent EGFR/PI3K-stimulated macropinocytosis for membrane repair."},"narrative":{"mechanistic_narrative":"ANO5 (TMEM16E) is an intracellular Ca2+-activated phospholipid scramblase of the TMEM16 family that governs membrane remodeling in muscle, bone, germ cells, and endothelium [PMID:26667038, PMID:30257928]. The protein is an integral membrane glycoprotein residing in intracellular vesicles and the sarcoplasmic/endoplasmic reticulum rather than the plasma membrane [PMID:17418107, PMID:29665321], and it carries a discrete scrambling domain homologous to TMEM16F that, when transplanted into TMEM16A, is sufficient to confer Ca2+-dependent phospholipid scrambling [PMID:26667038]. In reconstituted systems and ANO5-null muscle precursor cells, ANO5 produces Ca2+-dependent phosphatidylserine exposure and nonselective ionic currents, and these activities are required for muscle precursor cell fusion, with viral re-introduction rescuing both scrambling and fusion [PMID:30257928]. Beyond myogenesis, ANO5 supports plasma membrane repair by enabling ER-mediated clearance of cytosolic Ca2+ after focal injury, a function lost in patient myoblasts and restored by wild-type ANO5 or by limiting Ca2+ overload [PMID:31341644]. Recessive loss-of-function ANO5 mutations cause limb-girdle muscular dystrophy 2L and distal Miyoshi myopathy MMD3 through protein destabilization and defective membrane resealing [PMID:20096397, PMID:28489263], whereas gain-of-function gnathodiaphyseal dysplasia mutations constitutively activate scrambling at low cytosolic Ca2+ — opposite functional consequences depending on mutation class [PMID:29124309, PMID:32112655]. ANO5 also interacts through its N-terminus with the C-terminus of BVES at the ER membrane to drive myoblast differentiation [PMID:34963485], localizes to the sperm tail to support motility and fertilization [PMID:26667038], drives endothelial procoagulant phosphatidylserine externalization that promotes vessel-wall fibrin formation [PMID:36951953], and engages EGFR in a ligand-independent manner to stimulate PI3K and macropinocytosis during membrane repair [PMID:39794444].","teleology":[{"year":2007,"claim":"Established where the ANO5 protein resides, distinguishing it from plasma-membrane TMEM16 channels and framing its function as intracellular.","evidence":"Biochemical fractionation and immunohistochemistry with a GDD1-specific antibody","pmids":["17418107"],"confidence":"Medium","gaps":["Identity of the specific intracellular compartment beyond 'vesicles' not resolved","No functional activity assayed"]},{"year":2010,"claim":"Linked ANO5 to human disease and to a cellular defect, showing recessive loss-of-function mutations cause LGMD2L/MMD3 with impaired membrane resealing.","evidence":"Genetic analysis, NMD assays, membrane-resealing assays, and EM of sarcolemmal lesions in patient fibroblasts","pmids":["20096397"],"confidence":"High","gaps":["Molecular activity of ANO5 underlying repair not defined","Mechanism connecting mutation to repair failure unknown"]},{"year":2014,"claim":"Showed ANO5 is unstable and proteasomally degraded and lacks surface chloride-channel activity, attributing absent CaCC behavior to its intracellular distribution.","evidence":"Proteasome and PI3K-inhibitor rescue, domain-swap experiments, and CaCC electrophysiology","pmids":["23843187"],"confidence":"Medium","gaps":["Did not establish the genuine physiological activity of ANO5","Domain-swap effects on TMEM16A indirect"]},{"year":2015,"claim":"Defined the molecular activity by demonstrating ANO5 contains a functional Ca2+-dependent phospholipid scrambling domain, and uncovered a reproductive role in sperm.","evidence":"Chimeric TMEM16A/E proteins with scrambling assay; TMEM16E-deficient mice with sperm motility and IVF analysis","pmids":["26667038"],"confidence":"High","gaps":["Scrambling shown for chimera, not full-length native ANO5 in this study","Molecular target of ANO5 in sperm tail unknown"]},{"year":2015,"claim":"Tested whether ANO5 loss alone produces muscle disease in mice, finding no overt myopathy but altered lipid/complement gene expression.","evidence":"Ano5 knockout mouse with histology, force assays, echocardiography, and microarray","pmids":["26693275"],"confidence":"Medium","gaps":["Species/strain differences from human disease unexplained","Functional meaning of transcriptional changes not pursued"]},{"year":2017,"claim":"Confirmed native human ANO5 scrambling and current activity and demonstrated that a GDD mutation is gain-of-function, activating scrambling at low Ca2+.","evidence":"Mammalian overexpression with scrambling assay, patch-clamp, and site-directed mutagenesis (T513I)","pmids":["29124309"],"confidence":"High","gaps":["Only partial plasma-membrane localization on overexpression complicates physiological interpretation"]},{"year":2017,"claim":"Connected pathogenic mutations to protein loss in patient muscle, showing truncating mutations abolish and point mutations destabilize ANO5.","evidence":"Western blot with validated monoclonal antibody on fractionated patient muscle biopsies","pmids":["28489263"],"confidence":"Medium","gaps":["Degradation pathway in vivo not dissected","Residual function of destabilized mutants unquantified"]},{"year":2018,"claim":"Causally tied ANO5 scrambling/current activity to muscle precursor cell fusion via knockout and viral rescue.","evidence":"HEK293 reconstitution, Ano5-/- MPC fusion and PS-exposure assays, electrophysiology, and viral rescue","pmids":["30257928"],"confidence":"High","gaps":["How scrambling mechanistically drives fusion not resolved","Subcellular site of fusion-relevant scrambling unclear"]},{"year":2018,"claim":"Refined localization to the sarcoplasmic reticulum and showed truncated ANO5 forms aggregates.","evidence":"Immunofluorescence with validated antibody on muscle cryosections; truncated construct expression in C2C12 cells","pmids":["29665321"],"confidence":"Medium","gaps":["Aggregation pathogenicity not tested in vivo"]},{"year":2018,"claim":"Established a rabbit model recapitulating human dystrophy, providing an animal system where ANO5 loss reproduces disease unlike the mouse.","evidence":"CRISPR/Cas9 frame-disrupting indels in rabbits with CK, histology, and imaging","pmids":["29789544"],"confidence":"Medium","gaps":["Cellular mechanism not interrogated in this model"]},{"year":2019,"claim":"Defined the membrane-repair mechanism, showing ANO5 enables ER-mediated cytosolic Ca2+ clearance after injury.","evidence":"Patient myoblasts, focal laser injury repair assays, Ca2+ imaging, genetic and pharmacological rescue","pmids":["31341644"],"confidence":"High","gaps":["Molecular link between scrambling activity and ER Ca2+ handling not delineated"]},{"year":2019,"claim":"Implicated ANO5 in SR Ca2+ handling and nuclear positioning in myotubes, broadening its muscle role beyond repair.","evidence":"shRNA knockdown in C2C12 with immunofluorescence, western blot (Kif5b/DHPR/SERCA1), Ca2+ imaging, co-localization","pmids":["31680776"],"confidence":"Medium","gaps":["Whether protein-expression changes are direct or secondary unknown","Single knockdown system"]},{"year":2020,"claim":"Systematically partitioned disease alleles, showing MD mutations cause loss-of-function and GDD mutations gain-of-function scrambling.","evidence":"HEK293 scrambling assays across multiple site-directed patient variants","pmids":["32112655"],"confidence":"High","gaps":["Tissue-specific consequences of opposite activities not addressed"]},{"year":2021,"claim":"Identified BVES as a direct ER-membrane partner of ANO5 required for myoblast differentiation, adding a protein-interaction dimension.","evidence":"BioID2 proximity labeling, reciprocal Co-IP, domain mapping, co-localization, and CRISPR differentiation assays","pmids":["34963485"],"confidence":"High","gaps":["Functional consequence of the interaction on scrambling activity not tested"]},{"year":2021,"claim":"Produced a mouse out-of-frame deletion model reproducing mild-to-moderate LGMD2L independent of sarcolemmal chloride-channel activity.","evidence":"Knockout mouse with histology, force measures, chloride electrophysiology, and regeneration assays","pmids":["34633328"],"confidence":"Medium","gaps":["Cellular driver of progressive muscle loss not isolated"]},{"year":2021,"claim":"Demonstrated that a GDD gain-of-function knock-in recapitulates skeletal dysplasia with opposing osteoblast/osteoclast phenotypes.","evidence":"Cys360Tyr knock-in mouse with skeletal phenotyping and osteoblast/osteoclast cultures","pmids":["34841576"],"confidence":"Medium","gaps":["Mechanistic link between scrambling gain-of-function and bone cell behavior unresolved"]},{"year":2022,"claim":"Extended ANO5 function to bone homeostasis through Ca2+-dependent osteoblast and osteoclast differentiation.","evidence":"Ano5 knockout mice with calcium imaging, differentiation assays, signaling western blots, and PTH treatment","pmids":["35982081"],"confidence":"Medium","gaps":["How ANO5 controls intracellular Ca2+ transients in bone cells not defined"]},{"year":2022,"claim":"Revealed sex-dependent tissue involvement, with male mice showing muscle repair defects and females showing cardiomyopathy.","evidence":"Ano5-/- mice with post-exercise CK, laser-injury repair, and echocardiography, sex-stratified","pmids":["35020501"],"confidence":"Medium","gaps":["Basis of sexual dimorphism unknown","Cardiac mechanism not dissected"]},{"year":2023,"claim":"Established a vascular function: ANO5-dependent endothelial PS externalization drives vessel-wall procoagulant activity and thrombosis.","evidence":"Genetic screen, in vitro PS assays, intravital laser-injury thrombosis model, TMEM16E-null mice, benzbromarone inhibition","pmids":["36951953"],"confidence":"High","gaps":["Endothelial localization and Ca2+ trigger not detailed"]},{"year":2023,"claim":"Detailed the osteoclast arm, showing ANO5 loss suppresses RANKL-NF-kB signaling and osteoclastogenesis.","evidence":"Ano5-/- mice with TRAP/phalloidin staining, resorption assays, qRT-PCR, NF-kB western blots and pharmacological rescue","pmids":["36989132"],"confidence":"Medium","gaps":["Direct connection of scrambling to NF-kB signaling not shown"]},{"year":2025,"claim":"Connected ANO5 scrambling to a membrane-repair effector pathway via ligand-independent EGFR/PI3K signaling and macropinocytosis.","evidence":"Co-IP (TMEM16E-EGFR), PI3K activity assay, macropinocytosis and annexin-V internalization assays, amiloride inhibition, live imaging","pmids":["39794444"],"confidence":"Medium","gaps":["How PS externalization triggers EGFR remains unclear","Single-lab Co-IP for EGFR interaction"]},{"year":null,"claim":"How a single intracellular scramblase mechanistically produces such divergent tissue outcomes—muscle repair, sperm motility, bone remodeling, and endothelial coagulation—and the structural basis of its Ca2+-gated scrambling remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of ANO5 scrambling gate","Unified mechanism linking scrambling to downstream tissue-specific signaling absent","Reasons for mouse vs. rabbit/human disease discordance unexplained"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[3,6,8,13]},{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[6,8]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[0,11,14]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[0]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[6,18,20]}],"pathway":[{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[8,14]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[11]},{"term_id":"R-HSA-109582","term_label":"Hemostasis","supporting_discovery_ids":[18]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[1,13]}],"complexes":[],"partners":["BVES","EGFR"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q75V66","full_name":"Anoctamin-5","aliases":["Gnathodiaphyseal dysplasia 1 protein","Transmembrane protein 16E"],"length_aa":913,"mass_kda":107.2,"function":"Plays a role in plasma membrane repair in a process involving annexins (PubMed:33496727). Does not exhibit calcium-activated chloride channel (CaCC) activity","subcellular_location":"Endoplasmic reticulum membrane; Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q75V66/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ANO5","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ANO5","total_profiled":1310},"omim":[{"mim_id":"613319","title":"MIYOSHI MUSCULAR DYSTROPHY 3; MMD3","url":"https://www.omim.org/entry/613319"},{"mim_id":"611307","title":"MUSCULAR DYSTROPHY, LIMB-GIRDLE, AUTOSOMAL RECESSIVE 12; LGMDR12","url":"https://www.omim.org/entry/611307"},{"mim_id":"608662","title":"ANOCTAMIN 5; 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The GDD-causing mutant TMEM16E(gdd) exhibits lower stability than wild-type. TMEM16E does not exhibit cell-surface Ca2+-activated Cl- channel (CaCC) activity due to its intracellular vesicle distribution. A putative pore-forming domain of TMEM16E disrupts CaCC activity of TMEM16A via domain swapping.\",\n      \"method\": \"Proteasome inhibitor assays, PI3K inhibitor rescue, domain-swap experiments, electrophysiology (CaCC assay), site-directed mutagenesis\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro functional assays with mutagenesis and domain swapping, single lab\",\n      \"pmids\": [\"23843187\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TMEM16E carries a 35-amino-acid segment homologous to the scrambling domain in TMEM16F. When this segment replaced the corresponding region of TMEM16A, the chimeric molecule localized to the plasma membrane and supported Ca2+-dependent phospholipid scrambling, demonstrating that TMEM16E harbors a functional scrambling domain.\",\n      \"method\": \"Chimeric protein construction, plasma membrane localization assay, Ca2+-dependent phospholipid scrambling assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstitution of scrambling activity with chimeric protein and direct functional assay, multiple orthogonal readouts in one study\",\n      \"pmids\": [\"26667038\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TMEM16E is expressed in germ cells during spermatogenesis and localizes to the sperm tail. TMEM16E-deficient male mice show reduced sperm motility and inefficient fertilization of zona-intact eggs in vitro, while morphology, beating, mitochondrial function, capacitation, and zona pellucida binding are unaffected.\",\n      \"method\": \"TMEM16E-deficient mouse generation, immunolocalization, in vitro fertilization assay, sperm motility analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockout mouse model with defined cellular phenotype and direct localization, multiple readouts, single lab\",\n      \"pmids\": [\"26667038\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Genetic disruption of Ano5 in C57BL/6J mice does not cause overt skeletal or cardiac muscle pathology up to 18 months, no significant differences in force production or eccentric contraction force deficit, and cardiac hypertrophy after isoproterenol was indistinguishable from wild type. Microarray identified altered lipid metabolism and complement pathway genes in KO skeletal muscle.\",\n      \"method\": \"Ano5 knockout mouse generation, histology, force production assays, eccentric contraction assay, echocardiography, microarray\",\n      \"journal\": \"Skeletal muscle\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with multiple functional readouts, single lab; negative result for muscle pathology is mechanistically informative\",\n      \"pmids\": [\"26693275\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Human TMEM16E/ANO5 displays Ca2+-dependent phospholipid scrambling (PLS) and non-selective ionic currents when overexpressed in mammalian cells, with partial plasma membrane localization. The GDD-causing T513I mutation confers gain-of-function PLS and large ion currents even at low cytosolic Ca2+, whereas the equivalent mutation in TMEM16B paralog had little effect.\",\n      \"method\": \"Overexpression in mammalian cell lines, phospholipid scrambling assay, patch-clamp electrophysiology, site-directed mutagenesis\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct biochemical reconstitution of PLS and electrophysiology with mutagenesis, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"29124309\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ANO5 protein is expressed as a single 107 kDa polypeptide in human skeletal muscle. Truncating mutations (c.191dupA, c.1261C>T) abolish ANO5 expression entirely; non-truncating pathogenic point mutations reduce ANO5 expression in the membrane fraction, consistent with protein destabilization and degradation.\",\n      \"method\": \"Western blot with validated monoclonal antibody, tissue fractionation of patient muscle biopsies\",\n      \"journal\": \"Neuropathology and applied neurobiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — validated antibody, fractionation, multiple patient samples with different mutations; single lab\",\n      \"pmids\": [\"28489263\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"ANO5 overexpression confers Ca2+-dependent phospholipid scrambling and a nonselective ionic current to HEK-293 cells. Ano5-/- mouse muscle precursor cells (MPCs) exhibit defective cell fusion in culture (fewer nuclei per myotube), decreased Ca2+-dependent phosphatidylserine exposure, and reduced Ca2+-dependent outwardly rectifying ionic currents. Viral re-introduction of ANO5 rescues fusion, scrambling, and ionic currents.\",\n      \"method\": \"HEK-293 overexpression, phospholipid scrambling assay, patch-clamp electrophysiology, Ano5-/- MPC isolation, myotube fusion assay, viral rescue experiment\",\n      \"journal\": \"The Journal of general physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstitution in HEK cells, KO cell functional assay, viral rescue, multiple orthogonal methods; directly establishes ANO5-mediated scrambling and its role in muscle cell fusion\",\n      \"pmids\": [\"30257928\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Immunofluorescence staining of healthy muscle cryosections with a validated monoclonal antibody localizes ANO5 to the sarcoplasmic reticulum. A LGMD2L patient with the c.191dupA homozygous mutation showed no ANO5 signal. A truncated ANO5 peptide (first 121 aa) resulting from a novel splice variant forms aggregates when expressed in C2C12 cells.\",\n      \"method\": \"Immunofluorescence with validated monoclonal antibody (N421A/85), transfection of truncated ANO5 constructs into C2C12 cells, observation of aggregate formation\",\n      \"journal\": \"The journal of pathology. Clinical research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization with validated antibody, functional consequence (aggregate formation) shown in cell line; single lab\",\n      \"pmids\": [\"29665321\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CRISPR-mediated frame-disrupting ANO5 mutations in rabbits (exon 12/13 indels) lead to typical muscular dystrophy signs: increased serum CK, muscle necrosis, regeneration, fatty replacement, and fibrosis, establishing a rabbit animal model that recapitulates human disease.\",\n      \"method\": \"CRISPR/Cas9 knock-in in rabbits, serum CK measurement, histology, muscle imaging\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — defined loss-of-function model with multiple histological readouts, single lab\",\n      \"pmids\": [\"29789544\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"ANO5-deficient patient myoblasts (carrying c.2272C>T mutation causing protein degradation) show normal myogenesis but defective plasma membrane repair (PMR). The repair deficit is linked to impaired ER-mediated cytosolic Ca2+ clearance after focal injury. Wild-type ANO5 expression or pharmacological prevention of cytosolic Ca2+ overload rescues PMR in patient cells.\",\n      \"method\": \"Patient-derived myoblast lines, plasma membrane repair assay (focal laser injury), Ca2+ imaging, WT ANO5 rescue by transfection, pharmacological Ca2+ modulation\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — patient cells, multiple orthogonal methods (PMR assay, Ca2+ imaging, genetic rescue, pharmacological rescue), mechanistic link to ER Ca2+ clearance\",\n      \"pmids\": [\"31341644\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"ANO5 knockdown in C2C12 myotubes causes clustered/aggregated nuclei (nuclear positioning defect) associated with reduced Kif5b protein expression. ANO5 knockdown also impairs depolarization-induced Ca2+ transients and reduces SR Ca2+ storage, associated with reduced DHPR and SERCA1 protein expression and disrupted DHPR–RyR1 co-localization.\",\n      \"method\": \"shRNA knockdown in C2C12 myoblasts, immunofluorescence for nuclear positioning, western blot for Kif5b/DHPR/SERCA1, Ca2+ imaging, co-localization analysis\",\n      \"journal\": \"The Korean journal of physiology & pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — shRNA KD with defined cellular phenotypes and multiple protein readouts; single lab\",\n      \"pmids\": [\"31680776\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Using HEK293-based functional assays, MD-associated ANO5 mutations are associated with loss-of-function phospholipid scrambling, while GDD-associated mutations are associated with gain-of-function scrambling, demonstrating opposite effects on ANO5 activity depending on mutation class.\",\n      \"method\": \"HEK293 overexpression, phospholipid scrambling assay, site-directed mutagenesis of multiple patient-associated variants\",\n      \"journal\": \"Human mutation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct reconstitution of scrambling activity with multiple disease mutations, systematic functional characterization; single lab with multiple orthogonal variants tested\",\n      \"pmids\": [\"32112655\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"BVES is a novel interacting protein of ANO5 identified by proximity labeling (BioID2) and confirmed by co-immunoprecipitation. The N-terminus of ANO5 mediates interaction with the C-terminus of BVES. ANO5 and BVES co-localize at the endoplasmic reticulum membrane in muscle cells. Genome-editing disruption of either ANO5 or BVES significantly suppresses C2C12 myoblast differentiation.\",\n      \"method\": \"BioID2 proximity labeling, mass spectrometry, co-immunoprecipitation, co-localization by immunofluorescence, CRISPR/Cas9 disruption, myoblast differentiation assay\",\n      \"journal\": \"Cell & bioscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP confirming interaction, BioID2 identification, domain mapping, localization, and functional knockout phenotype; multiple orthogonal methods, single lab\",\n      \"pmids\": [\"34963485\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"A targeted out-of-frame deletion Ano5 mouse model shows progressive muscle loss, increased muscle weakness, and persistent myofiber regeneration without chronic inflammation, recapitulating mild-to-moderate LGMD2L. These features are not associated with changes in calcium-activated sarcolemmal chloride channel activity or impaired in vivo regenerative myogenesis.\",\n      \"method\": \"Knockout mouse generation, histology, grip/force measurements, chloride channel electrophysiology, regeneration assays\",\n      \"journal\": \"Journal of neuromuscular diseases\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse with multiple functional readouts, negative result for Cl- channel at sarcolemma is mechanistically informative; single lab\",\n      \"pmids\": [\"34633328\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Ablation of Ano5 in mice reduces intracellular calcium transients in osteoblasts and osteoclasts, leading to defective osteoblast and osteoclast differentiation. Ano5 KO mice exhibit low bone volume, abnormal calcium deposits, and reduced WNT/β-Catenin signaling in osteoblasts and RANKL-NFATc1 signaling in osteoclasts. Parathyroid hormone treatment enhances bone strength in KO mice.\",\n      \"method\": \"Ano5 knockout mouse model, calcium imaging, osteoblast/osteoclast differentiation assays, western blot for signaling pathways, PTH treatment in vivo\",\n      \"journal\": \"NPJ genomic medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse with Ca2+ imaging and pathway analysis, multiple orthogonal readouts; single lab\",\n      \"pmids\": [\"35982081\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Male Ano5-/- mice show elevated serum CK after exercise and defective plasma membrane repair after laser injury, while female Ano5-/- mice are indistinguishable from wild type by these measures. Female Ano5-/- mice exhibit features of cardiomyopathy by echocardiography despite normal skeletal muscle repair, demonstrating sex-dependent tissue involvement.\",\n      \"method\": \"Ano5-/- mouse model, serum CK measurement after exercise, laser injury membrane repair assay, echocardiography\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse with multiple functional readouts, sex-stratified analysis; single lab\",\n      \"pmids\": [\"35020501\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TMEM16E (ANO5) is required for endothelial cell procoagulant activity via phosphatidylserine externalization. In an intravital laser-injury thrombosis model, PS externalization is concentrated at the vessel wall. TMEM16E-null mice show reduced vessel-wall-dependent fibrin formation. The TMEM16 inhibitor benzbromarone prevents PS externalization, EC procoagulant activity, and protects mice from thrombosis.\",\n      \"method\": \"Focused genetic screen, in vitro PS externalization assay, intravital laser-injury mouse thrombosis model, TMEM16E-null mice, pharmacological inhibition with benzbromarone\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO mouse + pharmacological inhibition + in vivo model + in vitro assays, multiple orthogonal methods establishing procoagulant PS externalization function\",\n      \"pmids\": [\"36951953\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Ano5 deficiency impairs osteoclastogenesis: Ano5-/- mice exhibit inhibited formation of multinucleated osteoclasts, reduced TRAP activity, decreased expression of Nfatc1, c-Fos, and osteoclast genes, disrupted actin ring formation, and less mineral resorption. RANKL-induced NF-κB signaling is suppressed in Ano5-/- osteoclasts, and this can be partially rescued by NF-κB activator.\",\n      \"method\": \"Ano5-/- mouse model, TRAP staining, phalloidin staining for actin rings, bone resorption assay, qRT-PCR, western blot for NF-κB signaling, pharmacological NF-κB activation\",\n      \"journal\": \"Oral diseases\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse with multiple functional assays and pathway rescue; single lab\",\n      \"pmids\": [\"36989132\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TMEM16E activation promotes macropinocytosis essential for plasma membrane integrity. TMEM16E externalizes phosphatidylserine associated with resting growth factor receptors, interacts with and signals through EGFR in a ligand-independent manner, stimulates PI3K, and facilitates macropinocytosis and internalization of membrane-bound annexin V. TMEM16E is internalized during this process but returns to the plasma membrane.\",\n      \"method\": \"Co-immunoprecipitation (TMEM16E–EGFR interaction), PI3K activity assay, macropinocytosis assay, annexin V internalization assay, amiloride inhibition, live imaging\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, functional assays with pharmacological inhibitors, multiple cellular readouts; single lab\",\n      \"pmids\": [\"39794444\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Introduction of the GDD-causing p.Cys360Tyr mutation in Ano5 knock-in mice recapitulates GDD skeletal features (massive jawbones, bowing tibia, bone fragility, sclerosis). Knock-in osteoblasts show increased osteoblastogenesis and hypermineralized bone matrix. Knock-in osteoclasts show decreased osteoclastogenesis and disrupted actin ring formation.\",\n      \"method\": \"Knock-in mouse model (Cys360Tyr), skeletal phenotyping, calvaria-derived osteoblast cultures, bone marrow-derived macrophage osteoclast cultures, ALP/TRAP staining, phalloidin staining\",\n      \"journal\": \"Journal of bone and mineral research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knock-in mouse with multiple cellular assays; single lab\",\n      \"pmids\": [\"34841576\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ANO5/TMEM16E is an intracellular (sarcoplasmic reticulum/endoplasmic reticulum-resident) Ca2+-activated phospholipid scramblase that also generates nonselective ionic currents; its scrambling activity is required for muscle precursor cell fusion and plasma membrane repair (via regulation of ER-mediated cytosolic Ca2+ clearance), and loss-of-function mutations cause LGMD2L/MMD3 while gain-of-function GDD mutations constitutively activate scrambling—ANO5 also interacts with BVES at the ER membrane to regulate myoblast differentiation, localizes to the sperm tail to support motility, and at the endothelial cell surface drives procoagulant phosphatidylserine externalization and ligand-independent EGFR/PI3K-stimulated macropinocytosis for membrane repair.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ANO5 (TMEM16E) is an intracellular Ca2+-activated phospholipid scramblase of the TMEM16 family that governs membrane remodeling in muscle, bone, germ cells, and endothelium [#3, #8]. The protein is an integral membrane glycoprotein residing in intracellular vesicles and the sarcoplasmic/endoplasmic reticulum rather than the plasma membrane [#0, #9], and it carries a discrete scrambling domain homologous to TMEM16F that, when transplanted into TMEM16A, is sufficient to confer Ca2+-dependent phospholipid scrambling [#3]. In reconstituted systems and ANO5-null muscle precursor cells, ANO5 produces Ca2+-dependent phosphatidylserine exposure and nonselective ionic currents, and these activities are required for muscle precursor cell fusion, with viral re-introduction rescuing both scrambling and fusion [#8]. Beyond myogenesis, ANO5 supports plasma membrane repair by enabling ER-mediated clearance of cytosolic Ca2+ after focal injury, a function lost in patient myoblasts and restored by wild-type ANO5 or by limiting Ca2+ overload [#11]. Recessive loss-of-function ANO5 mutations cause limb-girdle muscular dystrophy 2L and distal Miyoshi myopathy MMD3 through protein destabilization and defective membrane resealing [#1, #7], whereas gain-of-function gnathodiaphyseal dysplasia mutations constitutively activate scrambling at low cytosolic Ca2+ — opposite functional consequences depending on mutation class [#6, #13]. ANO5 also interacts through its N-terminus with the C-terminus of BVES at the ER membrane to drive myoblast differentiation [#14], localizes to the sperm tail to support motility and fertilization [#4], drives endothelial procoagulant phosphatidylserine externalization that promotes vessel-wall fibrin formation [#18], and engages EGFR in a ligand-independent manner to stimulate PI3K and macropinocytosis during membrane repair [#20].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Established where the ANO5 protein resides, distinguishing it from plasma-membrane TMEM16 channels and framing its function as intracellular.\",\n      \"evidence\": \"Biochemical fractionation and immunohistochemistry with a GDD1-specific antibody\",\n      \"pmids\": [\"17418107\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identity of the specific intracellular compartment beyond 'vesicles' not resolved\", \"No functional activity assayed\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Linked ANO5 to human disease and to a cellular defect, showing recessive loss-of-function mutations cause LGMD2L/MMD3 with impaired membrane resealing.\",\n      \"evidence\": \"Genetic analysis, NMD assays, membrane-resealing assays, and EM of sarcolemmal lesions in patient fibroblasts\",\n      \"pmids\": [\"20096397\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular activity of ANO5 underlying repair not defined\", \"Mechanism connecting mutation to repair failure unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showed ANO5 is unstable and proteasomally degraded and lacks surface chloride-channel activity, attributing absent CaCC behavior to its intracellular distribution.\",\n      \"evidence\": \"Proteasome and PI3K-inhibitor rescue, domain-swap experiments, and CaCC electrophysiology\",\n      \"pmids\": [\"23843187\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not establish the genuine physiological activity of ANO5\", \"Domain-swap effects on TMEM16A indirect\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined the molecular activity by demonstrating ANO5 contains a functional Ca2+-dependent phospholipid scrambling domain, and uncovered a reproductive role in sperm.\",\n      \"evidence\": \"Chimeric TMEM16A/E proteins with scrambling assay; TMEM16E-deficient mice with sperm motility and IVF analysis\",\n      \"pmids\": [\"26667038\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Scrambling shown for chimera, not full-length native ANO5 in this study\", \"Molecular target of ANO5 in sperm tail unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Tested whether ANO5 loss alone produces muscle disease in mice, finding no overt myopathy but altered lipid/complement gene expression.\",\n      \"evidence\": \"Ano5 knockout mouse with histology, force assays, echocardiography, and microarray\",\n      \"pmids\": [\"26693275\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Species/strain differences from human disease unexplained\", \"Functional meaning of transcriptional changes not pursued\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Confirmed native human ANO5 scrambling and current activity and demonstrated that a GDD mutation is gain-of-function, activating scrambling at low Ca2+.\",\n      \"evidence\": \"Mammalian overexpression with scrambling assay, patch-clamp, and site-directed mutagenesis (T513I)\",\n      \"pmids\": [\"29124309\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Only partial plasma-membrane localization on overexpression complicates physiological interpretation\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Connected pathogenic mutations to protein loss in patient muscle, showing truncating mutations abolish and point mutations destabilize ANO5.\",\n      \"evidence\": \"Western blot with validated monoclonal antibody on fractionated patient muscle biopsies\",\n      \"pmids\": [\"28489263\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Degradation pathway in vivo not dissected\", \"Residual function of destabilized mutants unquantified\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Causally tied ANO5 scrambling/current activity to muscle precursor cell fusion via knockout and viral rescue.\",\n      \"evidence\": \"HEK293 reconstitution, Ano5-/- MPC fusion and PS-exposure assays, electrophysiology, and viral rescue\",\n      \"pmids\": [\"30257928\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How scrambling mechanistically drives fusion not resolved\", \"Subcellular site of fusion-relevant scrambling unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Refined localization to the sarcoplasmic reticulum and showed truncated ANO5 forms aggregates.\",\n      \"evidence\": \"Immunofluorescence with validated antibody on muscle cryosections; truncated construct expression in C2C12 cells\",\n      \"pmids\": [\"29665321\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Aggregation pathogenicity not tested in vivo\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Established a rabbit model recapitulating human dystrophy, providing an animal system where ANO5 loss reproduces disease unlike the mouse.\",\n      \"evidence\": \"CRISPR/Cas9 frame-disrupting indels in rabbits with CK, histology, and imaging\",\n      \"pmids\": [\"29789544\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cellular mechanism not interrogated in this model\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined the membrane-repair mechanism, showing ANO5 enables ER-mediated cytosolic Ca2+ clearance after injury.\",\n      \"evidence\": \"Patient myoblasts, focal laser injury repair assays, Ca2+ imaging, genetic and pharmacological rescue\",\n      \"pmids\": [\"31341644\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular link between scrambling activity and ER Ca2+ handling not delineated\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Implicated ANO5 in SR Ca2+ handling and nuclear positioning in myotubes, broadening its muscle role beyond repair.\",\n      \"evidence\": \"shRNA knockdown in C2C12 with immunofluorescence, western blot (Kif5b/DHPR/SERCA1), Ca2+ imaging, co-localization\",\n      \"pmids\": [\"31680776\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether protein-expression changes are direct or secondary unknown\", \"Single knockdown system\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Systematically partitioned disease alleles, showing MD mutations cause loss-of-function and GDD mutations gain-of-function scrambling.\",\n      \"evidence\": \"HEK293 scrambling assays across multiple site-directed patient variants\",\n      \"pmids\": [\"32112655\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tissue-specific consequences of opposite activities not addressed\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified BVES as a direct ER-membrane partner of ANO5 required for myoblast differentiation, adding a protein-interaction dimension.\",\n      \"evidence\": \"BioID2 proximity labeling, reciprocal Co-IP, domain mapping, co-localization, and CRISPR differentiation assays\",\n      \"pmids\": [\"34963485\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of the interaction on scrambling activity not tested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Produced a mouse out-of-frame deletion model reproducing mild-to-moderate LGMD2L independent of sarcolemmal chloride-channel activity.\",\n      \"evidence\": \"Knockout mouse with histology, force measures, chloride electrophysiology, and regeneration assays\",\n      \"pmids\": [\"34633328\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cellular driver of progressive muscle loss not isolated\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Demonstrated that a GDD gain-of-function knock-in recapitulates skeletal dysplasia with opposing osteoblast/osteoclast phenotypes.\",\n      \"evidence\": \"Cys360Tyr knock-in mouse with skeletal phenotyping and osteoblast/osteoclast cultures\",\n      \"pmids\": [\"34841576\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanistic link between scrambling gain-of-function and bone cell behavior unresolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extended ANO5 function to bone homeostasis through Ca2+-dependent osteoblast and osteoclast differentiation.\",\n      \"evidence\": \"Ano5 knockout mice with calcium imaging, differentiation assays, signaling western blots, and PTH treatment\",\n      \"pmids\": [\"35982081\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How ANO5 controls intracellular Ca2+ transients in bone cells not defined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Revealed sex-dependent tissue involvement, with male mice showing muscle repair defects and females showing cardiomyopathy.\",\n      \"evidence\": \"Ano5-/- mice with post-exercise CK, laser-injury repair, and echocardiography, sex-stratified\",\n      \"pmids\": [\"35020501\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Basis of sexual dimorphism unknown\", \"Cardiac mechanism not dissected\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Established a vascular function: ANO5-dependent endothelial PS externalization drives vessel-wall procoagulant activity and thrombosis.\",\n      \"evidence\": \"Genetic screen, in vitro PS assays, intravital laser-injury thrombosis model, TMEM16E-null mice, benzbromarone inhibition\",\n      \"pmids\": [\"36951953\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endothelial localization and Ca2+ trigger not detailed\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Detailed the osteoclast arm, showing ANO5 loss suppresses RANKL-NF-kB signaling and osteoclastogenesis.\",\n      \"evidence\": \"Ano5-/- mice with TRAP/phalloidin staining, resorption assays, qRT-PCR, NF-kB western blots and pharmacological rescue\",\n      \"pmids\": [\"36989132\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct connection of scrambling to NF-kB signaling not shown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Connected ANO5 scrambling to a membrane-repair effector pathway via ligand-independent EGFR/PI3K signaling and macropinocytosis.\",\n      \"evidence\": \"Co-IP (TMEM16E-EGFR), PI3K activity assay, macropinocytosis and annexin-V internalization assays, amiloride inhibition, live imaging\",\n      \"pmids\": [\"39794444\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How PS externalization triggers EGFR remains unclear\", \"Single-lab Co-IP for EGFR interaction\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single intracellular scramblase mechanistically produces such divergent tissue outcomes—muscle repair, sperm motility, bone remodeling, and endothelial coagulation—and the structural basis of its Ca2+-gated scrambling remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of ANO5 scrambling gate\", \"Unified mechanism linking scrambling to downstream tissue-specific signaling absent\", \"Reasons for mouse vs. rabbit/human disease discordance unexplained\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [3, 6, 8, 13]},\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [6, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [0, 11, 14]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [6, 18, 20]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [8, 14]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [11]},\n      {\"term_id\": \"R-HSA-109582\", \"supporting_discovery_ids\": [18]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [1, 13]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"BVES\", \"EGFR\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}