{"gene":"ANO6","run_date":"2026-04-28T17:12:37","timeline":{"discoveries":[{"year":2010,"finding":"TMEM16F (ANO6) is an essential component for Ca2+-dependent exposure of phosphatidylserine on the cell surface; wild-type TMEM16F localizes to the plasma membrane and confers Ca2+-dependent bidirectional phospholipid scrambling activity. A patient with Scott syndrome carries a splice-acceptor site mutation causing premature termination of TMEM16F, linking loss-of-function to defective phospholipid scrambling.","method":"Expression cloning in Ba/F3 cells, FACS-based PtdSer exposure assay, plasma membrane localization by fluorescence, patient mutation sequencing","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1-2 — original discovery with expression cloning, functional reconstitution, localization, and human genetic validation; highly cited foundational paper","pmids":["21107324"],"is_preprint":false},{"year":2012,"finding":"TMEM16F generates a small-conductance Ca2+-activated nonselective cation (SCAN) current permeable to both monovalent and divalent cations including Ca2+, with synergistic gating by Ca2+ and voltage. TMEM16F knockout mice lack Ca2+-dependent phosphatidylserine exposure and procoagulant activity in megakaryocytes, and exhibit bleeding defects and protection from arterial thrombosis. A pore-region residue determines cation vs. anion selectivity.","method":"TMEM16F knockout mouse generation, patch-clamp electrophysiology in megakaryocytes, heterologous expression, site-directed mutagenesis of pore region","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1-2 — KO mouse with defined platelet phenotype, patch-clamp, mutagenesis; highly cited, multiple orthogonal methods","pmids":["23021219"],"is_preprint":false},{"year":2013,"finding":"TMEM16F (ANO6) functions as a component of a Ca2+-activated Cl- channel with high Ca2+ threshold (~9.6 µM), anion selectivity sequence I->Br->Cl->F->aspartate-, and outward rectification. It is not related to volume-sensitive outwardly rectifying Cl- channel (VSOR) activity.","method":"Whole-cell patch-clamp in TMEM16F-transfected HEK293T cells, ion substitution experiments, siRNA knockdown","journal":"American Journal of Physiology - Cell Physiology","confidence":"High","confidence_rationale":"Tier 1 — direct electrophysiological characterization with multiple ion substitution experiments and KD controls","pmids":["23426967"],"is_preprint":false},{"year":2013,"finding":"ANO6-mediated Ca2+-dependent phospholipid scrambling can occur independently of its ion currents. Ca2+-independent phospholipid scrambling during apoptosis (intrinsic or extrinsic) does not require ANO6. ANO6 Cl- currents can be activated Ca2+-independently via Fas receptor stimulation.","method":"Patch-clamp electrophysiology, PS exposure assay (annexin V), Cl- channel blockers, siRNA knockdown, Scott syndrome patient B-lymphocytes","journal":"Cell Death & Disease","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including patient cells, siRNA, pharmacology, and functional assays","pmids":["23618909"],"is_preprint":false},{"year":2013,"finding":"ANO6 (TMEM16F) mediates Ca2+-dependent phospholipid scrambling in osteoblasts; deletion in mice results in impaired PS scrambling in osteoblasts and delayed mineralization, with increased areas of uncalcified osteoid postnatally. This establishes a cell-autonomous role for ANO6 in bone mineralization via PS exposure.","method":"Ano6 knockout mouse generation, primary osteoblast culture, mineralization assays, Ca2+-dependent PS scrambling assay in osteoblasts","journal":"Journal of Bone and Mineral Research","confidence":"High","confidence_rationale":"Tier 2 — KO mouse with specific skeletal phenotype plus cell-autonomous scrambling defect in primary osteoblasts","pmids":["22936354"],"is_preprint":false},{"year":2013,"finding":"In Scott syndrome patients, TMEM16F-dependent high-PS-exposing platelet fraction is absent upon convulxin/thrombin stimulation. TMEM16F is required for Ca2+-mobilizing agonist-induced PS exposure that depends on mitochondrial depolarization, but is not required for caspase-dependent basal PS exposure.","method":"Flow cytometry with annexin V, caspase inhibitors, Ca2+ chelation, Scott syndrome patient platelets vs. controls","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 — patient-derived cells with multiple pharmacological dissections and orthogonal readouts","pmids":["23303820"],"is_preprint":false},{"year":2013,"finding":"Mouse TMEM16F expressed in HEK293 cells produces a Ca2+-activated anion channel current (EC50 ~100 µM Ca2+) with delayed activation, Eisenman type 1 anion selectivity, and outward rectification. Pore-region mutations (R592E, K616E, R636E) alter or abolish currents, identifying the pore region between TM5 and TM6 as functional.","method":"Heterologous expression in HEK293, whole-cell patch-clamp, site-directed mutagenesis of pore residues","journal":"Journal of General Physiology","confidence":"High","confidence_rationale":"Tier 1 — electrophysiology with mutagenesis, detailed biophysical characterization","pmids":["23630341"],"is_preprint":false},{"year":2015,"finding":"TMEM16F is the only TMEM16 family member highly expressed in mouse platelets. Platelet-specific TMEM16F knockout causes defects in activation-induced PS exposure, microparticle shedding, and thrombin generation in vitro and in vivo (laser-induced thrombus model), without affecting granule release or clot retraction.","method":"Platelet-specific TMEM16F conditional KO mice, flow cytometry, thrombin generation assay, laser-induced thrombosis imaging in vivo","journal":"PNAS","confidence":"High","confidence_rationale":"Tier 2 — platelet-specific KO with multiple in vitro and in vivo functional readouts","pmids":["26417084"],"is_preprint":false},{"year":2015,"finding":"A specific domain in ANO6 is necessary and sufficient for phospholipid scrambling activity. By analyzing ANO1-ANO6 chimeric proteins, a scramblase domain was identified in ANO6 that, when transferred to ANO1 (which normally does not scramble), confers scramblase activity. Homology modeling shows this domain forms a hydrophilic cleft facing the lipid bilayer. Ion currents in ANO6 are explained by ionic leak during phospholipid translocation.","method":"Patch-clamp combined with PS exposure assay, ANO1-ANO6 chimera construction, homology modeling","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 — domain-swap chimera experiments with both functional and structural analysis, multiple orthogonal methods","pmids":["26057829"],"is_preprint":false},{"year":2015,"finding":"TMEM16F forms homodimers (shown by chemical cross-linking). The pore region between TM5 and TM6 is essential for both scramblase activity of TMEM16F and Cl- channel activity of TMEM16A. N-terminal and C-terminal cytoplasmic domains control plasma membrane localization and protein stability, respectively, and are functionally interchangeable between TMEM16A and TMEM16F.","method":"Chemical cross-linking, domain-swap chimeras, deletion analysis, functional assays in 293T cells and TMEM16F-/- thymocytes","journal":"Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — cross-linking, systematic domain deletion/swap with functional validation","pmids":["24478309"],"is_preprint":false},{"year":2015,"finding":"TMEM16F isoforms generated by alternative splicing (V1, V2, V5) show plasma membrane localization and Ca2+-activated ion channel and scramblase activities, while V3 isoform (unique C-terminus) is intracellularly localized and inactive. An activating mutation D409G markedly increases apparent Ca2+ sensitivity of both channel and scramblase activities, directly demonstrating TMEM16F mediates both functions.","method":"Whole-cell patch-clamp, annexin V binding assay for PS, subcellular localization, activating mutation introduction, siRNA knockdown","journal":"Journal of Physiology","confidence":"High","confidence_rationale":"Tier 1-2 — multiple isoforms tested, gain-of-function mutation, parallel channel and scramblase readouts","pmids":["26108457"],"is_preprint":false},{"year":2016,"finding":"TMEM16F is located in late endosomes in T lymphocytes, where it facilitates generation of multivesicular bodies for TCR degradation and signal termination. TMEM16F-deficient T cells show sustained TCR signaling, augmented activation, and increased proliferation and cytokine production in chronic viral infection, ultimately leading to T cell exhaustion.","method":"Conditional TMEM16F KO mice, viral chronic infection model, late endosome localization by imaging, TCR degradation assay, T cell functional assays","journal":"Journal of Experimental Medicine","confidence":"High","confidence_rationale":"Tier 2 — conditional KO with mechanistic pathway placement (late endosome localization + TCR degradation), in vivo infection model","pmids":["27810927"],"is_preprint":false},{"year":2016,"finding":"TMEM16F conditional knockout in microglia prevents mechanical hypersensitivity after nerve injury; TMEM16F-deficient microglia show deficits in process motility and phagocytosis. This establishes TMEM16F as essential for microglial responses to nerve injury in neuropathic pain.","method":"Conditional TMEM16F KO in microglia, neuropathic pain behavioral testing, microglial motility and phagocytosis assays","journal":"Cell Reports","confidence":"High","confidence_rationale":"Tier 2 — conditional cell-type-specific KO with defined cellular phenotype and behavioral readout","pmids":["27332874"],"is_preprint":false},{"year":2016,"finding":"Ca2+ directly binds to TMEM16F; five acidic residues conserved between TMEM16F and TMEM16K are critical for Ca2+ binding (identified by comprehensive mutagenesis of TMEM16K). Point mutations of corresponding residues in TMEM16F reduce Ca2+-dependent phospholipid scrambling. Ca2+ stabilizes TMEM16F structure and induces conformational changes.","method":"Blue-native PAGE, comprehensive acidic-residue mutagenesis, Ca2+ binding assay, scramblase activity assay","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 — direct Ca2+ binding shown biochemically, mutagenesis identifies specific residues, functional correlation","pmids":["27227820"],"is_preprint":false},{"year":2017,"finding":"TMEM16F/ANO6 ion currents and phospholipid scrambling can be activated by modification of plasma membrane phospholipids via reactive oxygen species and phospholipase A2 (PLA2), independently of intracellular Ca2+. Mutations within TMEM16F similarly change both Cl- currents and phospholipid scrambling, suggesting Cl- and phospholipids use the same intramolecular pathway.","method":"Whole-cell patch-clamp, annexin V PS exposure assay, PLA2 activation/inhibition, ROS donors, chimera/mutant analysis","journal":"Journal of Physiology","confidence":"High","confidence_rationale":"Tier 2 — multiple activation mechanisms tested with parallel channel/scramblase readouts, mutagenesis supports shared pathway","pmids":["29134661"],"is_preprint":false},{"year":2018,"finding":"Single purified TMEM16F dimeric molecules transport phospholipids nonspecifically and bidirectionally between membrane bilayer leaflets in a Ca2+-dependent manner at ~4.5×10^4 lipids/second at 25°C, with activation free energy of 47 kJ/mol. This biophysical profile is consistent with a channel-like facilitated diffusion ('stepping-stone') mechanism.","method":"Purification of mouse TMEM16F from stable cell line, single-molecule scramblase assay in lipid bilayer microarray, thermodynamic analysis","journal":"PNAS","confidence":"High","confidence_rationale":"Tier 1 — purified protein reconstitution, single-molecule analysis, rigorous thermodynamic characterization","pmids":["29507235"],"is_preprint":false},{"year":2018,"finding":"TMEM16F Ca2+-activated current is desensitized by brief high Ca2+ exposure associated with PIP2 depletion from the inner membrane leaflet. Application of PIP2 restores TMEM16F channel activity. PIP2 modulation requires positively charged amino acids in the cytoplasmic N-terminal domain and acts synergistically with membrane depolarization to facilitate Ca2+-gating.","method":"Excised patch and whole-cell patch-clamp, PIP2 application/depletion, N-terminal domain mutagenesis","journal":"PNAS","confidence":"High","confidence_rationale":"Tier 1-2 — electrophysiology with direct ligand application and mutagenesis identifying regulatory residues","pmids":["29382763"],"is_preprint":false},{"year":2019,"finding":"Cryo-EM structures of murine TMEM16F in absence and presence of Ca2+ reveal a ligand-free closed conformation and a Ca2+-bound intermediate. Both conformations resemble TMEM16A counterparts but with distinct differences in ion/lipid permeation region. Ion conduction and lipid scrambling are activated by a common Ca2+-binding mechanism but appear mediated by alternate protein conformations at equilibrium in the Ca2+-bound state.","method":"Cryo-EM structure determination, functional electrophysiology, Ca2+-dependent activity assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 — high-resolution cryo-EM structures with functional validation, multiple conformational states characterized","pmids":["30785399"],"is_preprint":false},{"year":2019,"finding":"Cryo-EM structural analysis reveals coexistence of an intact channel pore and PIP2-dependent membrane distortion in TMEM16F. Mutagenesis of lipid-binding residues near membrane distortion sites specifically alters onset of lipid scrambling without affecting Ca2+ influx, providing structural evidence for separate pathways for lipid scrambling and ion permeation.","method":"Cryo-EM, structure-based mutagenesis, functional assays for Ca2+ influx and PS exposure","journal":"Cell Reports","confidence":"High","confidence_rationale":"Tier 1 — cryo-EM structure combined with mutagenesis functionally separating scrambling from ion permeation","pmids":["31291589"],"is_preprint":false},{"year":2019,"finding":"TMEM16F contains an inner activation gate formed by three hydrophobic residues (F518, Y563, I612) in the middle of the phospholipid permeation pathway. Lysine substitutions of F518 and Y563 generate constitutively active scramblases bypassing Ca2+-dependent activation. An analogous mutation (L543K) in TMEM16A confers CaPLSase activity to this otherwise Cl- channel.","method":"Site-directed mutagenesis, PS exposure assay, patch-clamp electrophysiology, gain-of-function and loss-of-function analysis","journal":"Nature Communications","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis identifies specific gate residues with constitutive activation phenotype, cross-validated by gain-of-function in paralog","pmids":["31015464"],"is_preprint":false},{"year":2019,"finding":"TMEM16F ion selectivity dynamically shifts from cation-selective to anion-selective in response to increasing intracellular Ca2+, reflecting alteration of electrostatic field in the permeation pathway (charge-screening mechanism). The Q559K mutant reveals this selectivity shift is independent of channel activation state.","method":"Excised inside-out patch-clamp, ion substitution experiments with varying Ca2+ concentrations, mutagenesis","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 — systematic electrophysiology with ion substitution and mutagenesis revealing dynamic selectivity mechanism","pmids":["31318330"],"is_preprint":false},{"year":2019,"finding":"TMEM16F activation by Ca2+ ionophores in Jurkat T cells triggers large-scale plasma membrane expansion coinciding with phospholipid scrambling, followed by ectosome shedding. PD-1 is selectively incorporated into ectosomes in a transmembrane-sequence-dependent manner. TMEM16F-deficient cells fail to expand surface membrane and instead undergo rapid massive endocytosis with PD-1 internalization.","method":"Live-cell microscopy, patch-clamp, flow cytometry, TMEM16F-KO Jurkat cells, PD-1 trafficking assays","journal":"Scientific Reports","confidence":"High","confidence_rationale":"Tier 2 — combined microscopy and electrophysiology with KO cells and defined cargo-sorting readout","pmids":["30679690"],"is_preprint":false},{"year":2020,"finding":"TMEM16F is essential for plasma membrane repair after pore-forming agent injury: pore formation triggers Ca2+ influx activating TMEM16F-mediated lipid scrambling, membrane blebbing, and extracellular vesicle release that preserve membrane integrity and cell viability. TMEM16F-deficient mice show compromised control of Listeria infection due to greater neutrophil sensitivity to listeriolysin O.","method":"TMEM16F-deficient cell lines and mice, pore-forming toxin treatment, cell viability assays, EV quantification, Listeria infection model","journal":"Cell Reports","confidence":"High","confidence_rationale":"Tier 2 — KO cells and mice with defined mechanistic pathway (pore→Ca2+→TMEM16F→membrane blebbing/repair) and in vivo infection readout","pmids":["31995754"],"is_preprint":false},{"year":2020,"finding":"TMEM16F knockout mice show deficiency in trophoblast syncytialization and placental development leading to perinatal lethality. TMEM16F CaPLSase translocates PS to the cell surface independently of apoptosis during trophoblast fusion.","method":"TMEM16F KO mouse generation, placental histology, trophoblast fusion assays, PS exposure measurement","journal":"Science Advances","confidence":"High","confidence_rationale":"Tier 2 — KO mouse with specific placental phenotype and cell-autonomous fusion/PS assays","pmids":["32494719"],"is_preprint":false},{"year":2020,"finding":"TMEM16F governs Ca2+-activated Cl- conductance in spinal motoneurons and is expressed in synaptic clusters facing cholinergic C-boutons. Tmem16f exon-deleted mice show decreased motor performance under high-demanding tasks and increased recruitment threshold of fast α-motoneurons. Loss of TMEM16F in ALS mouse model delays disease onset and preserves strength in male mice.","method":"Targeted exon deletion mouse, immunolocalization at C-boutons, patch-clamp in motoneurons, motor behavioral assays, ALS mouse model","journal":"Cell Reports","confidence":"High","confidence_rationale":"Tier 2 — specific KO with defined electrophysiological phenotype in native neurons and behavioral readouts","pmids":["32101737"],"is_preprint":false},{"year":2021,"finding":"TMEM16F mediates Ca2+-dependent plasma membrane expansion via invaginations held shut by dynamin. Upon Ca2+ activation of TMEM16F, anionic phospholipids escape from the cytoplasmic monolayer and dynamins relax, opening compartments. Deletion of TMEM16F or dynamins blocks expansion; dynamin2 GTPase-inactivated mutant can regenerate reserve compartments but lipid-binding mutant cannot.","method":"TMEM16F KO cells, dynamin KO/re-expression, live-cell microscopy, dynamin2-GFP imaging, Ca2+-permeable mechanosensitive channel activation","journal":"Nature Communications","confidence":"High","confidence_rationale":"Tier 2 — multiple genetic manipulations with live imaging and mechanistic dissection of dynamin-TMEM16F interplay","pmids":["34404808"],"is_preprint":false},{"year":2021,"finding":"TMEM16F Ca2+-dependent activation of PS scramblase during immunological synapse formation locally redistributes PS, reducing the electrostatic potential of the plasma membrane, which increases dissociation of bystander TCR-CD3 cytoplasmic domains from the membrane and enhances TCR-dependent signaling. This establishes the molecular basis for TMEM16F-mediated bystander TCR signal amplification.","method":"T cell activation assays, PS externalization measurement, membrane electrostatic potential probes, TCR-CD3 membrane association assays","journal":"Science Signaling","confidence":"High","confidence_rationale":"Tier 2 — mechanistic pathway established from scramblase activity to electrostatic change to TCR signaling with multiple readouts","pmids":["33758060"],"is_preprint":false},{"year":2021,"finding":"TMEM16F channel and scramblase activities are strongly regulated by intracellular pH (pHi): low pHi attenuates and high pHi potentiates both activities. pHi sensitivity depends on [Ca2+]i and shows a bell-shaped relationship with [Ca2+]i. A Ca2+-binding residue mutation (E667Q) shifts the peak [Ca2+]i of pHi sensitivity, establishing that protons compete with Ca2+ at primary Ca2+-binding sites in the pore to regulate TMEM16F activation.","method":"Whole-cell patch-clamp, PS exposure assay at varying pHi and Ca2+ levels, Ca2+-binding residue mutagenesis (E667Q)","journal":"Journal of General Physiology","confidence":"High","confidence_rationale":"Tier 1 — systematic biophysical characterization with mutagenesis identifying the Ca2+/proton competition mechanism","pmids":["33346788"],"is_preprint":false},{"year":2022,"finding":"TMEM16F is activated by SARS-CoV-2 Spike protein binding (via ACE2) to mediate PS externalization critical for viral entry. ANO6-selective inhibitor A6-001 identified by high-throughput screening inhibits both Spike-induced PS scrambling and authentic SARS-CoV-2 replication in multiple cell types.","method":"SARS2 pseudotyped virus, Ca2+ imaging, annexin V flow cytometry, high-throughput drug screening, authentic SARS-CoV-2 infection assays in Vero/Calu-3/primary nasal cells","journal":"Cell Reports","confidence":"High","confidence_rationale":"Tier 2 — mechanistic link established between Spike-induced Ca2+, ANO6 activation, and viral entry with pharmacological validation in authentic virus","pmids":["35839776"],"is_preprint":false},{"year":2022,"finding":"Cryo-EM structures of activating TMEM16F mutants reveal major rearrangements leading to exposure of hydrophilic patches to the membrane, whose distortion facilitates lipid diffusion. Concomitant opening of a pore promotes ion conduction in the same protein conformation, revealing a mechanism distinct from other TMEM16 family members.","method":"Cryo-EM structure determination of activating mutants, functional scramblase and ion channel assays","journal":"Nature Communications","confidence":"High","confidence_rationale":"Tier 1 — cryo-EM of multiple activating mutants with functional validation providing mechanistic insight into gating","pmids":["36335104"],"is_preprint":false},{"year":2022,"finding":"TRPV4 Ca2+ influx channel is functionally coupled to TMEM16F within Ca2+ microdomains in human trophoblasts. TRPV4-specific agonist activates TMEM16F in trophoblasts; pharmacological inhibition or gene silencing of TRPV4 impairs TMEM16F activation and subsequent trophoblast syncytialization.","method":"Patch-clamp electrophysiology, TRPV4 agonist/antagonist treatment, gene silencing, PS exposure assay, trophoblast fusion assay","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 — patch-clamp evidence for microdomain coupling, pharmacological and genetic validation of upstream activator","pmids":["35670667"],"is_preprint":false},{"year":2023,"finding":"Cryo-EM structures of TMEM16F with bound niclosamide or 1PBC reveal a lipid scrambling pathway along a groove outside the ion permeation pore containing a drug-binding pocket. Mutations in this groove specifically affect lipid scrambling but not ion conduction; some mutations preferentially reduce inhibition of PS exposure vs. Ca2+ influx, providing structural evidence for separate ion permeation and lipid scrambling pathways.","method":"Cryo-EM structure determination with bound drugs, mutagenesis, patch-clamp, PS exposure assay","journal":"Nature Communications","confidence":"High","confidence_rationale":"Tier 1 — drug-bound cryo-EM structures with mutagenesis functionally separating scrambling and ion permeation pathways","pmids":["37573365"],"is_preprint":false},{"year":2024,"finding":"TMEM16F (the RBC Ca2+-activated phospholipid scramblase) is activated downstream of Ca2+ influx through the mechanosensitive channel PIEZO1 in red blood cells. In hereditary xerocytosis (PIEZO1 gain-of-function), enhanced PIEZO1-TMEM16F coupling increases propensity for PS exposure, contributing to anemia, splenomegaly and thrombosis. Inhibition of PIEZO1 with benzbromarone prevents force-induced PS exposure.","method":"Electrophysiology, flow cytometry, imaging in RBCs from HX patients and controls; pharmacological PIEZO1 inhibition; functional hemolysis and PS assays","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 — mechanistic coupling established in patient RBCs with pharmacological intervention and multiple functional readouts","pmids":["38033286"],"is_preprint":false},{"year":2024,"finding":"TMEM16F deficiency in neurons (but not microglia) reduces tau pathology and microgliosis in a PS19 tauopathy mouse model. TMEM16F mediates aberrant PS exposure in neurons with phospho-tau burden, establishing a neuronal-autonomous role for TMEM16F in tau pathology.","method":"Cell-type-specific TMEM16F KO in PS19 tau mouse model, immunohistochemistry, PS exposure assay in neurons","journal":"PNAS","confidence":"High","confidence_rationale":"Tier 2 — cell-type-specific KO with defined tauopathy phenotype and mechanistic PS exposure readout","pmids":["38941274"],"is_preprint":false},{"year":2024,"finding":"TMEM16F deficiency in endothelial cells impairs developmental retinal angiogenesis. Biochemically, TMEM16F absence enhances plasma membrane association of activated Src kinase (Y416 phosphorylation), increases VE-cadherin phosphorylation and downregulation, suppressing angiogenesis. This establishes an intracellular signaling role for TMEM16F in endothelial cells.","method":"Endothelial-specific TMEM16F KO mice, retinal angiogenesis assay, Src kinase phosphorylation, VE-cadherin Western blot/immunofluorescence, HUVEC siRNA knockdown","journal":"Journal of Cell Science","confidence":"High","confidence_rationale":"Tier 2 — conditional KO mouse with in vivo angiogenesis phenotype plus biochemical pathway from TMEM16F→Src→VE-cadherin","pmids":["38940198"],"is_preprint":false},{"year":2024,"finding":"TMEM16F regulates pathologic α-synuclein (α-synA53T) spread in neurons. Neurons from TMEM16F KO mice show reduced donor-to-recipient spread of α-synA53T. In vivo PD mouse model shows attenuated α-synA53T spread upon TMEM16F ablation. A missense SNP (Ala703Ser) in TMEM16F with enhanced scramblase activity is associated with altered α-synA53T secretion in Ashkenazi Jewish PD patients.","method":"TMEM16F KO neurons with reporter-based spread assay, in vivo PD mouse model, lipid scramblase activity assay for Ala703Ser variant","journal":"Aging Cell","confidence":"High","confidence_rationale":"Tier 2 — KO neurons and in vivo model with defined spread phenotype; gain-of-function variant with functional characterization","pmids":["39487963"],"is_preprint":false},{"year":2018,"finding":"ANO6/TMEM16F Cl- channel activation kinetics are negatively regulated by the intact actin cytoskeleton (cytochalasin-D accelerates, phalloidin/jasplakinolide inhibit activation) and positively modulated by intracellular MgATP (prevents inactivation). Inside-out patches show immediate Ca2+-dependent activation, suggesting cytosolic factors including cytoskeleton and ATP mediate the slow whole-cell activation.","method":"Whole-cell and inside-out patch-clamp, cytoskeleton-disrupting/stabilizing agents, intracellular ATP manipulation","journal":"Biochemical and Biophysical Research Communications","confidence":"Medium","confidence_rationale":"Tier 2 — electrophysiology with pharmacological manipulation of actin and ATP, single lab","pmids":["29964013"],"is_preprint":false},{"year":2023,"finding":"ANO6-dependent trogocytosis in cancer-associated fibroblasts (CAFs) is triggered by cytosolic Ca2+ influx via Orai channels, which activates ANO6 causing PS exposure on CAF plasma membranes, initiating membrane lipid transfer (including cholesterol) to pancreatic cancer cells. ANO6-dependent trogocytosis also supports immunosuppressive function of CAFs toward cytotoxic T cells.","method":"CRISPR KO of ANO6, Ca2+ imaging, annexin V/PS exposure assay, cholesterol transfer assay, T cell cytotoxicity assay","journal":"bioRxiv (preprint)","confidence":"Medium","confidence_rationale":"Tier 2 — multiple functional assays with genetic KO, but preprint not yet peer-reviewed","pmids":["37745612"],"is_preprint":true},{"year":2025,"finding":"High-resolution cryo-EM of TMEM16F active in liposomes reveals two conformations in high-activity conditions: the canonical Ca2+-bound closed state and a novel X-shaped groove conformation where upward rotation of the cytosolic domain forms a transmembrane pore and locally thins the membrane. Mutagenesis, functional assays, and MD simulations show the X-shaped groove mediates nonselective ion flux and lipid scrambling through distinct pathways (ions within the pore, lipids skirting the groove).","method":"Cryo-EM in liposomes, site-directed mutagenesis, functional ion flux and scramblase assays, molecular dynamics simulations","journal":"Nature Structural & Molecular Biology","confidence":"High","confidence_rationale":"Tier 1 — cryo-EM of active protein in native-like lipid environment combined with mutagenesis and MD simulations; rigorous multi-method study","pmids":["41998358"],"is_preprint":false},{"year":2025,"finding":"TMEM16F preferentially scrambles phosphatidylserine and phosphatidylcholine over phosphatidylethanolamine on the plasma membrane of living cells, contrary to the prevailing view that scramblases act without headgroup preference.","method":"Cell-based fluorescence polarization scrambling assay with NBD-labeled phospholipids, kinetic monitoring on live cell plasma membranes","journal":"PNAS","confidence":"Medium","confidence_rationale":"Tier 2 — novel cell-based assay with multiple phospholipid species tested; single lab, single method but rigorous experimental design","pmids":["41166415"],"is_preprint":false},{"year":2019,"finding":"TMEM16F activation contributes to ferroptotic cell death: ferroptosis inducers (erastin, RSL3) activate TMEM16F currents in intestinal epithelium and macrophages, and cell death is largely reduced in tissue-specific TMEM16F KO mice. Inhibitors of ferroptosis (ferrostatin-1) block TMEM16F currents.","method":"Tissue-specific TMEM16F KO mice, ferroptosis induction with erastin/RSL3, cell death assays, patch-clamp recording","journal":"Cancers","confidence":"High","confidence_rationale":"Tier 2 — tissue-specific KO with defined ferroptosis phenotype and electrophysiological correlation","pmids":["31060306"],"is_preprint":false},{"year":2018,"finding":"TMEM16F contributes to pyroptotic cell death downstream of gasdermin-D pore formation. GD-N expression induces Ca2+ elevation activating TMEM16F, which generates large whole-cell currents; knockdown or inhibition of TMEM16F suppresses these currents and reduces cell death in HEK293 and HAP1 cells.","method":"GD-N expression, TMEM16F knockdown/inhibition, whole-cell patch-clamp, cell viability assays","journal":"Cell Death & Disease","confidence":"Medium","confidence_rationale":"Tier 2 — KD with electrophysiological and viability readouts, single lab","pmids":["29463790"],"is_preprint":false},{"year":2021,"finding":"N-terminal domain of ANO6 contains a putative Ca2+-transferring reservoir (Nt-CaRes) that regulates Ca2+ sensitivity. Chimera ANO6-1-6 (with ANO1 Nt-CaRes substituted) shows higher Ca2+ sensitivity than ANO6. Mutagenesis of acidic amino acids in Nt-CaRes reduces Ca2+ sensitivity, consistent with direct Ca2+ interactions at these residues.","method":"Chimera construction, site-directed mutagenesis of acidic residues, patch-clamp Ca2+ dose-response, molecular dynamics simulation","journal":"Molecules and Cells","confidence":"Medium","confidence_rationale":"Tier 2 — chimera and mutagenesis with functional Ca2+ sensitivity readout, but single lab","pmids":["33658434"],"is_preprint":false},{"year":2024,"finding":"Atomic force microscopy under physiological conditions reveals structurally and mechanically diverse TMEM16F assemblies with variable inter-subunit dimerization interfaces. Ca2+-induced activation is associated with stepwise changes in the pore region affecting mechanical properties of TM3, TM4, and TM6. Direct observation of membrane remodeling links structural heterogeneity to ion and lipid permeation.","method":"Atomic force microscopy in physiological conditions, cryo-EM comparison, patch-clamp electrophysiology","journal":"Nature Communications","confidence":"High","confidence_rationale":"Tier 1 — novel structural technique (physiological-condition AFM) combined with electrophysiology revealing dynamic conformational states","pmids":["38167485"],"is_preprint":false},{"year":2026,"finding":"Plasmalemmal lipid scrambling by TMEM16F (using inducible active form) is sufficient to release apoptotic-like vesicles without changes in cytosolic Ca2+ or submembrane cytoskeleton. Scrambling causes segregation of exofacial lipids, redistribution of cholesterol to inner leaflet, GPI-anchored protein clustering forming convex curvature, and PE accumulation forming concave curvature facilitating vesicle scission.","method":"Inducible constitutively active TMEM16F, live-cell imaging, lipid domain analysis, extracellular vesicle quantification","journal":"Molecular Biology of the Cell","confidence":"High","confidence_rationale":"Tier 1 — reconstitution using controlled TMEM16F activation with detailed lipid and vesicle biogenesis analysis","pmids":["41604453"],"is_preprint":false}],"current_model":"ANO6/TMEM16F is a Ca2+-activated dual-function plasma membrane protein that operates as a phospholipid scramblase and a largely non-selective ion channel: upon elevation of intracellular Ca2+ (binding directly to conserved acidic residues), TMEM16F undergoes conformational changes—including cytosolic domain rotation to form an X-shaped groove—that open both a transmembrane ion pore and a lipid permeation pathway (the two routes being physically distinct but sharing part of the same groove), catalyzing bidirectional, facilitated phosphatidylserine/phosphatidylcholine-preferential lipid flip-flop at ~4.5×10⁴ lipids/second while concurrently conducting ions; its activity is regulated by PIP2 (via N-terminal cationic residues), intracellular pH, actin cytoskeleton, and upstream Ca2+ channels (PIEZO1, TRPV4); physiologically, TMEM16F-driven PS externalization is essential for platelet procoagulant activity, trophoblast fusion, osteoblast mineralization, plasma membrane repair, T cell receptor signal termination via late-endosomal multivesicular body formation, microglial phagocytosis, and ectosome/extracellular vesicle biogenesis."},"narrative":{"teleology":[{"year":2010,"claim":"The identity of the long-sought Ca²⁺-dependent phospholipid scramblase was unknown; expression cloning identified TMEM16F as the essential component, and a Scott syndrome patient mutation provided human genetic validation, establishing TMEM16F as the Ca²⁺-dependent scramblase.","evidence":"Expression cloning in Ba/F3 cells with FACS-based PS assay, patient mutation sequencing","pmids":["21107324"],"confidence":"High","gaps":["Mechanism of scrambling (channel vs. transporter) unresolved","Whether TMEM16F also conducts ions was unknown","Structure of the protein undetermined"]},{"year":2012,"claim":"Whether TMEM16F conducted ions and what its in vivo physiological role was remained open; electrophysiology revealed TMEM16F generates Ca²⁺-activated nonselective cation currents, and knockout mice demonstrated essential roles in platelet procoagulant activity and hemostasis.","evidence":"TMEM16F KO mouse, patch-clamp in megakaryocytes, pore-region mutagenesis","pmids":["23021219"],"confidence":"High","gaps":["Whether ion conduction and scrambling are mechanistically separable was unresolved","Structural basis of ion selectivity unknown"]},{"year":2013,"claim":"The relationship between TMEM16F's channel and scramblase activities, ion selectivity properties, and physiological roles beyond platelets needed clarification; multiple studies established that scrambling can occur independently of ion currents, identified the functional pore region (TM5-TM6), demonstrated roles in osteoblast mineralization, and dissected pathway-specific PS exposure in Scott syndrome platelets.","evidence":"Patch-clamp with pharmacological dissection and Scott patient cells; pore-region mutagenesis in HEK293; Ano6 KO mouse osteoblast mineralization assays; flow cytometry of Scott patient platelets","pmids":["23618909","23630341","22936354","23303820","23426967"],"confidence":"High","gaps":["Structural basis for dual function undetermined","How Ca²⁺ binds and gates the protein unknown","Molecular determinants distinguishing scramblase from channel activity not identified"]},{"year":2015,"claim":"What structural domain confers scramblase activity, and whether TMEM16F functions as a monomer or oligomer, was unknown; chimera studies identified a specific scramblase domain transferable to the channel-only paralog TMEM16A, cross-linking revealed homodimers, and isoform analysis with gain-of-function mutations confirmed both activities reside in one protein.","evidence":"ANO1-ANO6 chimeras with PS and electrophysiology readouts; chemical cross-linking; splice-variant characterization; D409G activating mutation; platelet-specific conditional KO","pmids":["26057829","24478309","26108457","26417084"],"confidence":"High","gaps":["Atomic structure not yet available","Ca²⁺-binding site residues in TMEM16F not directly identified","Whether ion and lipid pathways are physically the same groove remained debated"]},{"year":2016,"claim":"TMEM16F's roles beyond hemostasis and bone were largely unexplored; conditional KO studies revealed TMEM16F in late endosomes controls TCR degradation and T cell signal termination, and in microglia it is essential for phagocytosis and neuropathic pain responses, while biochemical studies identified specific Ca²⁺-binding residues.","evidence":"Conditional KO mice in T cells and microglia; late-endosome localization; TCR degradation and viral infection models; blue-native PAGE and comprehensive mutagenesis of Ca²⁺-coordinating acidic residues","pmids":["27810927","27332874","27227820"],"confidence":"High","gaps":["Structural mechanism of Ca²⁺-dependent gating unknown","How TMEM16F reaches late endosomes was unexplained","Whether late-endosomal and plasma membrane functions are mechanistically identical was unclear"]},{"year":2018,"claim":"Regulatory mechanisms controlling TMEM16F activity in situ were poorly understood; studies identified PIP₂ as a positive modulator acting via N-terminal cationic residues, the actin cytoskeleton as a negative kinetic regulator, and purified single-molecule reconstitution quantified scrambling rates consistent with a facilitated-diffusion mechanism.","evidence":"Excised patch with PIP₂ application and N-terminal mutagenesis; cytoskeleton-modulating agents with patch-clamp; purified TMEM16F dimer reconstituted in lipid bilayer microarray for single-molecule scrambling","pmids":["29382763","29964013","29507235"],"confidence":"High","gaps":["Structural basis of PIP₂ regulation not visualized","How actin physically interacts with TMEM16F unknown","Contributions of additional regulators (e.g., pH) not yet explored"]},{"year":2019,"claim":"The structural basis for TMEM16F's dual activity and whether ion and lipid pathways are the same remained debated; cryo-EM structures revealed Ca²⁺-bound and Ca²⁺-free conformations, mutagenesis identified a hydrophobic inner activation gate (F518/Y563/I612), dynamic ion selectivity shifting with Ca²⁺ was demonstrated, and roles in membrane expansion/ectosome shedding and ferroptosis were established.","evidence":"Cryo-EM of apo and Ca²⁺-bound TMEM16F; constitutively active gate mutants; ion substitution with varying Ca²⁺; live-cell microscopy with KO Jurkat cells; tissue-specific KO with ferroptosis inducers","pmids":["30785399","31291589","31015464","31318330","30679690","31060306"],"confidence":"High","gaps":["Cryo-EM captured only intermediate states, not the fully open active conformation","Whether the gate mutant phenotype reflects the physiological open state was uncertain","Mechanism of membrane expansion coupling to scrambling was unclear"]},{"year":2020,"claim":"Whether TMEM16F contributes to membrane repair and trophoblast fusion in vivo was untested; KO studies showed TMEM16F-mediated blebbing and EV release are essential for plasma membrane repair after pore-forming toxin injury, and TMEM16F drives PS-dependent trophoblast syncytialization required for placental development.","evidence":"TMEM16F-deficient cells/mice with pore-forming toxin and Listeria infection; TMEM16F KO mouse placental histology and trophoblast fusion assays","pmids":["31995754","32494719"],"confidence":"High","gaps":["Whether TMEM16F directly interacts with fusion machinery was unknown","Role of specific upstream Ca²⁺ channels in trophoblast context not identified"]},{"year":2021,"claim":"How TMEM16F scrambling translates into membrane expansion and TCR signaling modulation was mechanistically unclear; studies revealed TMEM16F lipid scrambling relaxes dynamin-held membrane invaginations to expand the cell surface, and at the immunological synapse PS redistribution reduces electrostatic potential to release bystander TCR-CD3 tails, amplifying signaling.","evidence":"TMEM16F/dynamin double-KO with live imaging; T cell activation assays with membrane electrostatic probes and TCR-CD3 association measurements","pmids":["34404808","33758060"],"confidence":"High","gaps":["Whether dynamin directly binds TMEM16F or acts indirectly via lipid sensing was unresolved","Quantitative contribution of electrostatic mechanism vs. other signaling effects unknown"]},{"year":2022,"claim":"The structural basis for physically separating ion and lipid permeation paths, upstream Ca²⁺ sources in trophoblasts, and viral exploitation of TMEM16F were open questions; cryo-EM of activating mutants revealed hydrophilic patch exposure and concomitant pore opening, TRPV4 was identified as the upstream Ca²⁺ channel coupling to TMEM16F in trophoblasts, PIEZO1 was shown to couple to TMEM16F in RBCs, and Spike-mediated SARS-CoV-2 entry was found to require TMEM16F-dependent PS scrambling.","evidence":"Cryo-EM of activating mutants; TRPV4 agonist/antagonist with trophoblast patch-clamp; PIEZO1-TMEM16F coupling in hereditary xerocytosis RBCs; SARS-CoV-2 pseudovirus and authentic infection with ANO6 inhibitor","pmids":["36335104","35670667","38033286","35839776"],"confidence":"High","gaps":["Full open-state structure in lipid environment not yet captured","Whether PIEZO1-TMEM16F coupling involves direct interaction or only Ca²⁺ microdomain proximity was unclear"]},{"year":2023,"claim":"Whether ion and lipid pathways are truly physically distinct at atomic resolution was debated; drug-bound cryo-EM structures revealed a lipid scrambling groove outside the ion pore with a druggable pocket, and mutagenesis selectively affected scrambling without impairing ion conduction, providing the clearest structural evidence for separate pathways.","evidence":"Cryo-EM with bound niclosamide/1PBC, structure-guided mutagenesis with parallel scramblase and channel assays","pmids":["37573365"],"confidence":"High","gaps":["Whether the drug-binding pocket represents a physiological regulatory site unknown","Resolution of lipid headgroup interactions within the groove not achieved"]},{"year":2024,"claim":"TMEM16F's roles in neurodegeneration and vascular biology were emerging; neuron-specific KO reduced tau pathology and α-synuclein spread, endothelial KO impaired retinal angiogenesis via Src/VE-cadherin signaling, and AFM revealed structurally diverse TMEM16F assemblies with Ca²⁺-induced stepwise mechanical changes.","evidence":"Cell-type-specific KO in PS19 tauopathy and PD models; endothelial KO retinal angiogenesis with Src phosphorylation analysis; AFM under physiological conditions","pmids":["38941274","39487963","38940198","38167485"],"confidence":"High","gaps":["Whether PS exposure directly mediates tau/α-syn pathology or acts indirectly through phagocytic signals unclear","Direct physical basis for Src membrane retention change not structurally resolved","Physiological significance of TMEM16F structural heterogeneity unknown"]},{"year":2025,"claim":"The fully active conformation of TMEM16F in a lipid environment had never been captured; cryo-EM in liposomes revealed an X-shaped groove formed by cytosolic domain rotation that simultaneously enables ion flux through a central pore and lipid scrambling along the groove periphery, while cell-based assays showed unexpected PS/PC preference over PE.","evidence":"Cryo-EM in liposomes with MD simulations and mutagenesis; live-cell fluorescence polarization scrambling assay with multiple lipid species","pmids":["41998358","41166415"],"confidence":"High","gaps":["Whether the X-shaped groove is the sole active conformation or one of multiple substates is uncertain","Molecular determinants of headgroup selectivity not identified","How lipid preference relates to physiological PS exposure vs. other lipid movements unknown"]},{"year":2026,"claim":"Whether lipid scrambling alone suffices for extracellular vesicle biogenesis without cytoskeletal or Ca²⁺ signaling changes was untested; inducible constitutively active TMEM16F demonstrated that scrambling alone drives lipid domain segregation, cholesterol redistribution, GPI-anchor clustering, PE-mediated concave curvature, and vesicle scission.","evidence":"Inducible constitutively active TMEM16F with live-cell lipid domain and EV analysis","pmids":["41604453"],"confidence":"High","gaps":["Whether this mechanism operates in physiological (non-overexpressed) contexts uncertain","Identity of scission machinery (if any) beyond lipid curvature not identified"]},{"year":null,"claim":"Key unresolved questions include the complete conformational trajectory from resting to fully active states in native membranes, the molecular basis of phospholipid headgroup selectivity, whether TMEM16F directly interacts with upstream Ca²⁺ channels or acts solely through Ca²⁺ microdomains, and how TMEM16F's scramblase activity is differentially harnessed across its many physiological contexts (hemostasis, fusion, neurodegeneration, immunity).","evidence":"","pmids":[],"confidence":"High","gaps":["Complete gating cycle at atomic resolution in native membranes not captured","Molecular basis for headgroup selectivity unidentified","Direct vs. microdomain-mediated coupling to upstream channels unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[1,2,6,10,20]},{"term_id":"GO:0140104","term_label":"molecular carrier activity","supporting_discovery_ids":[0,3,8,15,19]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[8,15,18,31,38,39]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,1,7,10,21,25]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[11]}],"pathway":[],"complexes":[],"partners":["PIEZO1","TRPV4","DNM2"],"other_free_text":[]},"mechanistic_narrative":"ANO6 (TMEM16F) is a Ca²⁺-activated plasma membrane protein that functions as both a phospholipid scramblase and a non-selective ion channel, serving as the principal mediator of Ca²⁺-dependent phosphatidylserine externalization across diverse cell types. Ca²⁺ binds directly to conserved acidic residues in the transmembrane domain, inducing conformational rearrangements—including formation of an X-shaped groove—that open physically distinct but structurally coupled pathways for bidirectional lipid translocation (~4.5×10⁴ lipids/s, preferring PS and PC) and ion permeation, with activity further modulated by PIP₂, intracellular pH, and the actin cytoskeleton [PMID:21107324, PMID:29507235, PMID:37573365, PMID:41998358, PMID:33346788]. Upstream Ca²⁺ sources including PIEZO1 and TRPV4 feed into TMEM16F activation in cell-type-specific contexts [PMID:38033286, PMID:35670667]. TMEM16F-driven PS exposure is essential for platelet procoagulant activity, trophoblast syncytialization, osteoblast mineralization, plasma membrane repair, microglial phagocytosis, ectosome/extracellular vesicle biogenesis, TCR signal modulation, and endothelial angiogenesis [PMID:23021219, PMID:32494719, PMID:22936354, PMID:31995754, PMID:27332874, PMID:41604453, PMID:33758060, PMID:38940198]. Loss-of-function mutations in ANO6 cause Scott syndrome, a bleeding disorder characterized by defective platelet phospholipid scrambling [PMID:21107324, PMID:23303820]."},"prefetch_data":{"uniprot":{"accession":"Q4KMQ2","full_name":"Anoctamin-6","aliases":["Small-conductance calcium-activated nonselective cation channel","SCAN channel","Transmembrane protein 16F"],"length_aa":910,"mass_kda":106.2,"function":"Small-conductance calcium-activated nonselective cation (SCAN) channel which acts as a regulator of phospholipid scrambling in platelets and osteoblasts (PubMed:20056604, PubMed:21107324, PubMed:21908539, PubMed:22006324, PubMed:22946059). Phospholipid scrambling results in surface exposure of phosphatidylserine which in platelets is essential to trigger the clotting system whereas in osteoblasts is essential for the deposition of hydroxyapatite during bone mineralization (By similarity). Has calcium-dependent phospholipid scramblase activity; scrambles phosphatidylserine, phosphatidylcholine and galactosylceramide (By similarity). Can generate outwardly rectifying chloride channel currents in airway epithelial cells and Jurkat T lymphocytes (By similarity) (Microbial infection) Upon SARS coronavirus-2/SARS-CoV-2 infection, is activated by spike protein which increases the amplitude of spontaneous Ca(2+) signals and is required for spike-mediated syncytia","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q4KMQ2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ANO6","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CANX","stoichiometry":0.2},{"gene":"HSP90B1","stoichiometry":0.2},{"gene":"VAMP3","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/ANO6","total_profiled":1310},"omim":[{"mim_id":"620706","title":"DEVELOPING BRAIN HOMEOBOX 2; DBX2","url":"https://www.omim.org/entry/620706"},{"mim_id":"608663","title":"ANOCTAMIN 6; ANO6","url":"https://www.omim.org/entry/608663"},{"mim_id":"608662","title":"ANOCTAMIN 5; ANO5","url":"https://www.omim.org/entry/608662"},{"mim_id":"262890","title":"SCOTT SYNDROME; SCTS","url":"https://www.omim.org/entry/262890"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Plasma membrane","reliability":"Approved"},{"location":"Cytosol","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ANO6"},"hgnc":{"alias_symbol":["DKFZp313M0720"],"prev_symbol":["TMEM16F"]},"alphafold":{"accession":"Q4KMQ2","domains":[{"cath_id":"-","chopping":"44-273","consensus_level":"medium","plddt":81.5953,"start":44,"end":273},{"cath_id":"-","chopping":"294-417_596-638_648-860","consensus_level":"medium","plddt":88.6588,"start":294,"end":860}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q4KMQ2","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q4KMQ2-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q4KMQ2-F1-predicted_aligned_error_v6.png","plddt_mean":81.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ANO6","jax_strain_url":"https://www.jax.org/strain/search?query=ANO6"},"sequence":{"accession":"Q4KMQ2","fasta_url":"https://rest.uniprot.org/uniprotkb/Q4KMQ2.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q4KMQ2/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q4KMQ2"}},"corpus_meta":[{"pmid":"21107324","id":"PMC_21107324","title":"Calcium-dependent phospholipid scrambling by TMEM16F.","date":"2010","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/21107324","citation_count":779,"is_preprint":false},{"pmid":"23021219","id":"PMC_23021219","title":"TMEM16F forms a Ca2+-activated cation channel required for lipid scrambling in platelets during blood coagulation.","date":"2012","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/23021219","citation_count":386,"is_preprint":false},{"pmid":"26417084","id":"PMC_26417084","title":"TMEM16F is required for phosphatidylserine exposure and microparticle release in activated mouse platelets.","date":"2015","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/26417084","citation_count":207,"is_preprint":false},{"pmid":"26057829","id":"PMC_26057829","title":"Identification of a lipid scrambling domain in ANO6/TMEM16F.","date":"2015","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/26057829","citation_count":155,"is_preprint":false},{"pmid":"30785399","id":"PMC_30785399","title":"Cryo-EM structures and functional characterization of the murine lipid scramblase TMEM16F.","date":"2019","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/30785399","citation_count":128,"is_preprint":false},{"pmid":"23426967","id":"PMC_23426967","title":"TMEM16F is a component of a Ca2+-activated Cl- channel but not a volume-sensitive outwardly rectifying Cl- channel.","date":"2013","source":"American journal of physiology. Cell physiology","url":"https://pubmed.ncbi.nlm.nih.gov/23426967","citation_count":111,"is_preprint":false},{"pmid":"22936354","id":"PMC_22936354","title":"Inactivation of anoctamin-6/Tmem16f, a regulator of phosphatidylserine scrambling in osteoblasts, leads to decreased mineral deposition in skeletal tissues.","date":"2013","source":"Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research","url":"https://pubmed.ncbi.nlm.nih.gov/22936354","citation_count":106,"is_preprint":false},{"pmid":"23303820","id":"PMC_23303820","title":"Both TMEM16F-dependent and TMEM16F-independent pathways contribute to phosphatidylserine exposure in platelet apoptosis and platelet activation.","date":"2013","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/23303820","citation_count":104,"is_preprint":false},{"pmid":"23630341","id":"PMC_23630341","title":"TMEM16F (Anoctamin 6), an anion channel of delayed Ca(2+) activation.","date":"2013","source":"The Journal of general physiology","url":"https://pubmed.ncbi.nlm.nih.gov/23630341","citation_count":99,"is_preprint":false},{"pmid":"23748496","id":"PMC_23748496","title":"Molecular functions of anoctamin 6 (TMEM16F): a chloride channel, cation channel, or phospholipid scramblase?","date":"2013","source":"Pflugers Archiv : European journal of physiology","url":"https://pubmed.ncbi.nlm.nih.gov/23748496","citation_count":89,"is_preprint":false},{"pmid":"32494719","id":"PMC_32494719","title":"TMEM16F phospholipid scramblase mediates trophoblast fusion and placental development.","date":"2020","source":"Science advances","url":"https://pubmed.ncbi.nlm.nih.gov/32494719","citation_count":88,"is_preprint":false},{"pmid":"31995754","id":"PMC_31995754","title":"Critical Role of Lipid Scramblase TMEM16F in Phosphatidylserine Exposure and Repair of Plasma Membrane after Pore Formation.","date":"2020","source":"Cell 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pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/23159814","citation_count":45,"is_preprint":false},{"pmid":"29463790","id":"PMC_29463790","title":"Contribution of TMEM16F to pyroptotic cell death.","date":"2018","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/29463790","citation_count":45,"is_preprint":false},{"pmid":"36335104","id":"PMC_36335104","title":"Structural basis for the activation of the lipid scramblase TMEM16F.","date":"2022","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/36335104","citation_count":45,"is_preprint":false},{"pmid":"32733436","id":"PMC_32733436","title":"TMEM16F Aggravates Neuronal Loss by Mediating Microglial Phagocytosis of Neurons in a Rat Experimental Cerebral Ischemia and Reperfusion Model.","date":"2020","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/32733436","citation_count":43,"is_preprint":false},{"pmid":"27535660","id":"PMC_27535660","title":"Ca2+ signals, cell 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A patient with Scott syndrome carries a splice-acceptor site mutation causing premature termination of TMEM16F, linking loss-of-function to defective phospholipid scrambling.\",\n      \"method\": \"Expression cloning in Ba/F3 cells, FACS-based PtdSer exposure assay, plasma membrane localization by fluorescence, patient mutation sequencing\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — original discovery with expression cloning, functional reconstitution, localization, and human genetic validation; highly cited foundational paper\",\n      \"pmids\": [\"21107324\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"TMEM16F generates a small-conductance Ca2+-activated nonselective cation (SCAN) current permeable to both monovalent and divalent cations including Ca2+, with synergistic gating by Ca2+ and voltage. TMEM16F knockout mice lack Ca2+-dependent phosphatidylserine exposure and procoagulant activity in megakaryocytes, and exhibit bleeding defects and protection from arterial thrombosis. A pore-region residue determines cation vs. anion selectivity.\",\n      \"method\": \"TMEM16F knockout mouse generation, patch-clamp electrophysiology in megakaryocytes, heterologous expression, site-directed mutagenesis of pore region\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — KO mouse with defined platelet phenotype, patch-clamp, mutagenesis; highly cited, multiple orthogonal methods\",\n      \"pmids\": [\"23021219\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"TMEM16F (ANO6) functions as a component of a Ca2+-activated Cl- channel with high Ca2+ threshold (~9.6 µM), anion selectivity sequence I->Br->Cl->F->aspartate-, and outward rectification. It is not related to volume-sensitive outwardly rectifying Cl- channel (VSOR) activity.\",\n      \"method\": \"Whole-cell patch-clamp in TMEM16F-transfected HEK293T cells, ion substitution experiments, siRNA knockdown\",\n      \"journal\": \"American Journal of Physiology - Cell Physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct electrophysiological characterization with multiple ion substitution experiments and KD controls\",\n      \"pmids\": [\"23426967\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ANO6-mediated Ca2+-dependent phospholipid scrambling can occur independently of its ion currents. Ca2+-independent phospholipid scrambling during apoptosis (intrinsic or extrinsic) does not require ANO6. ANO6 Cl- currents can be activated Ca2+-independently via Fas receptor stimulation.\",\n      \"method\": \"Patch-clamp electrophysiology, PS exposure assay (annexin V), Cl- channel blockers, siRNA knockdown, Scott syndrome patient B-lymphocytes\",\n      \"journal\": \"Cell Death & Disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including patient cells, siRNA, pharmacology, and functional assays\",\n      \"pmids\": [\"23618909\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ANO6 (TMEM16F) mediates Ca2+-dependent phospholipid scrambling in osteoblasts; deletion in mice results in impaired PS scrambling in osteoblasts and delayed mineralization, with increased areas of uncalcified osteoid postnatally. This establishes a cell-autonomous role for ANO6 in bone mineralization via PS exposure.\",\n      \"method\": \"Ano6 knockout mouse generation, primary osteoblast culture, mineralization assays, Ca2+-dependent PS scrambling assay in osteoblasts\",\n      \"journal\": \"Journal of Bone and Mineral Research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse with specific skeletal phenotype plus cell-autonomous scrambling defect in primary osteoblasts\",\n      \"pmids\": [\"22936354\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"In Scott syndrome patients, TMEM16F-dependent high-PS-exposing platelet fraction is absent upon convulxin/thrombin stimulation. TMEM16F is required for Ca2+-mobilizing agonist-induced PS exposure that depends on mitochondrial depolarization, but is not required for caspase-dependent basal PS exposure.\",\n      \"method\": \"Flow cytometry with annexin V, caspase inhibitors, Ca2+ chelation, Scott syndrome patient platelets vs. controls\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — patient-derived cells with multiple pharmacological dissections and orthogonal readouts\",\n      \"pmids\": [\"23303820\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Mouse TMEM16F expressed in HEK293 cells produces a Ca2+-activated anion channel current (EC50 ~100 µM Ca2+) with delayed activation, Eisenman type 1 anion selectivity, and outward rectification. Pore-region mutations (R592E, K616E, R636E) alter or abolish currents, identifying the pore region between TM5 and TM6 as functional.\",\n      \"method\": \"Heterologous expression in HEK293, whole-cell patch-clamp, site-directed mutagenesis of pore residues\",\n      \"journal\": \"Journal of General Physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — electrophysiology with mutagenesis, detailed biophysical characterization\",\n      \"pmids\": [\"23630341\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TMEM16F is the only TMEM16 family member highly expressed in mouse platelets. Platelet-specific TMEM16F knockout causes defects in activation-induced PS exposure, microparticle shedding, and thrombin generation in vitro and in vivo (laser-induced thrombus model), without affecting granule release or clot retraction.\",\n      \"method\": \"Platelet-specific TMEM16F conditional KO mice, flow cytometry, thrombin generation assay, laser-induced thrombosis imaging in vivo\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — platelet-specific KO with multiple in vitro and in vivo functional readouts\",\n      \"pmids\": [\"26417084\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"A specific domain in ANO6 is necessary and sufficient for phospholipid scrambling activity. By analyzing ANO1-ANO6 chimeric proteins, a scramblase domain was identified in ANO6 that, when transferred to ANO1 (which normally does not scramble), confers scramblase activity. Homology modeling shows this domain forms a hydrophilic cleft facing the lipid bilayer. Ion currents in ANO6 are explained by ionic leak during phospholipid translocation.\",\n      \"method\": \"Patch-clamp combined with PS exposure assay, ANO1-ANO6 chimera construction, homology modeling\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — domain-swap chimera experiments with both functional and structural analysis, multiple orthogonal methods\",\n      \"pmids\": [\"26057829\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TMEM16F forms homodimers (shown by chemical cross-linking). The pore region between TM5 and TM6 is essential for both scramblase activity of TMEM16F and Cl- channel activity of TMEM16A. N-terminal and C-terminal cytoplasmic domains control plasma membrane localization and protein stability, respectively, and are functionally interchangeable between TMEM16A and TMEM16F.\",\n      \"method\": \"Chemical cross-linking, domain-swap chimeras, deletion analysis, functional assays in 293T cells and TMEM16F-/- thymocytes\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — cross-linking, systematic domain deletion/swap with functional validation\",\n      \"pmids\": [\"24478309\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TMEM16F isoforms generated by alternative splicing (V1, V2, V5) show plasma membrane localization and Ca2+-activated ion channel and scramblase activities, while V3 isoform (unique C-terminus) is intracellularly localized and inactive. An activating mutation D409G markedly increases apparent Ca2+ sensitivity of both channel and scramblase activities, directly demonstrating TMEM16F mediates both functions.\",\n      \"method\": \"Whole-cell patch-clamp, annexin V binding assay for PS, subcellular localization, activating mutation introduction, siRNA knockdown\",\n      \"journal\": \"Journal of Physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple isoforms tested, gain-of-function mutation, parallel channel and scramblase readouts\",\n      \"pmids\": [\"26108457\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TMEM16F is located in late endosomes in T lymphocytes, where it facilitates generation of multivesicular bodies for TCR degradation and signal termination. TMEM16F-deficient T cells show sustained TCR signaling, augmented activation, and increased proliferation and cytokine production in chronic viral infection, ultimately leading to T cell exhaustion.\",\n      \"method\": \"Conditional TMEM16F KO mice, viral chronic infection model, late endosome localization by imaging, TCR degradation assay, T cell functional assays\",\n      \"journal\": \"Journal of Experimental Medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO with mechanistic pathway placement (late endosome localization + TCR degradation), in vivo infection model\",\n      \"pmids\": [\"27810927\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TMEM16F conditional knockout in microglia prevents mechanical hypersensitivity after nerve injury; TMEM16F-deficient microglia show deficits in process motility and phagocytosis. This establishes TMEM16F as essential for microglial responses to nerve injury in neuropathic pain.\",\n      \"method\": \"Conditional TMEM16F KO in microglia, neuropathic pain behavioral testing, microglial motility and phagocytosis assays\",\n      \"journal\": \"Cell Reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional cell-type-specific KO with defined cellular phenotype and behavioral readout\",\n      \"pmids\": [\"27332874\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Ca2+ directly binds to TMEM16F; five acidic residues conserved between TMEM16F and TMEM16K are critical for Ca2+ binding (identified by comprehensive mutagenesis of TMEM16K). Point mutations of corresponding residues in TMEM16F reduce Ca2+-dependent phospholipid scrambling. Ca2+ stabilizes TMEM16F structure and induces conformational changes.\",\n      \"method\": \"Blue-native PAGE, comprehensive acidic-residue mutagenesis, Ca2+ binding assay, scramblase activity assay\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct Ca2+ binding shown biochemically, mutagenesis identifies specific residues, functional correlation\",\n      \"pmids\": [\"27227820\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TMEM16F/ANO6 ion currents and phospholipid scrambling can be activated by modification of plasma membrane phospholipids via reactive oxygen species and phospholipase A2 (PLA2), independently of intracellular Ca2+. Mutations within TMEM16F similarly change both Cl- currents and phospholipid scrambling, suggesting Cl- and phospholipids use the same intramolecular pathway.\",\n      \"method\": \"Whole-cell patch-clamp, annexin V PS exposure assay, PLA2 activation/inhibition, ROS donors, chimera/mutant analysis\",\n      \"journal\": \"Journal of Physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple activation mechanisms tested with parallel channel/scramblase readouts, mutagenesis supports shared pathway\",\n      \"pmids\": [\"29134661\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Single purified TMEM16F dimeric molecules transport phospholipids nonspecifically and bidirectionally between membrane bilayer leaflets in a Ca2+-dependent manner at ~4.5×10^4 lipids/second at 25°C, with activation free energy of 47 kJ/mol. This biophysical profile is consistent with a channel-like facilitated diffusion ('stepping-stone') mechanism.\",\n      \"method\": \"Purification of mouse TMEM16F from stable cell line, single-molecule scramblase assay in lipid bilayer microarray, thermodynamic analysis\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — purified protein reconstitution, single-molecule analysis, rigorous thermodynamic characterization\",\n      \"pmids\": [\"29507235\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TMEM16F Ca2+-activated current is desensitized by brief high Ca2+ exposure associated with PIP2 depletion from the inner membrane leaflet. Application of PIP2 restores TMEM16F channel activity. PIP2 modulation requires positively charged amino acids in the cytoplasmic N-terminal domain and acts synergistically with membrane depolarization to facilitate Ca2+-gating.\",\n      \"method\": \"Excised patch and whole-cell patch-clamp, PIP2 application/depletion, N-terminal domain mutagenesis\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — electrophysiology with direct ligand application and mutagenesis identifying regulatory residues\",\n      \"pmids\": [\"29382763\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Cryo-EM structures of murine TMEM16F in absence and presence of Ca2+ reveal a ligand-free closed conformation and a Ca2+-bound intermediate. Both conformations resemble TMEM16A counterparts but with distinct differences in ion/lipid permeation region. Ion conduction and lipid scrambling are activated by a common Ca2+-binding mechanism but appear mediated by alternate protein conformations at equilibrium in the Ca2+-bound state.\",\n      \"method\": \"Cryo-EM structure determination, functional electrophysiology, Ca2+-dependent activity assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — high-resolution cryo-EM structures with functional validation, multiple conformational states characterized\",\n      \"pmids\": [\"30785399\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Cryo-EM structural analysis reveals coexistence of an intact channel pore and PIP2-dependent membrane distortion in TMEM16F. Mutagenesis of lipid-binding residues near membrane distortion sites specifically alters onset of lipid scrambling without affecting Ca2+ influx, providing structural evidence for separate pathways for lipid scrambling and ion permeation.\",\n      \"method\": \"Cryo-EM, structure-based mutagenesis, functional assays for Ca2+ influx and PS exposure\",\n      \"journal\": \"Cell Reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structure combined with mutagenesis functionally separating scrambling from ion permeation\",\n      \"pmids\": [\"31291589\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TMEM16F contains an inner activation gate formed by three hydrophobic residues (F518, Y563, I612) in the middle of the phospholipid permeation pathway. Lysine substitutions of F518 and Y563 generate constitutively active scramblases bypassing Ca2+-dependent activation. An analogous mutation (L543K) in TMEM16A confers CaPLSase activity to this otherwise Cl- channel.\",\n      \"method\": \"Site-directed mutagenesis, PS exposure assay, patch-clamp electrophysiology, gain-of-function and loss-of-function analysis\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis identifies specific gate residues with constitutive activation phenotype, cross-validated by gain-of-function in paralog\",\n      \"pmids\": [\"31015464\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TMEM16F ion selectivity dynamically shifts from cation-selective to anion-selective in response to increasing intracellular Ca2+, reflecting alteration of electrostatic field in the permeation pathway (charge-screening mechanism). The Q559K mutant reveals this selectivity shift is independent of channel activation state.\",\n      \"method\": \"Excised inside-out patch-clamp, ion substitution experiments with varying Ca2+ concentrations, mutagenesis\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — systematic electrophysiology with ion substitution and mutagenesis revealing dynamic selectivity mechanism\",\n      \"pmids\": [\"31318330\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TMEM16F activation by Ca2+ ionophores in Jurkat T cells triggers large-scale plasma membrane expansion coinciding with phospholipid scrambling, followed by ectosome shedding. PD-1 is selectively incorporated into ectosomes in a transmembrane-sequence-dependent manner. TMEM16F-deficient cells fail to expand surface membrane and instead undergo rapid massive endocytosis with PD-1 internalization.\",\n      \"method\": \"Live-cell microscopy, patch-clamp, flow cytometry, TMEM16F-KO Jurkat cells, PD-1 trafficking assays\",\n      \"journal\": \"Scientific Reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — combined microscopy and electrophysiology with KO cells and defined cargo-sorting readout\",\n      \"pmids\": [\"30679690\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TMEM16F is essential for plasma membrane repair after pore-forming agent injury: pore formation triggers Ca2+ influx activating TMEM16F-mediated lipid scrambling, membrane blebbing, and extracellular vesicle release that preserve membrane integrity and cell viability. TMEM16F-deficient mice show compromised control of Listeria infection due to greater neutrophil sensitivity to listeriolysin O.\",\n      \"method\": \"TMEM16F-deficient cell lines and mice, pore-forming toxin treatment, cell viability assays, EV quantification, Listeria infection model\",\n      \"journal\": \"Cell Reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO cells and mice with defined mechanistic pathway (pore→Ca2+→TMEM16F→membrane blebbing/repair) and in vivo infection readout\",\n      \"pmids\": [\"31995754\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TMEM16F knockout mice show deficiency in trophoblast syncytialization and placental development leading to perinatal lethality. TMEM16F CaPLSase translocates PS to the cell surface independently of apoptosis during trophoblast fusion.\",\n      \"method\": \"TMEM16F KO mouse generation, placental histology, trophoblast fusion assays, PS exposure measurement\",\n      \"journal\": \"Science Advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse with specific placental phenotype and cell-autonomous fusion/PS assays\",\n      \"pmids\": [\"32494719\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TMEM16F governs Ca2+-activated Cl- conductance in spinal motoneurons and is expressed in synaptic clusters facing cholinergic C-boutons. Tmem16f exon-deleted mice show decreased motor performance under high-demanding tasks and increased recruitment threshold of fast α-motoneurons. Loss of TMEM16F in ALS mouse model delays disease onset and preserves strength in male mice.\",\n      \"method\": \"Targeted exon deletion mouse, immunolocalization at C-boutons, patch-clamp in motoneurons, motor behavioral assays, ALS mouse model\",\n      \"journal\": \"Cell Reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — specific KO with defined electrophysiological phenotype in native neurons and behavioral readouts\",\n      \"pmids\": [\"32101737\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TMEM16F mediates Ca2+-dependent plasma membrane expansion via invaginations held shut by dynamin. Upon Ca2+ activation of TMEM16F, anionic phospholipids escape from the cytoplasmic monolayer and dynamins relax, opening compartments. Deletion of TMEM16F or dynamins blocks expansion; dynamin2 GTPase-inactivated mutant can regenerate reserve compartments but lipid-binding mutant cannot.\",\n      \"method\": \"TMEM16F KO cells, dynamin KO/re-expression, live-cell microscopy, dynamin2-GFP imaging, Ca2+-permeable mechanosensitive channel activation\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple genetic manipulations with live imaging and mechanistic dissection of dynamin-TMEM16F interplay\",\n      \"pmids\": [\"34404808\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TMEM16F Ca2+-dependent activation of PS scramblase during immunological synapse formation locally redistributes PS, reducing the electrostatic potential of the plasma membrane, which increases dissociation of bystander TCR-CD3 cytoplasmic domains from the membrane and enhances TCR-dependent signaling. This establishes the molecular basis for TMEM16F-mediated bystander TCR signal amplification.\",\n      \"method\": \"T cell activation assays, PS externalization measurement, membrane electrostatic potential probes, TCR-CD3 membrane association assays\",\n      \"journal\": \"Science Signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic pathway established from scramblase activity to electrostatic change to TCR signaling with multiple readouts\",\n      \"pmids\": [\"33758060\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TMEM16F channel and scramblase activities are strongly regulated by intracellular pH (pHi): low pHi attenuates and high pHi potentiates both activities. pHi sensitivity depends on [Ca2+]i and shows a bell-shaped relationship with [Ca2+]i. A Ca2+-binding residue mutation (E667Q) shifts the peak [Ca2+]i of pHi sensitivity, establishing that protons compete with Ca2+ at primary Ca2+-binding sites in the pore to regulate TMEM16F activation.\",\n      \"method\": \"Whole-cell patch-clamp, PS exposure assay at varying pHi and Ca2+ levels, Ca2+-binding residue mutagenesis (E667Q)\",\n      \"journal\": \"Journal of General Physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — systematic biophysical characterization with mutagenesis identifying the Ca2+/proton competition mechanism\",\n      \"pmids\": [\"33346788\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TMEM16F is activated by SARS-CoV-2 Spike protein binding (via ACE2) to mediate PS externalization critical for viral entry. ANO6-selective inhibitor A6-001 identified by high-throughput screening inhibits both Spike-induced PS scrambling and authentic SARS-CoV-2 replication in multiple cell types.\",\n      \"method\": \"SARS2 pseudotyped virus, Ca2+ imaging, annexin V flow cytometry, high-throughput drug screening, authentic SARS-CoV-2 infection assays in Vero/Calu-3/primary nasal cells\",\n      \"journal\": \"Cell Reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic link established between Spike-induced Ca2+, ANO6 activation, and viral entry with pharmacological validation in authentic virus\",\n      \"pmids\": [\"35839776\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Cryo-EM structures of activating TMEM16F mutants reveal major rearrangements leading to exposure of hydrophilic patches to the membrane, whose distortion facilitates lipid diffusion. Concomitant opening of a pore promotes ion conduction in the same protein conformation, revealing a mechanism distinct from other TMEM16 family members.\",\n      \"method\": \"Cryo-EM structure determination of activating mutants, functional scramblase and ion channel assays\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM of multiple activating mutants with functional validation providing mechanistic insight into gating\",\n      \"pmids\": [\"36335104\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TRPV4 Ca2+ influx channel is functionally coupled to TMEM16F within Ca2+ microdomains in human trophoblasts. TRPV4-specific agonist activates TMEM16F in trophoblasts; pharmacological inhibition or gene silencing of TRPV4 impairs TMEM16F activation and subsequent trophoblast syncytialization.\",\n      \"method\": \"Patch-clamp electrophysiology, TRPV4 agonist/antagonist treatment, gene silencing, PS exposure assay, trophoblast fusion assay\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — patch-clamp evidence for microdomain coupling, pharmacological and genetic validation of upstream activator\",\n      \"pmids\": [\"35670667\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Cryo-EM structures of TMEM16F with bound niclosamide or 1PBC reveal a lipid scrambling pathway along a groove outside the ion permeation pore containing a drug-binding pocket. Mutations in this groove specifically affect lipid scrambling but not ion conduction; some mutations preferentially reduce inhibition of PS exposure vs. Ca2+ influx, providing structural evidence for separate ion permeation and lipid scrambling pathways.\",\n      \"method\": \"Cryo-EM structure determination with bound drugs, mutagenesis, patch-clamp, PS exposure assay\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — drug-bound cryo-EM structures with mutagenesis functionally separating scrambling and ion permeation pathways\",\n      \"pmids\": [\"37573365\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TMEM16F (the RBC Ca2+-activated phospholipid scramblase) is activated downstream of Ca2+ influx through the mechanosensitive channel PIEZO1 in red blood cells. In hereditary xerocytosis (PIEZO1 gain-of-function), enhanced PIEZO1-TMEM16F coupling increases propensity for PS exposure, contributing to anemia, splenomegaly and thrombosis. Inhibition of PIEZO1 with benzbromarone prevents force-induced PS exposure.\",\n      \"method\": \"Electrophysiology, flow cytometry, imaging in RBCs from HX patients and controls; pharmacological PIEZO1 inhibition; functional hemolysis and PS assays\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic coupling established in patient RBCs with pharmacological intervention and multiple functional readouts\",\n      \"pmids\": [\"38033286\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TMEM16F deficiency in neurons (but not microglia) reduces tau pathology and microgliosis in a PS19 tauopathy mouse model. TMEM16F mediates aberrant PS exposure in neurons with phospho-tau burden, establishing a neuronal-autonomous role for TMEM16F in tau pathology.\",\n      \"method\": \"Cell-type-specific TMEM16F KO in PS19 tau mouse model, immunohistochemistry, PS exposure assay in neurons\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — cell-type-specific KO with defined tauopathy phenotype and mechanistic PS exposure readout\",\n      \"pmids\": [\"38941274\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TMEM16F deficiency in endothelial cells impairs developmental retinal angiogenesis. Biochemically, TMEM16F absence enhances plasma membrane association of activated Src kinase (Y416 phosphorylation), increases VE-cadherin phosphorylation and downregulation, suppressing angiogenesis. This establishes an intracellular signaling role for TMEM16F in endothelial cells.\",\n      \"method\": \"Endothelial-specific TMEM16F KO mice, retinal angiogenesis assay, Src kinase phosphorylation, VE-cadherin Western blot/immunofluorescence, HUVEC siRNA knockdown\",\n      \"journal\": \"Journal of Cell Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO mouse with in vivo angiogenesis phenotype plus biochemical pathway from TMEM16F→Src→VE-cadherin\",\n      \"pmids\": [\"38940198\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TMEM16F regulates pathologic α-synuclein (α-synA53T) spread in neurons. Neurons from TMEM16F KO mice show reduced donor-to-recipient spread of α-synA53T. In vivo PD mouse model shows attenuated α-synA53T spread upon TMEM16F ablation. A missense SNP (Ala703Ser) in TMEM16F with enhanced scramblase activity is associated with altered α-synA53T secretion in Ashkenazi Jewish PD patients.\",\n      \"method\": \"TMEM16F KO neurons with reporter-based spread assay, in vivo PD mouse model, lipid scramblase activity assay for Ala703Ser variant\",\n      \"journal\": \"Aging Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO neurons and in vivo model with defined spread phenotype; gain-of-function variant with functional characterization\",\n      \"pmids\": [\"39487963\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"ANO6/TMEM16F Cl- channel activation kinetics are negatively regulated by the intact actin cytoskeleton (cytochalasin-D accelerates, phalloidin/jasplakinolide inhibit activation) and positively modulated by intracellular MgATP (prevents inactivation). Inside-out patches show immediate Ca2+-dependent activation, suggesting cytosolic factors including cytoskeleton and ATP mediate the slow whole-cell activation.\",\n      \"method\": \"Whole-cell and inside-out patch-clamp, cytoskeleton-disrupting/stabilizing agents, intracellular ATP manipulation\",\n      \"journal\": \"Biochemical and Biophysical Research Communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — electrophysiology with pharmacological manipulation of actin and ATP, single lab\",\n      \"pmids\": [\"29964013\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ANO6-dependent trogocytosis in cancer-associated fibroblasts (CAFs) is triggered by cytosolic Ca2+ influx via Orai channels, which activates ANO6 causing PS exposure on CAF plasma membranes, initiating membrane lipid transfer (including cholesterol) to pancreatic cancer cells. ANO6-dependent trogocytosis also supports immunosuppressive function of CAFs toward cytotoxic T cells.\",\n      \"method\": \"CRISPR KO of ANO6, Ca2+ imaging, annexin V/PS exposure assay, cholesterol transfer assay, T cell cytotoxicity assay\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple functional assays with genetic KO, but preprint not yet peer-reviewed\",\n      \"pmids\": [\"37745612\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"High-resolution cryo-EM of TMEM16F active in liposomes reveals two conformations in high-activity conditions: the canonical Ca2+-bound closed state and a novel X-shaped groove conformation where upward rotation of the cytosolic domain forms a transmembrane pore and locally thins the membrane. Mutagenesis, functional assays, and MD simulations show the X-shaped groove mediates nonselective ion flux and lipid scrambling through distinct pathways (ions within the pore, lipids skirting the groove).\",\n      \"method\": \"Cryo-EM in liposomes, site-directed mutagenesis, functional ion flux and scramblase assays, molecular dynamics simulations\",\n      \"journal\": \"Nature Structural & Molecular Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM of active protein in native-like lipid environment combined with mutagenesis and MD simulations; rigorous multi-method study\",\n      \"pmids\": [\"41998358\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TMEM16F preferentially scrambles phosphatidylserine and phosphatidylcholine over phosphatidylethanolamine on the plasma membrane of living cells, contrary to the prevailing view that scramblases act without headgroup preference.\",\n      \"method\": \"Cell-based fluorescence polarization scrambling assay with NBD-labeled phospholipids, kinetic monitoring on live cell plasma membranes\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — novel cell-based assay with multiple phospholipid species tested; single lab, single method but rigorous experimental design\",\n      \"pmids\": [\"41166415\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TMEM16F activation contributes to ferroptotic cell death: ferroptosis inducers (erastin, RSL3) activate TMEM16F currents in intestinal epithelium and macrophages, and cell death is largely reduced in tissue-specific TMEM16F KO mice. Inhibitors of ferroptosis (ferrostatin-1) block TMEM16F currents.\",\n      \"method\": \"Tissue-specific TMEM16F KO mice, ferroptosis induction with erastin/RSL3, cell death assays, patch-clamp recording\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — tissue-specific KO with defined ferroptosis phenotype and electrophysiological correlation\",\n      \"pmids\": [\"31060306\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TMEM16F contributes to pyroptotic cell death downstream of gasdermin-D pore formation. GD-N expression induces Ca2+ elevation activating TMEM16F, which generates large whole-cell currents; knockdown or inhibition of TMEM16F suppresses these currents and reduces cell death in HEK293 and HAP1 cells.\",\n      \"method\": \"GD-N expression, TMEM16F knockdown/inhibition, whole-cell patch-clamp, cell viability assays\",\n      \"journal\": \"Cell Death & Disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KD with electrophysiological and viability readouts, single lab\",\n      \"pmids\": [\"29463790\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"N-terminal domain of ANO6 contains a putative Ca2+-transferring reservoir (Nt-CaRes) that regulates Ca2+ sensitivity. Chimera ANO6-1-6 (with ANO1 Nt-CaRes substituted) shows higher Ca2+ sensitivity than ANO6. Mutagenesis of acidic amino acids in Nt-CaRes reduces Ca2+ sensitivity, consistent with direct Ca2+ interactions at these residues.\",\n      \"method\": \"Chimera construction, site-directed mutagenesis of acidic residues, patch-clamp Ca2+ dose-response, molecular dynamics simulation\",\n      \"journal\": \"Molecules and Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — chimera and mutagenesis with functional Ca2+ sensitivity readout, but single lab\",\n      \"pmids\": [\"33658434\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Atomic force microscopy under physiological conditions reveals structurally and mechanically diverse TMEM16F assemblies with variable inter-subunit dimerization interfaces. Ca2+-induced activation is associated with stepwise changes in the pore region affecting mechanical properties of TM3, TM4, and TM6. Direct observation of membrane remodeling links structural heterogeneity to ion and lipid permeation.\",\n      \"method\": \"Atomic force microscopy in physiological conditions, cryo-EM comparison, patch-clamp electrophysiology\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — novel structural technique (physiological-condition AFM) combined with electrophysiology revealing dynamic conformational states\",\n      \"pmids\": [\"38167485\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Plasmalemmal lipid scrambling by TMEM16F (using inducible active form) is sufficient to release apoptotic-like vesicles without changes in cytosolic Ca2+ or submembrane cytoskeleton. Scrambling causes segregation of exofacial lipids, redistribution of cholesterol to inner leaflet, GPI-anchored protein clustering forming convex curvature, and PE accumulation forming concave curvature facilitating vesicle scission.\",\n      \"method\": \"Inducible constitutively active TMEM16F, live-cell imaging, lipid domain analysis, extracellular vesicle quantification\",\n      \"journal\": \"Molecular Biology of the Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstitution using controlled TMEM16F activation with detailed lipid and vesicle biogenesis analysis\",\n      \"pmids\": [\"41604453\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ANO6/TMEM16F is a Ca2+-activated dual-function plasma membrane protein that operates as a phospholipid scramblase and a largely non-selective ion channel: upon elevation of intracellular Ca2+ (binding directly to conserved acidic residues), TMEM16F undergoes conformational changes—including cytosolic domain rotation to form an X-shaped groove—that open both a transmembrane ion pore and a lipid permeation pathway (the two routes being physically distinct but sharing part of the same groove), catalyzing bidirectional, facilitated phosphatidylserine/phosphatidylcholine-preferential lipid flip-flop at ~4.5×10⁴ lipids/second while concurrently conducting ions; its activity is regulated by PIP2 (via N-terminal cationic residues), intracellular pH, actin cytoskeleton, and upstream Ca2+ channels (PIEZO1, TRPV4); physiologically, TMEM16F-driven PS externalization is essential for platelet procoagulant activity, trophoblast fusion, osteoblast mineralization, plasma membrane repair, T cell receptor signal termination via late-endosomal multivesicular body formation, microglial phagocytosis, and ectosome/extracellular vesicle biogenesis.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"ANO6 (TMEM16F) is a Ca²⁺-activated plasma membrane protein that functions as both a phospholipid scramblase and a non-selective ion channel, serving as the principal mediator of Ca²⁺-dependent phosphatidylserine externalization across diverse cell types. Ca²⁺ binds directly to conserved acidic residues in the transmembrane domain, inducing conformational rearrangements—including formation of an X-shaped groove—that open physically distinct but structurally coupled pathways for bidirectional lipid translocation (~4.5×10⁴ lipids/s, preferring PS and PC) and ion permeation, with activity further modulated by PIP₂, intracellular pH, and the actin cytoskeleton [PMID:21107324, PMID:29507235, PMID:37573365, PMID:41998358, PMID:33346788]. Upstream Ca²⁺ sources including PIEZO1 and TRPV4 feed into TMEM16F activation in cell-type-specific contexts [PMID:38033286, PMID:35670667]. TMEM16F-driven PS exposure is essential for platelet procoagulant activity, trophoblast syncytialization, osteoblast mineralization, plasma membrane repair, microglial phagocytosis, ectosome/extracellular vesicle biogenesis, TCR signal modulation, and endothelial angiogenesis [PMID:23021219, PMID:32494719, PMID:22936354, PMID:31995754, PMID:27332874, PMID:41604453, PMID:33758060, PMID:38940198]. Loss-of-function mutations in ANO6 cause Scott syndrome, a bleeding disorder characterized by defective platelet phospholipid scrambling [PMID:21107324, PMID:23303820].\",\n  \"teleology\": [\n    {\n      \"year\": 2010,\n      \"claim\": \"The identity of the long-sought Ca²⁺-dependent phospholipid scramblase was unknown; expression cloning identified TMEM16F as the essential component, and a Scott syndrome patient mutation provided human genetic validation, establishing TMEM16F as the Ca²⁺-dependent scramblase.\",\n      \"evidence\": \"Expression cloning in Ba/F3 cells with FACS-based PS assay, patient mutation sequencing\",\n      \"pmids\": [\"21107324\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of scrambling (channel vs. transporter) unresolved\", \"Whether TMEM16F also conducts ions was unknown\", \"Structure of the protein undetermined\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Whether TMEM16F conducted ions and what its in vivo physiological role was remained open; electrophysiology revealed TMEM16F generates Ca²⁺-activated nonselective cation currents, and knockout mice demonstrated essential roles in platelet procoagulant activity and hemostasis.\",\n      \"evidence\": \"TMEM16F KO mouse, patch-clamp in megakaryocytes, pore-region mutagenesis\",\n      \"pmids\": [\"23021219\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ion conduction and scrambling are mechanistically separable was unresolved\", \"Structural basis of ion selectivity unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"The relationship between TMEM16F's channel and scramblase activities, ion selectivity properties, and physiological roles beyond platelets needed clarification; multiple studies established that scrambling can occur independently of ion currents, identified the functional pore region (TM5-TM6), demonstrated roles in osteoblast mineralization, and dissected pathway-specific PS exposure in Scott syndrome platelets.\",\n      \"evidence\": \"Patch-clamp with pharmacological dissection and Scott patient cells; pore-region mutagenesis in HEK293; Ano6 KO mouse osteoblast mineralization assays; flow cytometry of Scott patient platelets\",\n      \"pmids\": [\"23618909\", \"23630341\", \"22936354\", \"23303820\", \"23426967\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for dual function undetermined\", \"How Ca²⁺ binds and gates the protein unknown\", \"Molecular determinants distinguishing scramblase from channel activity not identified\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"What structural domain confers scramblase activity, and whether TMEM16F functions as a monomer or oligomer, was unknown; chimera studies identified a specific scramblase domain transferable to the channel-only paralog TMEM16A, cross-linking revealed homodimers, and isoform analysis with gain-of-function mutations confirmed both activities reside in one protein.\",\n      \"evidence\": \"ANO1-ANO6 chimeras with PS and electrophysiology readouts; chemical cross-linking; splice-variant characterization; D409G activating mutation; platelet-specific conditional KO\",\n      \"pmids\": [\"26057829\", \"24478309\", \"26108457\", \"26417084\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic structure not yet available\", \"Ca²⁺-binding site residues in TMEM16F not directly identified\", \"Whether ion and lipid pathways are physically the same groove remained debated\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"TMEM16F's roles beyond hemostasis and bone were largely unexplored; conditional KO studies revealed TMEM16F in late endosomes controls TCR degradation and T cell signal termination, and in microglia it is essential for phagocytosis and neuropathic pain responses, while biochemical studies identified specific Ca²⁺-binding residues.\",\n      \"evidence\": \"Conditional KO mice in T cells and microglia; late-endosome localization; TCR degradation and viral infection models; blue-native PAGE and comprehensive mutagenesis of Ca²⁺-coordinating acidic residues\",\n      \"pmids\": [\"27810927\", \"27332874\", \"27227820\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural mechanism of Ca²⁺-dependent gating unknown\", \"How TMEM16F reaches late endosomes was unexplained\", \"Whether late-endosomal and plasma membrane functions are mechanistically identical was unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Regulatory mechanisms controlling TMEM16F activity in situ were poorly understood; studies identified PIP₂ as a positive modulator acting via N-terminal cationic residues, the actin cytoskeleton as a negative kinetic regulator, and purified single-molecule reconstitution quantified scrambling rates consistent with a facilitated-diffusion mechanism.\",\n      \"evidence\": \"Excised patch with PIP₂ application and N-terminal mutagenesis; cytoskeleton-modulating agents with patch-clamp; purified TMEM16F dimer reconstituted in lipid bilayer microarray for single-molecule scrambling\",\n      \"pmids\": [\"29382763\", \"29964013\", \"29507235\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of PIP₂ regulation not visualized\", \"How actin physically interacts with TMEM16F unknown\", \"Contributions of additional regulators (e.g., pH) not yet explored\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"The structural basis for TMEM16F's dual activity and whether ion and lipid pathways are the same remained debated; cryo-EM structures revealed Ca²⁺-bound and Ca²⁺-free conformations, mutagenesis identified a hydrophobic inner activation gate (F518/Y563/I612), dynamic ion selectivity shifting with Ca²⁺ was demonstrated, and roles in membrane expansion/ectosome shedding and ferroptosis were established.\",\n      \"evidence\": \"Cryo-EM of apo and Ca²⁺-bound TMEM16F; constitutively active gate mutants; ion substitution with varying Ca²⁺; live-cell microscopy with KO Jurkat cells; tissue-specific KO with ferroptosis inducers\",\n      \"pmids\": [\"30785399\", \"31291589\", \"31015464\", \"31318330\", \"30679690\", \"31060306\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cryo-EM captured only intermediate states, not the fully open active conformation\", \"Whether the gate mutant phenotype reflects the physiological open state was uncertain\", \"Mechanism of membrane expansion coupling to scrambling was unclear\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Whether TMEM16F contributes to membrane repair and trophoblast fusion in vivo was untested; KO studies showed TMEM16F-mediated blebbing and EV release are essential for plasma membrane repair after pore-forming toxin injury, and TMEM16F drives PS-dependent trophoblast syncytialization required for placental development.\",\n      \"evidence\": \"TMEM16F-deficient cells/mice with pore-forming toxin and Listeria infection; TMEM16F KO mouse placental histology and trophoblast fusion assays\",\n      \"pmids\": [\"31995754\", \"32494719\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether TMEM16F directly interacts with fusion machinery was unknown\", \"Role of specific upstream Ca²⁺ channels in trophoblast context not identified\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"How TMEM16F scrambling translates into membrane expansion and TCR signaling modulation was mechanistically unclear; studies revealed TMEM16F lipid scrambling relaxes dynamin-held membrane invaginations to expand the cell surface, and at the immunological synapse PS redistribution reduces electrostatic potential to release bystander TCR-CD3 tails, amplifying signaling.\",\n      \"evidence\": \"TMEM16F/dynamin double-KO with live imaging; T cell activation assays with membrane electrostatic probes and TCR-CD3 association measurements\",\n      \"pmids\": [\"34404808\", \"33758060\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether dynamin directly binds TMEM16F or acts indirectly via lipid sensing was unresolved\", \"Quantitative contribution of electrostatic mechanism vs. other signaling effects unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"The structural basis for physically separating ion and lipid permeation paths, upstream Ca²⁺ sources in trophoblasts, and viral exploitation of TMEM16F were open questions; cryo-EM of activating mutants revealed hydrophilic patch exposure and concomitant pore opening, TRPV4 was identified as the upstream Ca²⁺ channel coupling to TMEM16F in trophoblasts, PIEZO1 was shown to couple to TMEM16F in RBCs, and Spike-mediated SARS-CoV-2 entry was found to require TMEM16F-dependent PS scrambling.\",\n      \"evidence\": \"Cryo-EM of activating mutants; TRPV4 agonist/antagonist with trophoblast patch-clamp; PIEZO1-TMEM16F coupling in hereditary xerocytosis RBCs; SARS-CoV-2 pseudovirus and authentic infection with ANO6 inhibitor\",\n      \"pmids\": [\"36335104\", \"35670667\", \"38033286\", \"35839776\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full open-state structure in lipid environment not yet captured\", \"Whether PIEZO1-TMEM16F coupling involves direct interaction or only Ca²⁺ microdomain proximity was unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Whether ion and lipid pathways are truly physically distinct at atomic resolution was debated; drug-bound cryo-EM structures revealed a lipid scrambling groove outside the ion pore with a druggable pocket, and mutagenesis selectively affected scrambling without impairing ion conduction, providing the clearest structural evidence for separate pathways.\",\n      \"evidence\": \"Cryo-EM with bound niclosamide/1PBC, structure-guided mutagenesis with parallel scramblase and channel assays\",\n      \"pmids\": [\"37573365\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the drug-binding pocket represents a physiological regulatory site unknown\", \"Resolution of lipid headgroup interactions within the groove not achieved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"TMEM16F's roles in neurodegeneration and vascular biology were emerging; neuron-specific KO reduced tau pathology and α-synuclein spread, endothelial KO impaired retinal angiogenesis via Src/VE-cadherin signaling, and AFM revealed structurally diverse TMEM16F assemblies with Ca²⁺-induced stepwise mechanical changes.\",\n      \"evidence\": \"Cell-type-specific KO in PS19 tauopathy and PD models; endothelial KO retinal angiogenesis with Src phosphorylation analysis; AFM under physiological conditions\",\n      \"pmids\": [\"38941274\", \"39487963\", \"38940198\", \"38167485\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PS exposure directly mediates tau/α-syn pathology or acts indirectly through phagocytic signals unclear\", \"Direct physical basis for Src membrane retention change not structurally resolved\", \"Physiological significance of TMEM16F structural heterogeneity unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"The fully active conformation of TMEM16F in a lipid environment had never been captured; cryo-EM in liposomes revealed an X-shaped groove formed by cytosolic domain rotation that simultaneously enables ion flux through a central pore and lipid scrambling along the groove periphery, while cell-based assays showed unexpected PS/PC preference over PE.\",\n      \"evidence\": \"Cryo-EM in liposomes with MD simulations and mutagenesis; live-cell fluorescence polarization scrambling assay with multiple lipid species\",\n      \"pmids\": [\"41998358\", \"41166415\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the X-shaped groove is the sole active conformation or one of multiple substates is uncertain\", \"Molecular determinants of headgroup selectivity not identified\", \"How lipid preference relates to physiological PS exposure vs. other lipid movements unknown\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Whether lipid scrambling alone suffices for extracellular vesicle biogenesis without cytoskeletal or Ca²⁺ signaling changes was untested; inducible constitutively active TMEM16F demonstrated that scrambling alone drives lipid domain segregation, cholesterol redistribution, GPI-anchor clustering, PE-mediated concave curvature, and vesicle scission.\",\n      \"evidence\": \"Inducible constitutively active TMEM16F with live-cell lipid domain and EV analysis\",\n      \"pmids\": [\"41604453\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this mechanism operates in physiological (non-overexpressed) contexts uncertain\", \"Identity of scission machinery (if any) beyond lipid curvature not identified\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the complete conformational trajectory from resting to fully active states in native membranes, the molecular basis of phospholipid headgroup selectivity, whether TMEM16F directly interacts with upstream Ca²⁺ channels or acts solely through Ca²⁺ microdomains, and how TMEM16F's scramblase activity is differentially harnessed across its many physiological contexts (hemostasis, fusion, neurodegeneration, immunity).\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Complete gating cycle at atomic resolution in native membranes not captured\", \"Molecular basis for headgroup selectivity unidentified\", \"Direct vs. microdomain-mediated coupling to upstream channels unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [1, 2, 6, 10, 20]},\n      {\"term_id\": \"GO:0140104\", \"supporting_discovery_ids\": [0, 3, 8, 15, 19]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [8, 15, 18, 31, 38, 39]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 1, 7, 10, 21, 25]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0109582\", \"supporting_discovery_ids\": [1, 5, 7, 32]},\n      {\"term_id\": \"GO:0162582\", \"supporting_discovery_ids\": [11, 26, 34]},\n      {\"term_id\": \"GO:0168256\", \"supporting_discovery_ids\": [11, 12, 26, 28]},\n      {\"term_id\": \"GO:05357801\", \"supporting_discovery_ids\": [40, 41]},\n      {\"term_id\": \"GO:01266738\", \"supporting_discovery_ids\": [23, 34]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"PIEZO1\",\n      \"TRPV4\",\n      \"DNM2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\nWait, I need to fix the pathway term_ids - they should be Reactome IDs, not GO IDs. Let me correct:\n\n```json\n{\n  \"mechanistic_narrative\": \"ANO6 (TMEM16F) is a Ca²⁺-activated plasma membrane protein that functions as both a phospholipid scramblase and a non-selective ion channel, serving as the principal mediator of Ca²⁺-dependent phosphatidylserine externalization across diverse cell types. Ca²⁺ binds directly to conserved acidic residues in the transmembrane domain, inducing conformational rearrangements—including formation of an X-shaped groove—that open physically distinct but structurally coupled pathways for bidirectional lipid translocation (~4.5×10⁴ lipids/s, preferring PS and PC) and ion permeation, with activity further modulated by PIP₂ via N-terminal cationic residues, intracellular pH competing with Ca²⁺ at binding sites, and the actin cytoskeleton [PMID:21107324, PMID:29507235, PMID:37573365, PMID:41998358, PMID:29382763, PMID:33346788]. Upstream Ca²⁺ sources including PIEZO1 and TRPV4 couple to TMEM16F in cell-type-specific contexts to drive PS exposure essential for platelet procoagulant activity, trophoblast syncytialization, osteoblast mineralization, plasma membrane repair, microglial phagocytosis, TCR signal modulation, endothelial angiogenesis, and extracellular vesicle biogenesis [PMID:23021219, PMID:32494719, PMID:22936354, PMID:31995754, PMID:27332874, PMID:33758060, PMID:38940198, PMID:41604453, PMID:38033286, PMID:35670667]. Loss-of-function mutations in ANO6 cause Scott syndrome, a bleeding disorder characterized by defective platelet phospholipid scrambling [PMID:21107324, PMID:23303820].\",\n  \"teleology\": [\n    {\n      \"year\": 2010,\n      \"claim\": \"The identity of the long-sought Ca²⁺-dependent phospholipid scramblase was unknown; expression cloning identified TMEM16F as the essential component, and a Scott syndrome patient mutation provided human genetic validation, establishing TMEM16F as the Ca²⁺-dependent scramblase.\",\n      \"evidence\": \"Expression cloning in Ba/F3 cells with FACS-based PS assay, patient mutation sequencing\",\n      \"pmids\": [\"21107324\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of scrambling (channel vs. transporter) unresolved\", \"Whether TMEM16F also conducts ions was unknown\", \"Structure of the protein undetermined\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Whether TMEM16F conducted ions and what its in vivo physiological role was remained open; electrophysiology revealed TMEM16F generates Ca²⁺-activated nonselective cation currents, and knockout mice demonstrated essential roles in platelet procoagulant activity and hemostasis.\",\n      \"evidence\": \"TMEM16F KO mouse, patch-clamp in megakaryocytes, pore-region mutagenesis\",\n      \"pmids\": [\"23021219\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ion conduction and scrambling are mechanistically separable was unresolved\", \"Structural basis of ion selectivity unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"The relationship between TMEM16F's channel and scramblase activities, ion selectivity properties, and physiological roles beyond platelets needed clarification; multiple studies established that scrambling can occur independently of ion currents, identified the functional pore region (TM5-TM6), demonstrated roles in osteoblast mineralization, and dissected pathway-specific PS exposure in Scott syndrome platelets.\",\n      \"evidence\": \"Patch-clamp with pharmacological dissection and Scott patient cells; pore-region mutagenesis in HEK293; Ano6 KO mouse osteoblast mineralization assays; flow cytometry of Scott patient platelets\",\n      \"pmids\": [\"23618909\", \"23630341\", \"22936354\", \"23303820\", \"23426967\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for dual function undetermined\", \"How Ca²⁺ binds and gates the protein unknown\", \"Molecular determinants distinguishing scramblase from channel activity not identified\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"What structural domain confers scramblase activity, and whether TMEM16F functions as a monomer or oligomer, was unknown; chimera studies identified a specific scramblase domain transferable to the channel-only paralog TMEM16A, cross-linking revealed homodimers, and isoform analysis with gain-of-function mutations confirmed both activities reside in one protein.\",\n      \"evidence\": \"ANO1-ANO6 chimeras with PS and electrophysiology readouts; chemical cross-linking; splice-variant characterization; D409G activating mutation; platelet-specific conditional KO\",\n      \"pmids\": [\"26057829\", \"24478309\", \"26108457\", \"26417084\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic structure not yet available\", \"Ca²⁺-binding site residues in TMEM16F not directly identified\", \"Whether ion and lipid pathways are physically the same groove remained debated\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"TMEM16F's roles beyond hemostasis and bone were largely unexplored; conditional KO studies revealed TMEM16F in late endosomes controls TCR degradation and T cell signal termination, and in microglia it is essential for phagocytosis and neuropathic pain responses, while biochemical studies identified specific Ca²⁺-binding residues.\",\n      \"evidence\": \"Conditional KO mice in T cells and microglia; late-endosome localization; TCR degradation and viral infection models; blue-native PAGE and comprehensive mutagenesis of Ca²⁺-coordinating acidic residues\",\n      \"pmids\": [\"27810927\", \"27332874\", \"27227820\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural mechanism of Ca²⁺-dependent gating unknown\", \"How TMEM16F reaches late endosomes was unexplained\", \"Whether late-endosomal and plasma membrane functions are mechanistically identical was unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Regulatory mechanisms controlling TMEM16F activity in situ were poorly understood; studies identified PIP₂ as a positive modulator acting via N-terminal cationic residues, the actin cytoskeleton as a negative kinetic regulator, and purified single-molecule reconstitution quantified scrambling rates consistent with a facilitated-diffusion mechanism.\",\n      \"evidence\": \"Excised patch with PIP₂ application and N-terminal mutagenesis; cytoskeleton-modulating agents with patch-clamp; purified TMEM16F dimer reconstituted in lipid bilayer microarray for single-molecule scrambling\",\n      \"pmids\": [\"29382763\", \"29964013\", \"29507235\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of PIP₂ regulation not visualized\", \"How actin physically interacts with TMEM16F unknown\", \"Contributions of additional regulators (e.g., pH) not yet explored\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"The structural basis for TMEM16F's dual activity and whether ion and lipid pathways are the same remained debated; cryo-EM structures revealed Ca²⁺-bound and Ca²⁺-free conformations, mutagenesis identified a hydrophobic inner activation gate (F518/Y563/I612), dynamic ion selectivity shifting with Ca²⁺ was demonstrated, and roles in membrane expansion/ectosome shedding and ferroptosis were established.\",\n      \"evidence\": \"Cryo-EM of apo and Ca²⁺-bound TMEM16F; constitutively active gate mutants; ion substitution with varying Ca²⁺; live-cell microscopy with KO Jurkat cells; tissue-specific KO with ferroptosis inducers\",\n      \"pmids\": [\"30785399\", \"31291589\", \"31015464\", \"31318330\", \"30679690\", \"31060306\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cryo-EM captured only intermediate states, not the fully open active conformation\", \"Whether the gate mutant phenotype reflects the physiological open state was uncertain\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Whether TMEM16F contributes to membrane repair and trophoblast fusion in vivo was untested; KO studies showed TMEM16F-mediated blebbing and EV release are essential for plasma membrane repair after pore-forming toxin injury, and TMEM16F drives PS-dependent trophoblast syncytialization required for placental development.\",\n      \"evidence\": \"TMEM16F-deficient cells/mice with pore-forming toxin and Listeria infection; TMEM16F KO mouse placental histology and trophoblast fusion assays\",\n      \"pmids\": [\"31995754\", \"32494719\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether TMEM16F directly interacts with fusion machinery was unknown\", \"Role of specific upstream Ca²⁺ channels in trophoblast context not identified\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"How TMEM16F scrambling translates into membrane expansion and TCR signaling modulation was mechanistically unclear; studies revealed TMEM16F lipid scrambling relaxes dynamin-held membrane invaginations to expand the cell surface, and at the immunological synapse PS redistribution reduces electrostatic potential to release bystander TCR-CD3 tails, amplifying signaling.\",\n      \"evidence\": \"TMEM16F/dynamin double-KO with live imaging; T cell activation assays with membrane electrostatic probes and TCR-CD3 association measurements\",\n      \"pmids\": [\"34404808\", \"33758060\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether dynamin directly binds TMEM16F or acts indirectly via lipid sensing was unresolved\", \"Quantitative contribution of electrostatic mechanism vs. other signaling effects unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Upstream Ca²⁺ sources, structural basis for pathway separation, and viral exploitation of TMEM16F were open questions; cryo-EM of activating mutants revealed hydrophilic patch exposure and concomitant pore opening, TRPV4 was identified as the upstream Ca²⁺ channel coupling to TMEM16F in trophoblasts, PIEZO1 was shown to couple to TMEM16F in RBCs contributing to hereditary xerocytosis pathology, and SARS-CoV-2 Spike-mediated entry was found to require TMEM16F-dependent PS scrambling.\",\n      \"evidence\": \"Cryo-EM of activating mutants; TRPV4 agonist/antagonist with trophoblast patch-clamp; PIEZO1-TMEM16F coupling in hereditary xerocytosis RBCs; SARS-CoV-2 pseudovirus and authentic infection with ANO6 inhibitor\",\n      \"pmids\": [\"36335104\", \"35670667\", \"38033286\", \"35839776\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full open-state structure in lipid environment not yet captured\", \"Whether PIEZO1-TMEM16F coupling involves direct interaction or only Ca²⁺ microdomain proximity was unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Whether ion and lipid pathways are truly physically distinct at atomic resolution was debated; drug-bound cryo-EM structures revealed a lipid scrambling groove outside the ion pore with a druggable pocket, and mutagenesis selectively affected scrambling without impairing ion conduction, providing the clearest structural evidence for separate pathways.\",\n      \"evidence\": \"Cryo-EM with bound niclosamide/1PBC, structure-guided mutagenesis with parallel scramblase and channel assays\",\n      \"pmids\": [\"37573365\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the drug-binding pocket represents a physiological regulatory site unknown\", \"Resolution of lipid headgroup interactions within the groove not achieved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"TMEM16F's roles in neurodegeneration and vascular biology were emerging; neuron-specific KO reduced tau pathology and α-synuclein spread in disease models, endothelial KO impaired retinal angiogenesis via Src/VE-cadherin signaling, and AFM revealed structurally diverse TMEM16F assemblies with Ca²⁺-induced stepwise mechanical changes.\",\n      \"evidence\": \"Cell-type-specific KO in PS19 tauopathy and PD mouse models; endothelial KO retinal angiogenesis with Src phosphorylation analysis; AFM under physiological conditions with cryo-EM comparison\",\n      \"pmids\": [\"38941274\", \"39487963\", \"38940198\", \"38167485\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PS exposure directly mediates tau/α-syn pathology or acts indirectly through phagocytic signals unclear\", \"Direct physical basis for Src membrane retention change not structurally resolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"The fully active conformation of TMEM16F in a lipid environment had never been captured; cryo-EM in liposomes revealed an X-shaped groove formed by cytosolic domain rotation that simultaneously enables ion flux through a central pore and lipid scrambling along the groove periphery, while cell-based assays showed unexpected PS/PC preference over PE.\",\n      \"evidence\": \"Cryo-EM in liposomes with MD simulations and mutagenesis; live-cell fluorescence polarization scrambling assay with multiple lipid species\",\n      \"pmids\": [\"41998358\", \"41166415\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the X-shaped groove is the sole active conformation or one of multiple substates is uncertain\", \"Molecular determinants of headgroup selectivity not identified\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Whether lipid scrambling alone suffices for extracellular vesicle biogenesis without cytoskeletal or Ca²⁺ signaling changes was untested; inducible constitutively active TMEM16F demonstrated that scrambling alone drives lipid domain segregation, cholesterol redistribution, GPI-anchor clustering, and PE-mediated curvature leading to vesicle scission.\",\n      \"evidence\": \"Inducible constitutively active TMEM16F with live-cell lipid domain and EV analysis\",\n      \"pmids\": [\"41604453\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this mechanism operates at physiological expression levels uncertain\", \"Identity of scission machinery beyond lipid curvature not identified\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the complete conformational trajectory from resting to fully active states in native membranes, the molecular basis of phospholipid headgroup selectivity, whether TMEM16F directly interacts with upstream Ca²⁺ channels or acts solely through Ca²⁺ microdomains, and how TMEM16F's scramblase activity is differentially harnessed across its many physiological contexts.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Complete gating cycle at atomic resolution in native membranes not captured\", \"Molecular basis for headgroup selectivity unidentified\", \"Direct vs. microdomain-mediated coupling to upstream channels unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [1, 2, 6, 10, 20]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [0, 8, 15, 19, 31, 38, 39]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 1, 7, 10, 21, 25]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-109582\", \"supporting_discovery_ids\": [1, 5, 7, 32]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [11, 26, 34]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [11, 12, 26, 28]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [40, 41]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [23, 34]},\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [0, 8, 15, 39]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [21, 22, 44]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"PIEZO1\",\n      \"TRPV4\",\n      \"DNM2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}