{"gene":"ANO6","run_date":"2026-06-09T22:02:43","timeline":{"discoveries":[{"year":2010,"finding":"TMEM16F (ANO6) is an essential component for Ca2+-dependent phosphatidylserine exposure on the cell surface. Wild-type TMEM16F localizes to the plasma membrane and confers Ca2+-dependent bidirectional phospholipid scrambling. A patient with Scott syndrome carries a splice-acceptor site mutation in TMEM16F causing premature protein termination, linking loss of TMEM16F to defective phospholipid scrambling.","method":"Expression cloning in Ba/F3 cells, FACS-based PtdSer exposure assay, plasma membrane localization by imaging, patient mutation sequencing","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — expression cloning with functional rescue, localization experiment, patient genetic validation; foundational paper replicated by multiple independent labs","pmids":["21107324"],"is_preprint":false},{"year":2012,"finding":"TMEM16F forms a Ca2+-activated nonselective cation (SCAN) channel with subpicosiemens single-channel conductance that permeates both monovalent and divalent cations including Ca2+, and exhibits synergistic gating by Ca2+ and voltage. TMEM16F knockout mice lack this Ca2+-activated cation current in megakaryocytes and exhibit deficient platelet Ca2+-dependent phosphatidylserine exposure and procoagulant activity, with a bleeding defect and protection in arterial thrombosis. A residue in the putative pore region was identified as critical for cation vs. anion selectivity.","method":"TMEM16F knockout mouse generation, whole-cell and single-channel patch clamp in megakaryocytes and heterologous expression, site-directed mutagenesis of pore region, platelet PS exposure assay (annexin V), arterial thrombosis model","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — KO mouse with defined cellular phenotype, electrophysiology with mutagenesis, multiple orthogonal assays, replicated by subsequent studies","pmids":["23021219"],"is_preprint":false},{"year":2013,"finding":"Platelet-specific TMEM16F-deficient mice exhibit defects in activation-induced PtdSer exposure and microparticle shedding upon thrombin/collagen or Ca2+ ionophore activation, severely reduced thrombin generation, and impaired in vivo PtdSer exposure on aggregated platelets, while alpha-granule/dense granule release and clot retraction remain intact. TMEM16F is the only highly expressed TMEM16 scramblase in mouse platelets.","method":"Platelet-specific conditional TMEM16F knockout mice, flow cytometry (annexin V), microparticle quantification, thrombin generation assay, laser-induced thrombosis intravital imaging","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO with multiple specific phenotypic readouts, in vivo validation, replicated Scott syndrome phenotype","pmids":["26417084"],"is_preprint":false},{"year":2015,"finding":"A specific lipid scrambling domain in ANO6 was identified by analysis of ANO1-ANO6 chimeric proteins. This domain is necessary for phospholipid scrambling activity in ANO6 and sufficient to confer scrambling function on ANO1 (which normally does not scramble). Homology modeling shows the scramblase domain forms a hydrophilic cleft facing the lipid bilayer. Ionic currents associated with ANO6 are explained by ionic leak during phospholipid translocation.","method":"Patch clamp recording combined with PtdSer scrambling assay, chimeric ANO1-ANO6 protein analysis, homology modeling","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — chimeric protein domain-swap experiment with both electrophysiology and scrambling readouts, structural modeling validation","pmids":["26057829"],"is_preprint":false},{"year":2015,"finding":"TMEM16F forms homodimers (shown by chemical cross-linking). The cytoplasmic N-terminal and C-terminal regions are essential for plasma membrane localization and protein stability, respectively, and can be exchanged between TMEM16A and TMEM16F. The pore region (between TM5 and TM6) is essential for both the Cl- channel activity of TMEM16A and the phospholipid scramblase activity of TMEM16F, as shown by point mutations.","method":"Chemical cross-linking, successive deletion analysis, domain swapping between TMEM16A and TMEM16F, pore-region point mutagenesis, Ca2+-dependent Cl- channel and scramblase assays in 293T cells and TMEM16F-/- thymocytes","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstitution-style chimera/mutagenesis with dual functional readouts (channel and scramblase), cross-linking for dimerization, single lab","pmids":["24478309"],"is_preprint":false},{"year":2015,"finding":"TMEM16F ion channel and phospholipid scramblase activities are directly coupled: four isoforms from alternative splicing show correlated scramblase and channel activities, knockdown reduces both, and an activating mutation (D409G) markedly increases Ca2+ sensitivity of both ion channel and PtdSer scrambling, including scrambling at rest.","method":"Whole-cell patch clamp, annexin V binding assay for PtdSer, siRNA knockdown, activating point mutation (D409G) introduction, four splice isoform characterization","journal":"The Journal of physiology","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — activating mutation with dual functional readouts, multiple isoforms, gain- and loss-of-function, single lab","pmids":["26108457"],"is_preprint":false},{"year":2013,"finding":"ANO6 is required for Ca2+-dependent PtdSer scrambling in osteoblasts; deletion causes impaired Ca2+-dependent PS scrambling in primary osteoblasts, delayed mineralization in vitro, and reduced skeletal size/deformities with increased uncalcified osteoid in vivo, indicating a cell-autonomous function in bone mineralization.","method":"Ano6 knockout mice, primary osteoblast culture with PS scrambling assay, bone histology, in vivo skeletal analysis","journal":"Journal of bone and mineral research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — KO mouse with cell-autonomous validation in primary cells and in vivo phenotype, PS scrambling directly measured","pmids":["22936354"],"is_preprint":false},{"year":2019,"finding":"Cryo-EM structures of murine TMEM16F in absence and presence of Ca2+ define a ligand-free closed conformation and a Ca2+-bound intermediate. Both conformations resemble their counterparts in scrambling-incompetent mTMEM16A but with distinct differences in the ion/lipid permeation region. Functional data demonstrate the relationship between ion conduction and lipid scrambling, suggesting both functions are mediated by alternate protein conformations at equilibrium in the Ca2+-bound state.","method":"Cryo-EM structure determination (two states), functional scrambling and ion conduction assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM structures with functional characterization, two conformational states resolved, single lab","pmids":["30785399"],"is_preprint":false},{"year":2019,"finding":"Cryo-EM structural analysis of TMEM16F reveals coexistence of an intact channel pore and PIP2-dependent protein conformation changes leading to membrane distortion. Tightly bound lipids are slanted correlating with membrane distortion. Structure-based mutagenesis of lipid-binding residues or residues near membrane distortion sites specifically alters the onset of lipid scrambling but not Ca2+ influx, providing evidence for separate pathways for lipid scrambling and ion permeation.","method":"Cryo-EM structure determination, structure-based mutagenesis, electrophysiology, lipid scrambling assay","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM with mutagenesis distinguishing two functional readouts, single lab","pmids":["31291589"],"is_preprint":false},{"year":2019,"finding":"An inner activation gate in the phospholipid permeation pathway of TMEM16F, formed by three hydrophobic residues F518, Y563, and I612, controls Ca2+-dependent gating. Disruption of this gate profoundly alters phospholipid permeation. Lysine substitutions of F518 and Y563 produce constitutively active Ca2+-independent scramblases. Analogous mutation L543K in TMEM16A is sufficient to confer CaPLSase activity to the Ca2+-activated Cl- channel.","method":"Site-directed mutagenesis, patch clamp electrophysiology, phospholipid scrambling assay, gain-of-function constitutively active mutants","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis with mechanistic insight validated by constitutively active phenotype and cross-paralogue transfer of function","pmids":["31015464"],"is_preprint":false},{"year":2018,"finding":"Purified monomeric/dimeric TMEM16F is a Ca2+-dependent phospholipid scramblase at the single-molecule level, transporting phospholipids nonspecifically between membrane bilayer leaflets. Thermodynamic analysis shows TMEM16F transports ~4.5 × 10^4 lipids/second at 25°C with activation free energy of 47 kJ/mol, consistent with channel-like facilitated diffusion (stepping-stone model).","method":"Purified TMEM16F protein, single-molecule scramblase assay using microarray with asymmetrically distributed phospholipids, fluorescence monitoring of translocation, thermodynamic analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution at single-molecule level with thermodynamic characterization, purified protein","pmids":["29507235"],"is_preprint":false},{"year":2018,"finding":"PIP2 regulates Ca2+-gating of TMEM16F. Brief exposure to high intracellular Ca2+ desensitizes the TMEM16F Ca2+-response associated with PIP2 depletion from the inner leaflet. Application of PIP2 restores TMEM16F channel activity. PIP2 modulation requires positively charged amino acids in the cytoplasmic N-terminal domain. PIP2 works synergistically with membrane depolarization to facilitate Ca2+-gating.","method":"Patch clamp recording of TMEM16F Ca2+-activated current, PIP2 depletion and reconstitution, mutagenesis of N-terminal positively charged residues","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — electrophysiology with pharmacological and mutagenesis validation of PIP2 binding site, multiple approaches","pmids":["29382763"],"is_preprint":false},{"year":2016,"finding":"Ca2+ directly binds TMEM16F to stabilize its structure and activate phospholipid scrambling. Comprehensive mutagenesis of acidic residues in TMEM16F identified conserved residues (homologous to those in TMEM16K) critical for Ca2+ binding; point mutations of these residues reduce Ca2+-dependent phospholipid scrambling activity.","method":"Comprehensive acidic residue mutagenesis, blue-native PAGE to detect Ca2+-dependent stable complex, phospholipid scrambling assay","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis of putative Ca2+-binding residues with direct scrambling assay readout, structural validation by native PAGE","pmids":["27227820"],"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. The concomitant opening of a pore promotes ion conduction in the same protein conformation, establishing an activation mechanism distinct from other TMEM16 family members.","method":"Screen of activating mutants, cryo-EM structure determination, functional ion conduction and scrambling assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM of activated mutants with functional validation, mechanistic basis of activation established","pmids":["36335104"],"is_preprint":false},{"year":2023,"finding":"Cryo-EM structures of TMEM16F with bound niclosamide or 1PBC identify a drug-binding pocket within a groove harboring a lipid trail outside the ion permeation pore, constituting a separate lipid scrambling pathway. Mutations of two residues in this groove specifically affect lipid scrambling but not ion permeation. Mutations in the drug pocket reduce inhibition of either Ca2+ influx or PS exposure differentially, providing structural evidence for separate ion and lipid permeation pathways.","method":"Cryo-EM structure determination with drug-bound states, site-directed mutagenesis, electrophysiology, PS exposure assay (annexin V/flow cytometry)","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM with bound inhibitors plus mutagenesis with two orthogonal functional readouts, single lab","pmids":["37573365"],"is_preprint":false},{"year":2026,"finding":"High-resolution cryo-EM structures of active TMEM16F in liposomes reveal two conformations in high-activity conditions: the canonical Ca2+-bound closed state and an active state where upward rotation of the cytosolic domain creates an X-shaped groove forming a transmembrane pore and locally thins the membrane. Mutagenesis, functional assays, and MD simulations demonstrate that the X-shaped groove mediates nonselective ion flux and lipid scrambling through distinct pathways: ions move within the protein-delimited pore while lipids skirt the X-shaped groove.","method":"Cryo-EM in liposomes (native-like environment), site-directed mutagenesis, ion conduction and scrambling functional assays, molecular dynamics simulations","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM of active state in native-like lipid environment, mutagenesis, MD simulations, orthogonal functional assays in single rigorous study","pmids":["41998358"],"is_preprint":false},{"year":2020,"finding":"TMEM16F, a Ca2+-activated phospholipid scramblase, plays an essential role in placental trophoblast fusion (syncytialization) by translocating PS to the cell surface independent of apoptosis. TMEM16F knockout mice exhibit deficiency in trophoblast syncytialization and placental development leading to perinatal lethality.","method":"TMEM16F knockout mice, placental histology, PS exposure assay, syncytialization quantification","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 2 / Moderate — KO mouse with specific developmental phenotype and PS scrambling mechanistic link, multiple readouts","pmids":["32494719"],"is_preprint":false},{"year":2020,"finding":"TMEM16F is required for plasma membrane repair after injury by pore-forming agents. Upon pore formation and subsequent Ca2+ influx, TMEM16F induces rapid lipid scrambling, membrane blebbing, extracellular vesicle release, preserved membrane integrity, and increased cell viability. TMEM16F-deficient mice show compromised control of Listeria monocytogenes infection associated with greater sensitivity of neutrophils to listeriolysin O.","method":"TMEM16F-deficient mice, pore-forming toxin treatment, Ca2+ imaging, membrane integrity assays, cell viability, in vivo bacterial infection model","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO mouse with in vitro and in vivo validation, mechanistic Ca2+-TMEM16F-membrane repair pathway established","pmids":["31995754"],"is_preprint":false},{"year":2016,"finding":"TMEM16F is the dominant lipid scramblase in T lymphocytes. TMEM16F is located in late endosomes where it facilitates generation of multivesicular bodies for TCR degradation and signal termination. TMEM16F deficiency results in sustained TCR signaling and augmented T cell activation, ultimately causing T cell exhaustion in chronic viral infection.","method":"TMEM16F-deficient T cells, TCR signaling assays, multivesicular body quantification, late endosome localization imaging, viral infection model (in vivo), flow cytometry for T cell activation markers","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Moderate — KO T cells with subcellular localization data, TCR signaling readouts, in vivo infection model, mechanistic pathway established","pmids":["27810927"],"is_preprint":false},{"year":2016,"finding":"TMEM16F is expressed in synaptic clusters facing pre-synaptic cholinergic C-boutons in α-motoneurons of the spinal cord and governs a Ca2+-activated Cl- conductance in these neurons. Conditional Tmem16f deletion decreases motor performance under high-demanding tasks by increasing recruitment threshold of fast α-motoneurons. Loss of TMEM16F in an ALS mouse model reduces activity-dependent stress markers, delays disease onset, and preserves muscular strength in male mice.","method":"Conditional Tmem16f exon deletion, whole-cell patch clamp in spinal motoneurons, immunofluorescence localization at C-boutons, motor behavior testing, ALS mouse model (male mice only)","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Moderate — conditional KO with electrophysiological and behavioral phenotypes, localization to specific synaptic compartment, disease model validation","pmids":["32101737"],"is_preprint":false},{"year":2016,"finding":"TMEM16F conditional ablation in microglia prevents development of mechanical hypersensitivity after nerve injury. In absence of TMEM16F, microglia display deficits in process motility and phagocytosis, and loss of GABA immunoreactivity upon injury is spared.","method":"Conditional TMEM16F knockout in microglia, nerve injury model, mechanical hypersensitivity behavioral assay, microglial process motility imaging, phagocytosis assay, GABA immunostaining","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Moderate — conditional KO with specific cellular and behavioral phenotypes, multiple readouts","pmids":["27332874"],"is_preprint":false},{"year":2021,"finding":"Ca2+-dependent activation of TMEM16F at the immunological synapse reduces the electrostatic potential of the plasma membrane by locally redistributing phosphatidylserine, which increases dissociation of bystander TCR-CD3 cytoplasmic domains from the plasma membrane (via reduced electrostatic interactions) and enhances TCR-dependent signaling and T cell activation.","method":"Electrostatic membrane potential measurements, PS redistribution imaging, TCR-CD3 membrane binding assays, T cell activation assays, TMEM16F-dependent signaling experiments","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 2 / Moderate — mechanistic pathway established with electrostatic measurements and TCR-CD3 dissociation assays, multiple orthogonal methods","pmids":["33758060"],"is_preprint":false},{"year":2019,"finding":"TMEM16F activation by Ca2+ ionophores in Jurkat T cells triggers large-scale surface membrane expansion concurrent with phospholipid scrambling. Continued activation leads to shedding of ectosomes containing PD-1, which is selectively incorporated based on its transmembrane sequence. Cells lacking TMEM16F fail to expand surface membrane and instead undergo rapid massive endocytosis with PD-1 internalization.","method":"Microscopy and patch clamp recording in Jurkat T cells, Ca2+ ionophore treatment, ectosome isolation, PD-1 trafficking analysis, TMEM16F-deficient cells","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — combined microscopy and electrophysiology, TMEM16F-deficient comparison, single lab, functional consequence of membrane expansion established","pmids":["30679690"],"is_preprint":false},{"year":2021,"finding":"TMEM16F and dynamins cooperatively control large expansive plasma membrane reservoirs. Ca2+ activation of TMEM16F causes anionic phospholipids to escape from cytoplasmic monolayer (lipid scrambling), allowing dynamin-held invaginations to open and membrane to expand. Deletion of TMEM16F or dynamins blocks membrane expansion. Dynamin2-GFP punctae rapidly dissipate from compartments during TMEM16F activation.","method":"TMEM16F and dynamin deletion/expression, live cell microscopy, TMEM16F activation via Ca2+-permeable mechanosensitive channels, GFP-dynamin imaging, membrane expansion assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic deletion of both components, live imaging of dynamin dynamics, mechanosensitive channel-Ca2+-TMEM16F pathway defined","pmids":["34404808"],"is_preprint":false},{"year":2022,"finding":"ANO6/TMEM16F-mediated cell surface exposure of phosphatidylserine is critical for SARS-CoV-2 entry. SARS-CoV-2 Spike pseudotyped virus evokes cytosolic Ca2+ elevation and ANO6-dependent PS externalization in ACE2/TMPRSS2-positive cells. ANO6-selective inhibitor A6-001 inhibits SARS-CoV-2-induced PS scrambling and viral replication.","method":"Spike pseudovirus infection, Ca2+ imaging, annexin V PS exposure assay, high-throughput inhibitor screening, authentic SARS-CoV-2 replication assay in multiple cell lines including primary human nasal epithelial cells","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Moderate — Ca2+ and PS exposure assays, ANO6-selective pharmacological inhibition, authentic virus replication validated in multiple cell systems","pmids":["35839776"],"is_preprint":false},{"year":2022,"finding":"Ca2+ influx through TRPV4 channel is critical for TMEM16F activation and subsequent human trophoblast fusion. TRPV4 and TMEM16F are functionally coupled within Ca2+ microdomains in trophoblasts. Pharmacological inhibition or gene silencing of TRPV4 hinders TMEM16F activation and trophoblast syncytialization.","method":"Patch clamp electrophysiology, TRPV4 pharmacological agonist/antagonist, siRNA knockdown, trophoblast syncytialization quantification, Ca2+ microdomain imaging","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Moderate — electrophysiology with pharmacology and genetic knockdown, functional coupling within Ca2+ microdomains established","pmids":["35670667"],"is_preprint":false},{"year":2024,"finding":"TMEM16F is the long-sought RBC Ca2+-activated phospholipid scramblase (CaPLSase), activated by Ca2+ influx through the mechanosensitive channel PIEZO1 in red blood cells. PIEZO1-TMEM16F functional coupling is enhanced in hereditary xerocytosis RBCs carrying PIEZO1 gain-of-function mutations, leading to increased PS exposure propensity linked to HX clinical manifestations. PIEZO1 inhibition with GsMTx-4 or benzbromarone prevents force-induced PS exposure and hemolysis in HX RBCs.","method":"RBC patch clamp, Ca2+ imaging, annexin V PS exposure assay, PIEZO1 pharmacological inhibitors, primary RBCs from HX patients with PIEZO1 gain-of-function mutations, hemolysis assays, TMEM16F identification in RBCs","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — patient RBCs with gain-of-function PIEZO1 mutations, pharmacological PIEZO1 inhibition, multiple orthogonal assays establishing PIEZO1-TMEM16F functional coupling","pmids":["38033286"],"is_preprint":false},{"year":2021,"finding":"TMEM16F lipid scrambling and ion channel activities are strongly regulated by intracellular pH (pHi). Low pHi attenuates and high pHi potentiates TMEM16F channel and scramblase activation. pHi sensitivity depends on [Ca2+]i and shows a bell-shaped relationship. Mutation of a Ca2+-binding residue (E667Q) dramatically shifts the pHi-sensitivity peak, indicating that protons and Ca2+ compete for the primary Ca2+-binding residues in the pore, providing the molecular basis of pHi regulation.","method":"Whole-cell patch clamp, phospholipid scrambling assay, intracellular pH manipulation, Ca2+-binding site mutagenesis (E667Q), biophysical characterization","journal":"The Journal of general physiology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis of Ca2+-binding site with dual functional readouts, competition mechanism established, single lab","pmids":["33346788"],"is_preprint":false},{"year":2013,"finding":"ANO6 Cl- currents can be activated by increase in cytosolic Ca2+, or Ca2+-independently by Fas receptor stimulation. Ca2+-dependent PS scrambling induced by ANO6 does not require Cl- currents. Ca2+-independent PS scrambling due to extrinsic (FasL) or intrinsic (ABT-737) apoptosis does not require ANO6. ANO6 is necessary for Ca2+-dependent PS scrambling but not by increasing intracellular Ca2+.","method":"Whole-cell patch clamp in normal and Scott syndrome B-lymphocytes, ionomycin and FasL stimulation, Cl- channel blockers, siRNA knockdown, ABT-737 apoptosis induction, annexin V PS assay","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 2 / Strong — patient Scott syndrome cells as natural KO, pharmacological and siRNA approaches, multiple stimuli distinguishing Ca2+-dependent vs independent pathways","pmids":["23618909"],"is_preprint":false},{"year":2017,"finding":"TMEM16F Cl- currents and phospholipid scrambling can be activated by modification of plasma membrane phospholipids through reactive oxygen species and phospholipase A2 (PLA2), independent of intracellular Ca2+. Mutations within TMEM16F or TMEM16A/F chimeras similarly change both Cl- currents and phospholipid scrambling, suggesting the same intramolecular pathway for Cl- and phospholipids. Only TMEM16F (not TMEM16A) can expose PtdSer to the outer membrane leaflet.","method":"Overexpression of TMEM16A and TMEM16F, PLA2 activation/inhibition, ROS induction, PtdSer exposure (annexin V), whole-cell patch clamp, TMEM16A/F chimera mutagenesis","journal":"The Journal of physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — chimera mutagenesis with dual functional readouts, pharmacological manipulation, single lab","pmids":["29134661"],"is_preprint":false},{"year":2018,"finding":"The actin cytoskeleton negatively regulates ANO6/TMEM16F anion channel activity: cytochalasin-D disruption accelerates activation kinetics, while phalloidin/jasplakinolide inhibit ANO6 currents. Intracellular MgATP decelerates activation of whole-cell ANO6 currents and prevents inactivation. Inside-out patches show immediate Ca2+-dependent activation, indicating that cytosolic factors (including actin and ATP) modulate the delay and inactivation of ANO6.","method":"Whole-cell and inside-out patch clamp, cytochalasin-D, phalloidin, jasplakinolide actin manipulation, MgATP concentration variation","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological manipulation of actin with electrophysiological readout, inside-out vs whole-cell comparison, single lab","pmids":["29964013"],"is_preprint":false},{"year":2019,"finding":"TMEM16F undergoes a dynamic change in ion selectivity in response to changes in intracellular Ca2+ concentration: at higher Ca2+ levels, Cl- permeability relative to Na+ (PCl-/PNa+) increases. This dynamic selectivity shift is independent of channel activation state and is indicative of a charge-screening mechanism in the permeation pathway.","method":"Excised inside-out patch clamp with Q559K mutant (no current rundown), systematic variation of intracellular Ca2+ concentration, ion substitution experiments","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — electrophysiology with defined mutant lacking rundown, systematic Ca2+ concentration series, single lab","pmids":["31318330"],"is_preprint":false},{"year":2013,"finding":"Human TMEM16F expressed in HEK293T cells is an essential component of a Ca2+-activated Cl- channel with strong outward rectification. Cl- is the permeant ion with anion selectivity sequence I- > Br- > Cl- > F- > aspartate-. Ca2+ concentration for half-maximal activation is 9.6 μM, distinctly higher than for TMEM16A/B. TMEM16F is not related to volume-sensitive outwardly rectifying Cl- channel (VSOR) activity.","method":"Whole-cell patch clamp in HEK293T cells, ion substitution, niflumic acid pharmacology, TMEM16F overexpression and knockdown","journal":"American journal of physiology. Cell physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — electrophysiology with ion substitution and pharmacology, negative result (no VSOR role) explicitly established, single lab","pmids":["23426967"],"is_preprint":false},{"year":2013,"finding":"Mouse TMEM16F expressed in HEK293 cells generates an outwardly rectifying Ca2+-activated Cl- current activated with a delay of several minutes. A significant Na+ current is also activated with PNa = 0.3 PCl. EC50 is ~100 μM intracellular Ca2+. Pore region mutant R592E abolishes the current; K616E reduces relative iodide permeability; R636E alters anion selectivity, establishing the pore region as functionally critical.","method":"Whole-cell patch clamp in HEK293 cells, site-directed mutagenesis of pore region residues (R592E, K616E, R636E), ion selectivity measurements","journal":"The Journal of general physiology","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — pore mutagenesis with electrophysiology, multiple mutants with distinct phenotypes, single lab","pmids":["23630341"],"is_preprint":false},{"year":2019,"finding":"TMEM16F activation by Ca2+ triggers large-scale surface membrane expansion in Jurkat T cells. Cells lacking TMEM16F undergo rapid massive endocytosis with PD-1 internalization instead of expansion. The T cell co-receptor PD-1 is selectively incorporated into TMEM16F-dependent ectosomes based on its transmembrane sequence.","method":"Confocal microscopy, patch clamp, Ca2+ ionophore activation, TMEM16F-deficient cell comparison, ectosome characterization, PD-1 transmembrane domain mutants","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live imaging and electrophysiology combined with TMEM16F-deficient cells, transmembrane sequence dependency established, single lab","pmids":["30679690"],"is_preprint":false},{"year":2018,"finding":"TMEM16F contributes to pyroptotic cell death downstream of gasdermin-D pore formation. GD-N (N-terminal cleavage product of gasdermin D) expression enhances basal Ca2+ levels and induces TMEM16F-dependent large whole-cell currents and cell death in HEK293 and HAP1 cells. Knockdown or inhibition of TMEM16F suppresses GD-N-induced whole-cell currents.","method":"GD-N expression in HEK293 and HAP1 cells, whole-cell patch clamp, TMEM16F knockdown (siRNA), TMEM16F inhibitors, Ca2+ imaging, cell death assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockdown with electrophysiological and cell death readouts, endogenous TMEM16F required, single lab","pmids":["29463790"],"is_preprint":false},{"year":2019,"finding":"TMEM16F is activated during ferroptosis induced by erastin or RSL3 (inhibitor of GPX4). Cell death was largely reduced in intestinal epithelium and peritoneal macrophages from tissue-specific TMEM16F knockout mice. Ferrostatin-1 and TMEM16F inhibitors block both TMEM16F currents and ferroptotic cell death.","method":"Tissue-specific TMEM16F knockout mice, erastin/RSL3-induced ferroptosis, whole-cell patch clamp, cell viability assays, ferrostatin-1 inhibition","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — tissue-specific KO mouse with electrophysiological and cell death readouts, pharmacological validation, single lab","pmids":["31060306"],"is_preprint":false},{"year":2017,"finding":"CFTR enhances ANO6 activity, probably through translocation of signaling proteins to the plasma membrane. ANO6 produces outwardly rectifying Cl- currents and scrambles plasma membrane phospholipids when activated by increased cytosolic ROS and consecutive peroxidation of plasma membrane lipids. In ANO6 knockout mice, apoptotic cells in the intestinal epithelium are strongly reduced, supporting ANO6's role in cell death cooperatively with CFTR.","method":"ANO6 knockout mice, ROS donor treatment, whole-cell patch clamp, PS exposure assay, TUNEL staining of intestinal epithelium, CFTR and ANO6 co-expression in airway cells","journal":"Pflugers Archiv : European journal of physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse with in vivo cell death readout, electrophysiology and PS assay in vitro, single lab","pmids":["28875346"],"is_preprint":false},{"year":2012,"finding":"ANO6 functions as a Ca2+-activated Cl- channel in mouse dendritic cells (DCs) as demonstrated by siRNA knockdown. ANO6 is activated by chemokine receptor CCR7 ligation with CCL21. Knockdown of ANO6 reduces chemokine-induced migration of both immature and LPS-matured DCs.","method":"Whole-cell patch clamp in mouse bone marrow-derived DCs, siRNA knockdown of ANO6, RT-PCR and Western blot, CCL21 stimulation, migration assay","journal":"Cellular physiology and biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA knockdown with electrophysiological and migration readouts, chemokine receptor activation demonstrated, single lab","pmids":["23159814"],"is_preprint":false},{"year":2025,"finding":"TMEM16F mediates ER stress/calcium-induced PS externalization in tumor cells, which suppresses antitumor immunity. TMEM16F-KO tumor cells (CRISPR/Cas9) did not suppress tumorigenicity in immune-deficient mice (NOD/SCID or RAG-KO) but did in immune-competent mice, demonstrating that TMEM16F-dependent PS exposure promotes immune evasion through a T cell-dependent mechanism. TMEM16F-KO specifically abrogated ER stress/calcium-induced PS externalization without affecting cell-intrinsic proliferation.","method":"CRISPR/Cas9 knockout in EO771 breast cancer cells, orthotopic transplantation in immune-competent and immune-deficient mice, PS exposure assay, ER stress induction","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR KO with in vivo immune-competent vs immune-deficient comparison, mechanistic distinction from apoptotic PS exposure, single lab","pmids":["41198619"],"is_preprint":false},{"year":2024,"finding":"TMEM16F deficiency in endothelial cells (ECKO mice) causes prolonged tail bleeding and significantly smaller IVC thrombi, demonstrating that endothelial TMEM16F function is essential for normal hemostasis. Niclosamide (PLS inhibitor) prevents pathological Ca2+ signals and PS translocation in endothelial cells exposed to extracellular histones.","method":"Tamoxifen-inducible endothelial-specific Cdh5-Cre TMEM16F knockout mice, tail bleeding time, IVC stenosis thrombosis model, live-cell Ca2+ imaging, annexin V PS exposure flow cytometry, niclosamide pharmacology","journal":"Shock (Augusta, Ga.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell-type-specific KO mouse with multiple hemostasis readouts, pharmacological validation, single lab","pmids":["39874534"],"is_preprint":false},{"year":2024,"finding":"TMEM16F deficiency in neurons (but not microglia) reduces tauopathy and microgliosis in PS19 (P301S tau) mice. TMEM16F mediates aberrant phosphatidylserine exposure in neurons burdened with phospho-tau, linking neuronal TMEM16F-mediated PS scrambling to propagation of tau pathology.","method":"Neuron-specific and microglia-specific TMEM16F conditional knockout in PS19 tau mice, tauopathy quantification (AT8 staining), microgliosis assessment, PS exposure assay in neurons","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell-type-specific KO in disease model, PS exposure mechanistically linked to tau pathology spread, single lab","pmids":["38941274"],"is_preprint":false},{"year":2024,"finding":"TMEM16F deficiency impairs developmental retinal angiogenesis. TMEM16F knockdown enhances plasma membrane association of activated Src kinase, increases VE-cadherin phosphorylation and downregulation, and suppresses angiogenesis. This establishes an intracellular signaling function for TMEM16F in endothelial cells, separate from its canonical lipid scrambling role.","method":"Endothelial TMEM16F conditional KO mice (retinal angiogenesis), HUVEC siRNA knockdown, Src kinase phosphorylation assay (pY416), VE-cadherin expression and phosphorylation, in vitro angiogenesis assays","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse plus siRNA with biochemical pathway dissection, in vitro and in vivo angiogenesis readouts, single lab","pmids":["38940198"],"is_preprint":false},{"year":2025,"finding":"TMEM16F preferentially scrambles phosphatidylserine and phosphatidylcholine over phosphatidylethanolamine on the plasma membrane of living cells, contradicting the prevailing view of non-selective scrambling. This phospholipid headgroup preference was established using a fluorescence polarization-based cell scrambling assay with NBD-labeled phospholipids.","method":"Fluorescence polarization (FP) assay with NBD-labeled phospholipids for kinetic monitoring of scrambling on plasma membrane of living cells, TMEM16F-expressing vs control cells","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — novel cell-based assay with multiple phospholipid species side-by-side, single lab, challenges prior consensus","pmids":["41166415"],"is_preprint":false},{"year":2024,"finding":"TMEM16F-mediated PS scrambling polarizes macrophages to an immunosuppressive M2 phenotype, promoting TGF-β1 secretion and regulatory T cell expansion that suppresses cytotoxic lymphocytes. Genetic ablation of TMEM16F abolished PS exposure, reprogrammed the tumor microenvironment toward immune activation, and suppressed tumor growth across cancer models.","method":"TMEM16F genetic ablation across cancer models, macrophage polarization assays (M1/M2 markers), TGF-β1 ELISA, regulatory T cell quantification, cytotoxic T cell activity, tumor growth measurement","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic ablation with multiple immunological readouts, pharmacological scramblase inhibition validation, single lab","pmids":["41100671"],"is_preprint":false},{"year":2024,"finding":"TMEM16F regulates pathological α-synuclein (α-synA53T) spread in neurons. TMEM16F-knockout neurons show reduced spread of pathological α-synA53T in vitro and in vivo in a PD mouse model. A missense mutation Ala703Ser identified in Ashkenazi Jewish PD patients confers enhanced lipid scramblase activity and is associated with altered regulation of α-synA53T extracellular secretion in cellular models.","method":"TMEM16F knockout neurons with reporter system for donor/recipient α-syn spread, in vivo PD mouse model (α-synA53T injection), SNP identification in patient cohort, lipid scramblase activity assay, cellular secretion assays","journal":"Aging cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO neurons in vitro and in vivo plus human variant with enhanced scramblase activity, multiple readouts, single lab","pmids":["39487963"],"is_preprint":false},{"year":2025,"finding":"Lipid scrambling via TMEM16F is sufficient to induce extracellular vesicle formation and release without changes in cytosolic calcium or cytoskeleton. Scrambling causes segregation of exofacial lipids, redistribution of cholesterol to inner leaflet, clustering of GPI-linked proteins creating convex curvature, and PE accumulation creating concave curvature that facilitates vesicle scission.","method":"Inducible active TMEM16F expression, vesicle isolation and quantification, lipid distribution assays, cholesterol and GPI-protein imaging, PE distribution imaging","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — inducible active scramblase with multiple lipid distribution and vesicle biogenesis readouts, mechanistic model established, single lab","pmids":["41604453"],"is_preprint":false},{"year":2021,"finding":"The Ca2+ sensitivity of ANO6 is partially regulated by a putative Ca2+-binding reservoir at the N-terminal domain (Nt-CaRes). An ANO6-1-6 chimera with Nt-CaRes replaced by the corresponding ANO1 domain showed higher Ca2+ sensitivity. Mutations of Ca2+-interacting acidic residues in ANO6 Nt-CaRes resulted in reduced Ca2+ sensitivity, indicating direct Ca2+ interactions at this reservoir contribute to differential Ca2+ sensitivity between ANO1 and ANO6.","method":"Chimera construction (ANO6-1-6), N-terminal domain point mutagenesis, whole-cell patch clamp Ca2+ sensitivity measurement, molecular dynamics simulation of Ca2+ interactions","journal":"Molecules and cells","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — chimera and mutagenesis with electrophysiological readout, MD simulation support, single lab","pmids":["33658434"],"is_preprint":false},{"year":2023,"finding":"ANO6 expressed in cancer-associated fibroblasts (CAFs) is required for trogocytosis, a process by which PDAC cells acquire lipids (including cholesterol) from CAF plasma membranes. During trogocytosis, cancer cell-CAF synapse-like contacts induce cytosolic Ca2+ influx in CAFs via Orai channels, activating ANO6 and causing PS exposure on CAF plasma membranes that initiates lipid transfer to PDAC cells. ANO6-dependent trogocytosis also supports immunosuppressive function of CAFs toward cytotoxic T cells by enabling excess cholesterol transfer.","method":"CAF-PDAC co-culture trogocytosis assays, ANO6 knockdown/inhibition, Ca2+ imaging, PS exposure assay, lipid (cholesterol) transfer quantification, T cell cytotoxicity assays, Orai channel pharmacology","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mechanistic pathway established with Ca2+ imaging, PS assay, and lipid transfer readouts; preprint status","pmids":["37745612"],"is_preprint":true}],"current_model":"ANO6/TMEM16F is a Ca2+-activated plasma membrane protein that functions as both a phospholipid scramblase and a non-selective ion channel: upon elevation of intracellular Ca2+ (binding directly to conserved acidic residues in the transmembrane domain), TMEM16F undergoes conformational changes—including formation of an X-shaped groove/hydrophilic pore—that allow bidirectional, facilitated diffusion of phospholipids (with preference for PS and PC over PE) across the bilayer at ~4.5×10^4 lipids/second, while simultaneously conducting non-selective ion currents through a distinct but overlapping pathway; this Ca2+-dependent scrambling activity is further regulated by PIP2, intracellular pH, the actin cytoskeleton, and upstream Ca2+-influx channels (PIEZO1, TRPV4), and underlies diverse cellular functions including platelet procoagulant activity and blood coagulation, bone mineralization by osteoblasts, trophoblast syncytialization, plasma membrane repair after pore-forming toxin injury, TCR signal termination via late-endosomal multivesicular body formation, microglial phagocytosis, ectosome/extracellular vesicle release, and viral (SARS-CoV-2) entry facilitation."},"narrative":{"mechanistic_narrative":"ANO6/TMEM16F is a Ca2+-activated, plasma-membrane phospholipid scramblase that couples elevation of intracellular Ca2+ to bidirectional translocation of phospholipids—exposing phosphatidylserine on the cell surface—and to non-selective ion conduction, thereby driving membrane-remodeling events across many cell types [PMID:21107324, PMID:23021219, PMID:29507235]. Ca2+ binds directly to conserved acidic residues in the transmembrane domain to stabilize the protein and activate scrambling, with an N-terminal Ca2+ reservoir tuning sensitivity [PMID:27227820, PMID:33658434], while protons compete at these same sites to give intracellular pH-dependent regulation [PMID:33346788]; activity is further modulated by PIP2 at a cytoplasmic basic motif and by the actin cytoskeleton [PMID:29382763, PMID:29964013]. As a single-molecule scramblase the purified protein moves ~4.5×10^4 lipids/second by channel-like facilitated diffusion with headgroup preference for PS and PC over PE [PMID:29507235, PMID:41166415]. Cryo-EM and mutagenesis define an inner hydrophobic gate (F518/Y563/I612) and an active conformation in which upward rotation of the cytosolic domain creates an X-shaped groove that thins the membrane, establishing that lipids skirt this groove while ions pass through an adjacent protein-delimited pore as distinct but overlapping permeation pathways [PMID:31015464, PMID:37573365, PMID:41998358]. Upstream Ca2+-influx channels including PIEZO1 and TRPV4 are functionally coupled to TMEM16F within Ca2+ microdomains to gate its activation [PMID:35670667, PMID:38033286]. Through Ca2+-dependent PS exposure and membrane expansion TMEM16F mediates platelet procoagulant activity and blood coagulation [PMID:23021219, PMID:26417084], osteoblast bone mineralization [PMID:22936354], trophoblast syncytialization [PMID:32494719, PMID:35670667], plasma membrane repair after pore-forming toxin injury [PMID:31995754], and ectosome/extracellular-vesicle release [PMID:41604453]; in T cells it terminates TCR signaling via late-endosomal multivesicular bodies while at the synapse it can also tune the membrane electrostatic potential that controls TCR-CD3 engagement [PMID:27810927, PMID:33758060]. Loss-of-function mutation of TMEM16F underlies the human bleeding disorder Scott syndrome through defective phospholipid scrambling [PMID:21107324].","teleology":[{"year":2010,"claim":"Establishing the molecular identity of the long-sought Ca2+-dependent phospholipid scramblase resolved which protein exposes PS on activated cells and tied it to human disease.","evidence":"expression cloning in Ba/F3 cells, FACS PS-exposure assay, and Scott syndrome patient mutation sequencing","pmids":["21107324"],"confidence":"High","gaps":["mechanism of lipid translocation undefined","relationship to ion conduction unknown"]},{"year":2012,"claim":"Defining TMEM16F as a Ca2+-activated nonselective cation channel and demonstrating a platelet bleeding/thrombosis phenotype in knockout mice linked the protein's electrical activity to procoagulant PS exposure in vivo.","evidence":"knockout mouse, patch clamp in megakaryocytes and heterologous cells, pore mutagenesis, platelet PS assays, arterial thrombosis model","pmids":["23021219"],"confidence":"High","gaps":["whether channel and scramblase share one pathway unresolved","direct Ca2+-binding site not yet mapped"]},{"year":2013,"claim":"Platelet-specific and human Scott-syndrome studies confirmed TMEM16F is the dominant platelet scramblase required for procoagulant PS exposure and microparticle shedding while separating Ca2+-dependent from apoptotic PS scrambling.","evidence":"platelet-conditional knockout mice with thrombin generation and intravital imaging; Scott syndrome B-lymphocyte patch clamp with FasL/ABT-737 stimuli","pmids":["26417084","23618909"],"confidence":"High","gaps":["does not define the structural basis of scrambling","Ca2+-independent apoptotic scramblase identity unaddressed"]},{"year":2013,"claim":"Heterologous electrophysiology characterized TMEM16F's outwardly rectifying Ca2+-activated Cl- conductance, its low Ca2+ sensitivity relative to TMEM16A/B, and the pore residues governing ion selectivity.","evidence":"whole-cell patch clamp in HEK cells, ion substitution, pharmacology, pore-region mutagenesis (R592E, K616E, R636E)","pmids":["23426967","23630341"],"confidence":"Medium","gaps":["overexpression may not reflect native conductance","structural pore not resolved"]},{"year":2015,"claim":"Domain-swap and mutagenesis work localized a discrete scramblase domain and the shared pore region, and demonstrated that channel and scramblase activities are coupled and co-regulated by a single activating mutation.","evidence":"ANO1-ANO6 chimeras, pore-region point mutagenesis, dimerization cross-linking, splice-isoform and D409G activating-mutant analysis with dual scrambling/channel readouts","pmids":["26057829","24478309","26108457"],"confidence":"High","gaps":["whether ions and lipids use identical or merely overlapping paths unresolved","no atomic structure yet"]},{"year":2016,"claim":"Identifying the conserved acidic Ca2+-binding residues established how Ca2+ directly stabilizes and activates the scramblase.","evidence":"comprehensive acidic-residue mutagenesis, blue-native PAGE for Ca2+-dependent complex, scrambling assay","pmids":["27227820"],"confidence":"High","gaps":["conformational consequences of Ca2+ binding not visualized","stoichiometry of Ca2+ binding not defined here"]},{"year":2018,"claim":"Single-molecule reconstitution with purified protein and the PIP2-regulation study quantified scrambling as channel-like facilitated lipid diffusion and identified an inner-leaflet lipid cofactor controlling Ca2+ gating.","evidence":"purified TMEM16F single-molecule microarray scramblase assay with thermodynamic analysis; patch clamp with PIP2 depletion/reconstitution and N-terminal basic-residue mutagenesis","pmids":["29507235","29382763"],"confidence":"High","gaps":["physiological PIP2 dynamics during native activation unclear","lipid selectivity not addressed at single-molecule level"]},{"year":2019,"claim":"Cryo-EM of closed and Ca2+-bound states plus gate and lipid-binding mutagenesis defined the activation conformations and provided structural evidence that lipid scrambling and ion conduction proceed through separate but adjacent pathways.","evidence":"cryo-EM of two conformational states, structure-based mutagenesis of an inner gate (F518/Y563/I612) and lipid/membrane-distortion residues, paired ion and scrambling assays","pmids":["30785399","31291589","31015464"],"confidence":"High","gaps":["fully active scrambling-competent conformation not yet captured","dynamics of gating not directly observed"]},{"year":2022,"claim":"Cryo-EM of activating mutants captured the rearrangement exposing hydrophilic membrane patches and concomitant pore opening, defining a TMEM16F-specific activation mechanism.","evidence":"activating-mutant screen, cryo-EM, functional ion and scrambling assays","pmids":["36335104"],"confidence":"High","gaps":["mutant conformation may differ from wild-type active state","lipid trajectory not directly imaged"]},{"year":2023,"claim":"Drug-bound cryo-EM structures localized a scrambling groove with a lipid trail outside the ion pore, giving structural proof of separable lipid and ion permeation pathways and a druggable pocket.","evidence":"cryo-EM with niclosamide/1PBC, groove and pocket mutagenesis, electrophysiology and PS exposure assays","pmids":["37573365"],"confidence":"High","gaps":["inhibitor selectivity across paralogues not fully defined","lipid occupancy in the groove inferred not directly resolved during transport"]},{"year":2026,"claim":"High-resolution cryo-EM of active TMEM16F in liposomes resolved an X-shaped groove that forms a transmembrane pore and thins the membrane, demonstrating ions pass through the protein pore while lipids skirt the groove.","evidence":"cryo-EM in native-like liposomes, mutagenesis, ion/scrambling assays, molecular dynamics simulations","pmids":["41998358"],"confidence":"High","gaps":["temporal order of pore opening versus membrane thinning not established","lipid headgroup preference mechanism not structurally explained"]},{"year":2024,"claim":"Identifying upstream Ca2+ sources (PIEZO1 in red cells, TRPV4 in trophoblasts, Orai in fibroblasts) and biophysical regulators (pH competition, ion-selectivity shifts, actin) showed how TMEM16F is gated within physiological Ca2+ microdomains.","evidence":"RBC and trophoblast patch clamp with channel inhibition/knockdown, Ca2+-binding-site pH-competition mutagenesis (E667Q), inside-out actin pharmacology, dynamic-selectivity recordings","pmids":["38033286","35670667","33346788","31318330","29964013"],"confidence":"High","gaps":["molecular tethering of TMEM16F to upstream channels unknown","spatial organization of microdomains not resolved"]},{"year":2024,"claim":"Cell-type-specific knockouts mapped TMEM16F PS-scrambling and membrane-expansion functions onto diverse physiological and pathological processes spanning development, immunity, repair, vesicle release, and disease.","evidence":"conditional/tissue-specific knockouts and CRISPR ablation across osteoblasts, trophoblasts, T cells, microglia, motoneurons, endothelium, tumor cells, and neurons; membrane-repair, ectosome, EV-release, and SARS-CoV-2 entry assays","pmids":["22936354","32494719","31995754","27810927","33758060","30679690","34404808","35839776","32101737","27332874","39874534","38941274","41198619","41100671","39487963","46"],"confidence":"Medium","gaps":["many roles share the single PS-scrambling mechanism but downstream effectors differ by cell type","non-canonical signaling roles (e.g. endothelial Src/VE-cadherin) lack defined molecular linkage"]},{"year":null,"claim":"How TMEM16F achieves headgroup preference for PS and PC and how a single conformational change orchestrates the temporal coordination of ion conduction, lipid scrambling, and membrane curvature in living cells remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["structural basis of PS/PC over PE selectivity not defined","in vivo coupling of scrambling to vesicle scission and curvature generation incompletely mapped","proposed non-scramblase signaling functions mechanistically unexplained"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,3,10,43]},{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[1,32,33]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[10,11,46]},{"term_id":"GO:0140299","term_label":"molecular sensor activity","supporting_discovery_ids":[12,27]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,4,29]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[18]}],"pathway":[{"term_id":"R-HSA-109582","term_label":"Hemostasis","supporting_discovery_ids":[1,2,40]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[18,21,44]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[35,36,37]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[22,46]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[21,42]}],"complexes":[],"partners":["PIEZO1","TRPV4","DNM2","CFTR","ORAI1"],"other_free_text":[]}},"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":790,"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":392,"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":211,"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":156,"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":130,"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":112,"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":106,"is_preprint":false},{"pmid":"23630341","id":"PMC_23630341","title":"TMEM16F (Anoctamin 6), an anion channel of delayed Ca(2+) 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Wild-type TMEM16F localizes to the plasma membrane and confers Ca2+-dependent bidirectional phospholipid scrambling. A patient with Scott syndrome carries a splice-acceptor site mutation in TMEM16F causing premature protein termination, linking loss of TMEM16F to defective phospholipid scrambling.\",\n      \"method\": \"Expression cloning in Ba/F3 cells, FACS-based PtdSer exposure assay, plasma membrane localization by imaging, patient mutation sequencing\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — expression cloning with functional rescue, localization experiment, patient genetic validation; foundational paper replicated by multiple independent labs\",\n      \"pmids\": [\"21107324\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"TMEM16F forms a Ca2+-activated nonselective cation (SCAN) channel with subpicosiemens single-channel conductance that permeates both monovalent and divalent cations including Ca2+, and exhibits synergistic gating by Ca2+ and voltage. TMEM16F knockout mice lack this Ca2+-activated cation current in megakaryocytes and exhibit deficient platelet Ca2+-dependent phosphatidylserine exposure and procoagulant activity, with a bleeding defect and protection in arterial thrombosis. A residue in the putative pore region was identified as critical for cation vs. anion selectivity.\",\n      \"method\": \"TMEM16F knockout mouse generation, whole-cell and single-channel patch clamp in megakaryocytes and heterologous expression, site-directed mutagenesis of pore region, platelet PS exposure assay (annexin V), arterial thrombosis model\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — KO mouse with defined cellular phenotype, electrophysiology with mutagenesis, multiple orthogonal assays, replicated by subsequent studies\",\n      \"pmids\": [\"23021219\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Platelet-specific TMEM16F-deficient mice exhibit defects in activation-induced PtdSer exposure and microparticle shedding upon thrombin/collagen or Ca2+ ionophore activation, severely reduced thrombin generation, and impaired in vivo PtdSer exposure on aggregated platelets, while alpha-granule/dense granule release and clot retraction remain intact. TMEM16F is the only highly expressed TMEM16 scramblase in mouse platelets.\",\n      \"method\": \"Platelet-specific conditional TMEM16F knockout mice, flow cytometry (annexin V), microparticle quantification, thrombin generation assay, laser-induced thrombosis intravital imaging\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO with multiple specific phenotypic readouts, in vivo validation, replicated Scott syndrome phenotype\",\n      \"pmids\": [\"26417084\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"A specific lipid scrambling domain in ANO6 was identified by analysis of ANO1-ANO6 chimeric proteins. This domain is necessary for phospholipid scrambling activity in ANO6 and sufficient to confer scrambling function on ANO1 (which normally does not scramble). Homology modeling shows the scramblase domain forms a hydrophilic cleft facing the lipid bilayer. Ionic currents associated with ANO6 are explained by ionic leak during phospholipid translocation.\",\n      \"method\": \"Patch clamp recording combined with PtdSer scrambling assay, chimeric ANO1-ANO6 protein analysis, homology modeling\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — chimeric protein domain-swap experiment with both electrophysiology and scrambling readouts, structural modeling validation\",\n      \"pmids\": [\"26057829\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TMEM16F forms homodimers (shown by chemical cross-linking). The cytoplasmic N-terminal and C-terminal regions are essential for plasma membrane localization and protein stability, respectively, and can be exchanged between TMEM16A and TMEM16F. The pore region (between TM5 and TM6) is essential for both the Cl- channel activity of TMEM16A and the phospholipid scramblase activity of TMEM16F, as shown by point mutations.\",\n      \"method\": \"Chemical cross-linking, successive deletion analysis, domain swapping between TMEM16A and TMEM16F, pore-region point mutagenesis, Ca2+-dependent Cl- channel and scramblase assays in 293T cells and TMEM16F-/- thymocytes\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstitution-style chimera/mutagenesis with dual functional readouts (channel and scramblase), cross-linking for dimerization, single lab\",\n      \"pmids\": [\"24478309\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TMEM16F ion channel and phospholipid scramblase activities are directly coupled: four isoforms from alternative splicing show correlated scramblase and channel activities, knockdown reduces both, and an activating mutation (D409G) markedly increases Ca2+ sensitivity of both ion channel and PtdSer scrambling, including scrambling at rest.\",\n      \"method\": \"Whole-cell patch clamp, annexin V binding assay for PtdSer, siRNA knockdown, activating point mutation (D409G) introduction, four splice isoform characterization\",\n      \"journal\": \"The Journal of physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — activating mutation with dual functional readouts, multiple isoforms, gain- and loss-of-function, single lab\",\n      \"pmids\": [\"26108457\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ANO6 is required for Ca2+-dependent PtdSer scrambling in osteoblasts; deletion causes impaired Ca2+-dependent PS scrambling in primary osteoblasts, delayed mineralization in vitro, and reduced skeletal size/deformities with increased uncalcified osteoid in vivo, indicating a cell-autonomous function in bone mineralization.\",\n      \"method\": \"Ano6 knockout mice, primary osteoblast culture with PS scrambling assay, bone histology, in vivo skeletal analysis\",\n      \"journal\": \"Journal of bone and mineral research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse with cell-autonomous validation in primary cells and in vivo phenotype, PS scrambling directly measured\",\n      \"pmids\": [\"22936354\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Cryo-EM structures of murine TMEM16F in absence and presence of Ca2+ define a ligand-free closed conformation and a Ca2+-bound intermediate. Both conformations resemble their counterparts in scrambling-incompetent mTMEM16A but with distinct differences in the ion/lipid permeation region. Functional data demonstrate the relationship between ion conduction and lipid scrambling, suggesting both functions are mediated by alternate protein conformations at equilibrium in the Ca2+-bound state.\",\n      \"method\": \"Cryo-EM structure determination (two states), functional scrambling and ion conduction assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structures with functional characterization, two conformational states resolved, single lab\",\n      \"pmids\": [\"30785399\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Cryo-EM structural analysis of TMEM16F reveals coexistence of an intact channel pore and PIP2-dependent protein conformation changes leading to membrane distortion. Tightly bound lipids are slanted correlating with membrane distortion. Structure-based mutagenesis of lipid-binding residues or residues near membrane distortion sites specifically alters the onset of lipid scrambling but not Ca2+ influx, providing evidence for separate pathways for lipid scrambling and ion permeation.\",\n      \"method\": \"Cryo-EM structure determination, structure-based mutagenesis, electrophysiology, lipid scrambling assay\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM with mutagenesis distinguishing two functional readouts, single lab\",\n      \"pmids\": [\"31291589\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"An inner activation gate in the phospholipid permeation pathway of TMEM16F, formed by three hydrophobic residues F518, Y563, and I612, controls Ca2+-dependent gating. Disruption of this gate profoundly alters phospholipid permeation. Lysine substitutions of F518 and Y563 produce constitutively active Ca2+-independent scramblases. Analogous mutation L543K in TMEM16A is sufficient to confer CaPLSase activity to the Ca2+-activated Cl- channel.\",\n      \"method\": \"Site-directed mutagenesis, patch clamp electrophysiology, phospholipid scrambling assay, gain-of-function constitutively active mutants\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis with mechanistic insight validated by constitutively active phenotype and cross-paralogue transfer of function\",\n      \"pmids\": [\"31015464\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Purified monomeric/dimeric TMEM16F is a Ca2+-dependent phospholipid scramblase at the single-molecule level, transporting phospholipids nonspecifically between membrane bilayer leaflets. Thermodynamic analysis shows TMEM16F transports ~4.5 × 10^4 lipids/second at 25°C with activation free energy of 47 kJ/mol, consistent with channel-like facilitated diffusion (stepping-stone model).\",\n      \"method\": \"Purified TMEM16F protein, single-molecule scramblase assay using microarray with asymmetrically distributed phospholipids, fluorescence monitoring of translocation, thermodynamic analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution at single-molecule level with thermodynamic characterization, purified protein\",\n      \"pmids\": [\"29507235\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PIP2 regulates Ca2+-gating of TMEM16F. Brief exposure to high intracellular Ca2+ desensitizes the TMEM16F Ca2+-response associated with PIP2 depletion from the inner leaflet. Application of PIP2 restores TMEM16F channel activity. PIP2 modulation requires positively charged amino acids in the cytoplasmic N-terminal domain. PIP2 works synergistically with membrane depolarization to facilitate Ca2+-gating.\",\n      \"method\": \"Patch clamp recording of TMEM16F Ca2+-activated current, PIP2 depletion and reconstitution, mutagenesis of N-terminal positively charged residues\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — electrophysiology with pharmacological and mutagenesis validation of PIP2 binding site, multiple approaches\",\n      \"pmids\": [\"29382763\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Ca2+ directly binds TMEM16F to stabilize its structure and activate phospholipid scrambling. Comprehensive mutagenesis of acidic residues in TMEM16F identified conserved residues (homologous to those in TMEM16K) critical for Ca2+ binding; point mutations of these residues reduce Ca2+-dependent phospholipid scrambling activity.\",\n      \"method\": \"Comprehensive acidic residue mutagenesis, blue-native PAGE to detect Ca2+-dependent stable complex, phospholipid scrambling assay\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis of putative Ca2+-binding residues with direct scrambling assay readout, structural validation by native PAGE\",\n      \"pmids\": [\"27227820\"],\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. The concomitant opening of a pore promotes ion conduction in the same protein conformation, establishing an activation mechanism distinct from other TMEM16 family members.\",\n      \"method\": \"Screen of activating mutants, cryo-EM structure determination, functional ion conduction and scrambling assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM of activated mutants with functional validation, mechanistic basis of activation established\",\n      \"pmids\": [\"36335104\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Cryo-EM structures of TMEM16F with bound niclosamide or 1PBC identify a drug-binding pocket within a groove harboring a lipid trail outside the ion permeation pore, constituting a separate lipid scrambling pathway. Mutations of two residues in this groove specifically affect lipid scrambling but not ion permeation. Mutations in the drug pocket reduce inhibition of either Ca2+ influx or PS exposure differentially, providing structural evidence for separate ion and lipid permeation pathways.\",\n      \"method\": \"Cryo-EM structure determination with drug-bound states, site-directed mutagenesis, electrophysiology, PS exposure assay (annexin V/flow cytometry)\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM with bound inhibitors plus mutagenesis with two orthogonal functional readouts, single lab\",\n      \"pmids\": [\"37573365\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"High-resolution cryo-EM structures of active TMEM16F in liposomes reveal two conformations in high-activity conditions: the canonical Ca2+-bound closed state and an active state where upward rotation of the cytosolic domain creates an X-shaped groove forming a transmembrane pore and locally thins the membrane. Mutagenesis, functional assays, and MD simulations demonstrate that the X-shaped groove mediates nonselective ion flux and lipid scrambling through distinct pathways: ions move within the protein-delimited pore while lipids skirt the X-shaped groove.\",\n      \"method\": \"Cryo-EM in liposomes (native-like environment), site-directed mutagenesis, ion conduction and scrambling functional assays, molecular dynamics simulations\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM of active state in native-like lipid environment, mutagenesis, MD simulations, orthogonal functional assays in single rigorous study\",\n      \"pmids\": [\"41998358\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TMEM16F, a Ca2+-activated phospholipid scramblase, plays an essential role in placental trophoblast fusion (syncytialization) by translocating PS to the cell surface independent of apoptosis. TMEM16F knockout mice exhibit deficiency in trophoblast syncytialization and placental development leading to perinatal lethality.\",\n      \"method\": \"TMEM16F knockout mice, placental histology, PS exposure assay, syncytialization quantification\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse with specific developmental phenotype and PS scrambling mechanistic link, multiple readouts\",\n      \"pmids\": [\"32494719\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TMEM16F is required for plasma membrane repair after injury by pore-forming agents. Upon pore formation and subsequent Ca2+ influx, TMEM16F induces rapid lipid scrambling, membrane blebbing, extracellular vesicle release, preserved membrane integrity, and increased cell viability. TMEM16F-deficient mice show compromised control of Listeria monocytogenes infection associated with greater sensitivity of neutrophils to listeriolysin O.\",\n      \"method\": \"TMEM16F-deficient mice, pore-forming toxin treatment, Ca2+ imaging, membrane integrity assays, cell viability, in vivo bacterial infection model\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO mouse with in vitro and in vivo validation, mechanistic Ca2+-TMEM16F-membrane repair pathway established\",\n      \"pmids\": [\"31995754\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TMEM16F is the dominant lipid scramblase in T lymphocytes. TMEM16F is located in late endosomes where it facilitates generation of multivesicular bodies for TCR degradation and signal termination. TMEM16F deficiency results in sustained TCR signaling and augmented T cell activation, ultimately causing T cell exhaustion in chronic viral infection.\",\n      \"method\": \"TMEM16F-deficient T cells, TCR signaling assays, multivesicular body quantification, late endosome localization imaging, viral infection model (in vivo), flow cytometry for T cell activation markers\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO T cells with subcellular localization data, TCR signaling readouts, in vivo infection model, mechanistic pathway established\",\n      \"pmids\": [\"27810927\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TMEM16F is expressed in synaptic clusters facing pre-synaptic cholinergic C-boutons in α-motoneurons of the spinal cord and governs a Ca2+-activated Cl- conductance in these neurons. Conditional Tmem16f deletion decreases motor performance under high-demanding tasks by increasing recruitment threshold of fast α-motoneurons. Loss of TMEM16F in an ALS mouse model reduces activity-dependent stress markers, delays disease onset, and preserves muscular strength in male mice.\",\n      \"method\": \"Conditional Tmem16f exon deletion, whole-cell patch clamp in spinal motoneurons, immunofluorescence localization at C-boutons, motor behavior testing, ALS mouse model (male mice only)\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with electrophysiological and behavioral phenotypes, localization to specific synaptic compartment, disease model validation\",\n      \"pmids\": [\"32101737\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TMEM16F conditional ablation in microglia prevents development of mechanical hypersensitivity after nerve injury. In absence of TMEM16F, microglia display deficits in process motility and phagocytosis, and loss of GABA immunoreactivity upon injury is spared.\",\n      \"method\": \"Conditional TMEM16F knockout in microglia, nerve injury model, mechanical hypersensitivity behavioral assay, microglial process motility imaging, phagocytosis assay, GABA immunostaining\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with specific cellular and behavioral phenotypes, multiple readouts\",\n      \"pmids\": [\"27332874\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Ca2+-dependent activation of TMEM16F at the immunological synapse reduces the electrostatic potential of the plasma membrane by locally redistributing phosphatidylserine, which increases dissociation of bystander TCR-CD3 cytoplasmic domains from the plasma membrane (via reduced electrostatic interactions) and enhances TCR-dependent signaling and T cell activation.\",\n      \"method\": \"Electrostatic membrane potential measurements, PS redistribution imaging, TCR-CD3 membrane binding assays, T cell activation assays, TMEM16F-dependent signaling experiments\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic pathway established with electrostatic measurements and TCR-CD3 dissociation assays, multiple orthogonal methods\",\n      \"pmids\": [\"33758060\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TMEM16F activation by Ca2+ ionophores in Jurkat T cells triggers large-scale surface membrane expansion concurrent with phospholipid scrambling. Continued activation leads to shedding of ectosomes containing PD-1, which is selectively incorporated based on its transmembrane sequence. Cells lacking TMEM16F fail to expand surface membrane and instead undergo rapid massive endocytosis with PD-1 internalization.\",\n      \"method\": \"Microscopy and patch clamp recording in Jurkat T cells, Ca2+ ionophore treatment, ectosome isolation, PD-1 trafficking analysis, TMEM16F-deficient cells\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — combined microscopy and electrophysiology, TMEM16F-deficient comparison, single lab, functional consequence of membrane expansion established\",\n      \"pmids\": [\"30679690\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TMEM16F and dynamins cooperatively control large expansive plasma membrane reservoirs. Ca2+ activation of TMEM16F causes anionic phospholipids to escape from cytoplasmic monolayer (lipid scrambling), allowing dynamin-held invaginations to open and membrane to expand. Deletion of TMEM16F or dynamins blocks membrane expansion. Dynamin2-GFP punctae rapidly dissipate from compartments during TMEM16F activation.\",\n      \"method\": \"TMEM16F and dynamin deletion/expression, live cell microscopy, TMEM16F activation via Ca2+-permeable mechanosensitive channels, GFP-dynamin imaging, membrane expansion assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic deletion of both components, live imaging of dynamin dynamics, mechanosensitive channel-Ca2+-TMEM16F pathway defined\",\n      \"pmids\": [\"34404808\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ANO6/TMEM16F-mediated cell surface exposure of phosphatidylserine is critical for SARS-CoV-2 entry. SARS-CoV-2 Spike pseudotyped virus evokes cytosolic Ca2+ elevation and ANO6-dependent PS externalization in ACE2/TMPRSS2-positive cells. ANO6-selective inhibitor A6-001 inhibits SARS-CoV-2-induced PS scrambling and viral replication.\",\n      \"method\": \"Spike pseudovirus infection, Ca2+ imaging, annexin V PS exposure assay, high-throughput inhibitor screening, authentic SARS-CoV-2 replication assay in multiple cell lines including primary human nasal epithelial cells\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Ca2+ and PS exposure assays, ANO6-selective pharmacological inhibition, authentic virus replication validated in multiple cell systems\",\n      \"pmids\": [\"35839776\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Ca2+ influx through TRPV4 channel is critical for TMEM16F activation and subsequent human trophoblast fusion. TRPV4 and TMEM16F are functionally coupled within Ca2+ microdomains in trophoblasts. Pharmacological inhibition or gene silencing of TRPV4 hinders TMEM16F activation and trophoblast syncytialization.\",\n      \"method\": \"Patch clamp electrophysiology, TRPV4 pharmacological agonist/antagonist, siRNA knockdown, trophoblast syncytialization quantification, Ca2+ microdomain imaging\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — electrophysiology with pharmacology and genetic knockdown, functional coupling within Ca2+ microdomains established\",\n      \"pmids\": [\"35670667\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TMEM16F is the long-sought RBC Ca2+-activated phospholipid scramblase (CaPLSase), activated by Ca2+ influx through the mechanosensitive channel PIEZO1 in red blood cells. PIEZO1-TMEM16F functional coupling is enhanced in hereditary xerocytosis RBCs carrying PIEZO1 gain-of-function mutations, leading to increased PS exposure propensity linked to HX clinical manifestations. PIEZO1 inhibition with GsMTx-4 or benzbromarone prevents force-induced PS exposure and hemolysis in HX RBCs.\",\n      \"method\": \"RBC patch clamp, Ca2+ imaging, annexin V PS exposure assay, PIEZO1 pharmacological inhibitors, primary RBCs from HX patients with PIEZO1 gain-of-function mutations, hemolysis assays, TMEM16F identification in RBCs\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — patient RBCs with gain-of-function PIEZO1 mutations, pharmacological PIEZO1 inhibition, multiple orthogonal assays establishing PIEZO1-TMEM16F functional coupling\",\n      \"pmids\": [\"38033286\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TMEM16F lipid scrambling and ion channel activities are strongly regulated by intracellular pH (pHi). Low pHi attenuates and high pHi potentiates TMEM16F channel and scramblase activation. pHi sensitivity depends on [Ca2+]i and shows a bell-shaped relationship. Mutation of a Ca2+-binding residue (E667Q) dramatically shifts the pHi-sensitivity peak, indicating that protons and Ca2+ compete for the primary Ca2+-binding residues in the pore, providing the molecular basis of pHi regulation.\",\n      \"method\": \"Whole-cell patch clamp, phospholipid scrambling assay, intracellular pH manipulation, Ca2+-binding site mutagenesis (E667Q), biophysical characterization\",\n      \"journal\": \"The Journal of general physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis of Ca2+-binding site with dual functional readouts, competition mechanism established, single lab\",\n      \"pmids\": [\"33346788\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ANO6 Cl- currents can be activated by increase in cytosolic Ca2+, or Ca2+-independently by Fas receptor stimulation. Ca2+-dependent PS scrambling induced by ANO6 does not require Cl- currents. Ca2+-independent PS scrambling due to extrinsic (FasL) or intrinsic (ABT-737) apoptosis does not require ANO6. ANO6 is necessary for Ca2+-dependent PS scrambling but not by increasing intracellular Ca2+.\",\n      \"method\": \"Whole-cell patch clamp in normal and Scott syndrome B-lymphocytes, ionomycin and FasL stimulation, Cl- channel blockers, siRNA knockdown, ABT-737 apoptosis induction, annexin V PS assay\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — patient Scott syndrome cells as natural KO, pharmacological and siRNA approaches, multiple stimuli distinguishing Ca2+-dependent vs independent pathways\",\n      \"pmids\": [\"23618909\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TMEM16F Cl- currents and phospholipid scrambling can be activated by modification of plasma membrane phospholipids through reactive oxygen species and phospholipase A2 (PLA2), independent of intracellular Ca2+. Mutations within TMEM16F or TMEM16A/F chimeras similarly change both Cl- currents and phospholipid scrambling, suggesting the same intramolecular pathway for Cl- and phospholipids. Only TMEM16F (not TMEM16A) can expose PtdSer to the outer membrane leaflet.\",\n      \"method\": \"Overexpression of TMEM16A and TMEM16F, PLA2 activation/inhibition, ROS induction, PtdSer exposure (annexin V), whole-cell patch clamp, TMEM16A/F chimera mutagenesis\",\n      \"journal\": \"The Journal of physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — chimera mutagenesis with dual functional readouts, pharmacological manipulation, single lab\",\n      \"pmids\": [\"29134661\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The actin cytoskeleton negatively regulates ANO6/TMEM16F anion channel activity: cytochalasin-D disruption accelerates activation kinetics, while phalloidin/jasplakinolide inhibit ANO6 currents. Intracellular MgATP decelerates activation of whole-cell ANO6 currents and prevents inactivation. Inside-out patches show immediate Ca2+-dependent activation, indicating that cytosolic factors (including actin and ATP) modulate the delay and inactivation of ANO6.\",\n      \"method\": \"Whole-cell and inside-out patch clamp, cytochalasin-D, phalloidin, jasplakinolide actin manipulation, MgATP concentration variation\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological manipulation of actin with electrophysiological readout, inside-out vs whole-cell comparison, single lab\",\n      \"pmids\": [\"29964013\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TMEM16F undergoes a dynamic change in ion selectivity in response to changes in intracellular Ca2+ concentration: at higher Ca2+ levels, Cl- permeability relative to Na+ (PCl-/PNa+) increases. This dynamic selectivity shift is independent of channel activation state and is indicative of a charge-screening mechanism in the permeation pathway.\",\n      \"method\": \"Excised inside-out patch clamp with Q559K mutant (no current rundown), systematic variation of intracellular Ca2+ concentration, ion substitution experiments\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — electrophysiology with defined mutant lacking rundown, systematic Ca2+ concentration series, single lab\",\n      \"pmids\": [\"31318330\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Human TMEM16F expressed in HEK293T cells is an essential component of a Ca2+-activated Cl- channel with strong outward rectification. Cl- is the permeant ion with anion selectivity sequence I- > Br- > Cl- > F- > aspartate-. Ca2+ concentration for half-maximal activation is 9.6 μM, distinctly higher than for TMEM16A/B. TMEM16F is not related to volume-sensitive outwardly rectifying Cl- channel (VSOR) activity.\",\n      \"method\": \"Whole-cell patch clamp in HEK293T cells, ion substitution, niflumic acid pharmacology, TMEM16F overexpression and knockdown\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — electrophysiology with ion substitution and pharmacology, negative result (no VSOR role) explicitly established, single lab\",\n      \"pmids\": [\"23426967\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Mouse TMEM16F expressed in HEK293 cells generates an outwardly rectifying Ca2+-activated Cl- current activated with a delay of several minutes. A significant Na+ current is also activated with PNa = 0.3 PCl. EC50 is ~100 μM intracellular Ca2+. Pore region mutant R592E abolishes the current; K616E reduces relative iodide permeability; R636E alters anion selectivity, establishing the pore region as functionally critical.\",\n      \"method\": \"Whole-cell patch clamp in HEK293 cells, site-directed mutagenesis of pore region residues (R592E, K616E, R636E), ion selectivity measurements\",\n      \"journal\": \"The Journal of general physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — pore mutagenesis with electrophysiology, multiple mutants with distinct phenotypes, single lab\",\n      \"pmids\": [\"23630341\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TMEM16F activation by Ca2+ triggers large-scale surface membrane expansion in Jurkat T cells. Cells lacking TMEM16F undergo rapid massive endocytosis with PD-1 internalization instead of expansion. The T cell co-receptor PD-1 is selectively incorporated into TMEM16F-dependent ectosomes based on its transmembrane sequence.\",\n      \"method\": \"Confocal microscopy, patch clamp, Ca2+ ionophore activation, TMEM16F-deficient cell comparison, ectosome characterization, PD-1 transmembrane domain mutants\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live imaging and electrophysiology combined with TMEM16F-deficient cells, transmembrane sequence dependency established, single lab\",\n      \"pmids\": [\"30679690\"],\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 (N-terminal cleavage product of gasdermin D) expression enhances basal Ca2+ levels and induces TMEM16F-dependent large whole-cell currents and cell death in HEK293 and HAP1 cells. Knockdown or inhibition of TMEM16F suppresses GD-N-induced whole-cell currents.\",\n      \"method\": \"GD-N expression in HEK293 and HAP1 cells, whole-cell patch clamp, TMEM16F knockdown (siRNA), TMEM16F inhibitors, Ca2+ imaging, cell death assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockdown with electrophysiological and cell death readouts, endogenous TMEM16F required, single lab\",\n      \"pmids\": [\"29463790\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TMEM16F is activated during ferroptosis induced by erastin or RSL3 (inhibitor of GPX4). Cell death was largely reduced in intestinal epithelium and peritoneal macrophages from tissue-specific TMEM16F knockout mice. Ferrostatin-1 and TMEM16F inhibitors block both TMEM16F currents and ferroptotic cell death.\",\n      \"method\": \"Tissue-specific TMEM16F knockout mice, erastin/RSL3-induced ferroptosis, whole-cell patch clamp, cell viability assays, ferrostatin-1 inhibition\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — tissue-specific KO mouse with electrophysiological and cell death readouts, pharmacological validation, single lab\",\n      \"pmids\": [\"31060306\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CFTR enhances ANO6 activity, probably through translocation of signaling proteins to the plasma membrane. ANO6 produces outwardly rectifying Cl- currents and scrambles plasma membrane phospholipids when activated by increased cytosolic ROS and consecutive peroxidation of plasma membrane lipids. In ANO6 knockout mice, apoptotic cells in the intestinal epithelium are strongly reduced, supporting ANO6's role in cell death cooperatively with CFTR.\",\n      \"method\": \"ANO6 knockout mice, ROS donor treatment, whole-cell patch clamp, PS exposure assay, TUNEL staining of intestinal epithelium, CFTR and ANO6 co-expression in airway cells\",\n      \"journal\": \"Pflugers Archiv : European journal of physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse with in vivo cell death readout, electrophysiology and PS assay in vitro, single lab\",\n      \"pmids\": [\"28875346\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ANO6 functions as a Ca2+-activated Cl- channel in mouse dendritic cells (DCs) as demonstrated by siRNA knockdown. ANO6 is activated by chemokine receptor CCR7 ligation with CCL21. Knockdown of ANO6 reduces chemokine-induced migration of both immature and LPS-matured DCs.\",\n      \"method\": \"Whole-cell patch clamp in mouse bone marrow-derived DCs, siRNA knockdown of ANO6, RT-PCR and Western blot, CCL21 stimulation, migration assay\",\n      \"journal\": \"Cellular physiology and biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA knockdown with electrophysiological and migration readouts, chemokine receptor activation demonstrated, single lab\",\n      \"pmids\": [\"23159814\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TMEM16F mediates ER stress/calcium-induced PS externalization in tumor cells, which suppresses antitumor immunity. TMEM16F-KO tumor cells (CRISPR/Cas9) did not suppress tumorigenicity in immune-deficient mice (NOD/SCID or RAG-KO) but did in immune-competent mice, demonstrating that TMEM16F-dependent PS exposure promotes immune evasion through a T cell-dependent mechanism. TMEM16F-KO specifically abrogated ER stress/calcium-induced PS externalization without affecting cell-intrinsic proliferation.\",\n      \"method\": \"CRISPR/Cas9 knockout in EO771 breast cancer cells, orthotopic transplantation in immune-competent and immune-deficient mice, PS exposure assay, ER stress induction\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO with in vivo immune-competent vs immune-deficient comparison, mechanistic distinction from apoptotic PS exposure, single lab\",\n      \"pmids\": [\"41198619\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TMEM16F deficiency in endothelial cells (ECKO mice) causes prolonged tail bleeding and significantly smaller IVC thrombi, demonstrating that endothelial TMEM16F function is essential for normal hemostasis. Niclosamide (PLS inhibitor) prevents pathological Ca2+ signals and PS translocation in endothelial cells exposed to extracellular histones.\",\n      \"method\": \"Tamoxifen-inducible endothelial-specific Cdh5-Cre TMEM16F knockout mice, tail bleeding time, IVC stenosis thrombosis model, live-cell Ca2+ imaging, annexin V PS exposure flow cytometry, niclosamide pharmacology\",\n      \"journal\": \"Shock (Augusta, Ga.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-type-specific KO mouse with multiple hemostasis readouts, pharmacological validation, single lab\",\n      \"pmids\": [\"39874534\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TMEM16F deficiency in neurons (but not microglia) reduces tauopathy and microgliosis in PS19 (P301S tau) mice. TMEM16F mediates aberrant phosphatidylserine exposure in neurons burdened with phospho-tau, linking neuronal TMEM16F-mediated PS scrambling to propagation of tau pathology.\",\n      \"method\": \"Neuron-specific and microglia-specific TMEM16F conditional knockout in PS19 tau mice, tauopathy quantification (AT8 staining), microgliosis assessment, PS exposure assay in neurons\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-type-specific KO in disease model, PS exposure mechanistically linked to tau pathology spread, single lab\",\n      \"pmids\": [\"38941274\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TMEM16F deficiency impairs developmental retinal angiogenesis. TMEM16F knockdown enhances plasma membrane association of activated Src kinase, increases VE-cadherin phosphorylation and downregulation, and suppresses angiogenesis. This establishes an intracellular signaling function for TMEM16F in endothelial cells, separate from its canonical lipid scrambling role.\",\n      \"method\": \"Endothelial TMEM16F conditional KO mice (retinal angiogenesis), HUVEC siRNA knockdown, Src kinase phosphorylation assay (pY416), VE-cadherin expression and phosphorylation, in vitro angiogenesis assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse plus siRNA with biochemical pathway dissection, in vitro and in vivo angiogenesis readouts, single lab\",\n      \"pmids\": [\"38940198\"],\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, contradicting the prevailing view of non-selective scrambling. This phospholipid headgroup preference was established using a fluorescence polarization-based cell scrambling assay with NBD-labeled phospholipids.\",\n      \"method\": \"Fluorescence polarization (FP) assay with NBD-labeled phospholipids for kinetic monitoring of scrambling on plasma membrane of living cells, TMEM16F-expressing vs control cells\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — novel cell-based assay with multiple phospholipid species side-by-side, single lab, challenges prior consensus\",\n      \"pmids\": [\"41166415\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TMEM16F-mediated PS scrambling polarizes macrophages to an immunosuppressive M2 phenotype, promoting TGF-β1 secretion and regulatory T cell expansion that suppresses cytotoxic lymphocytes. Genetic ablation of TMEM16F abolished PS exposure, reprogrammed the tumor microenvironment toward immune activation, and suppressed tumor growth across cancer models.\",\n      \"method\": \"TMEM16F genetic ablation across cancer models, macrophage polarization assays (M1/M2 markers), TGF-β1 ELISA, regulatory T cell quantification, cytotoxic T cell activity, tumor growth measurement\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic ablation with multiple immunological readouts, pharmacological scramblase inhibition validation, single lab\",\n      \"pmids\": [\"41100671\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TMEM16F regulates pathological α-synuclein (α-synA53T) spread in neurons. TMEM16F-knockout neurons show reduced spread of pathological α-synA53T in vitro and in vivo in a PD mouse model. A missense mutation Ala703Ser identified in Ashkenazi Jewish PD patients confers enhanced lipid scramblase activity and is associated with altered regulation of α-synA53T extracellular secretion in cellular models.\",\n      \"method\": \"TMEM16F knockout neurons with reporter system for donor/recipient α-syn spread, in vivo PD mouse model (α-synA53T injection), SNP identification in patient cohort, lipid scramblase activity assay, cellular secretion assays\",\n      \"journal\": \"Aging cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO neurons in vitro and in vivo plus human variant with enhanced scramblase activity, multiple readouts, single lab\",\n      \"pmids\": [\"39487963\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Lipid scrambling via TMEM16F is sufficient to induce extracellular vesicle formation and release without changes in cytosolic calcium or cytoskeleton. Scrambling causes segregation of exofacial lipids, redistribution of cholesterol to inner leaflet, clustering of GPI-linked proteins creating convex curvature, and PE accumulation creating concave curvature that facilitates vesicle scission.\",\n      \"method\": \"Inducible active TMEM16F expression, vesicle isolation and quantification, lipid distribution assays, cholesterol and GPI-protein imaging, PE distribution imaging\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — inducible active scramblase with multiple lipid distribution and vesicle biogenesis readouts, mechanistic model established, single lab\",\n      \"pmids\": [\"41604453\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The Ca2+ sensitivity of ANO6 is partially regulated by a putative Ca2+-binding reservoir at the N-terminal domain (Nt-CaRes). An ANO6-1-6 chimera with Nt-CaRes replaced by the corresponding ANO1 domain showed higher Ca2+ sensitivity. Mutations of Ca2+-interacting acidic residues in ANO6 Nt-CaRes resulted in reduced Ca2+ sensitivity, indicating direct Ca2+ interactions at this reservoir contribute to differential Ca2+ sensitivity between ANO1 and ANO6.\",\n      \"method\": \"Chimera construction (ANO6-1-6), N-terminal domain point mutagenesis, whole-cell patch clamp Ca2+ sensitivity measurement, molecular dynamics simulation of Ca2+ interactions\",\n      \"journal\": \"Molecules and cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — chimera and mutagenesis with electrophysiological readout, MD simulation support, single lab\",\n      \"pmids\": [\"33658434\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ANO6 expressed in cancer-associated fibroblasts (CAFs) is required for trogocytosis, a process by which PDAC cells acquire lipids (including cholesterol) from CAF plasma membranes. During trogocytosis, cancer cell-CAF synapse-like contacts induce cytosolic Ca2+ influx in CAFs via Orai channels, activating ANO6 and causing PS exposure on CAF plasma membranes that initiates lipid transfer to PDAC cells. ANO6-dependent trogocytosis also supports immunosuppressive function of CAFs toward cytotoxic T cells by enabling excess cholesterol transfer.\",\n      \"method\": \"CAF-PDAC co-culture trogocytosis assays, ANO6 knockdown/inhibition, Ca2+ imaging, PS exposure assay, lipid (cholesterol) transfer quantification, T cell cytotoxicity assays, Orai channel pharmacology\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic pathway established with Ca2+ imaging, PS assay, and lipid transfer readouts; preprint status\",\n      \"pmids\": [\"37745612\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"ANO6/TMEM16F is a Ca2+-activated plasma membrane protein that functions as both a phospholipid scramblase and a non-selective ion channel: upon elevation of intracellular Ca2+ (binding directly to conserved acidic residues in the transmembrane domain), TMEM16F undergoes conformational changes—including formation of an X-shaped groove/hydrophilic pore—that allow bidirectional, facilitated diffusion of phospholipids (with preference for PS and PC over PE) across the bilayer at ~4.5×10^4 lipids/second, while simultaneously conducting non-selective ion currents through a distinct but overlapping pathway; this Ca2+-dependent scrambling activity is further regulated by PIP2, intracellular pH, the actin cytoskeleton, and upstream Ca2+-influx channels (PIEZO1, TRPV4), and underlies diverse cellular functions including platelet procoagulant activity and blood coagulation, bone mineralization by osteoblasts, trophoblast syncytialization, plasma membrane repair after pore-forming toxin injury, TCR signal termination via late-endosomal multivesicular body formation, microglial phagocytosis, ectosome/extracellular vesicle release, and viral (SARS-CoV-2) entry facilitation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ANO6/TMEM16F is a Ca2+-activated, plasma-membrane phospholipid scramblase that couples elevation of intracellular Ca2+ to bidirectional translocation of phospholipids—exposing phosphatidylserine on the cell surface—and to non-selective ion conduction, thereby driving membrane-remodeling events across many cell types [#0, #1, #10]. Ca2+ binds directly to conserved acidic residues in the transmembrane domain to stabilize the protein and activate scrambling, with an N-terminal Ca2+ reservoir tuning sensitivity [#12, #47], while protons compete at these same sites to give intracellular pH-dependent regulation [#27]; activity is further modulated by PIP2 at a cytoplasmic basic motif and by the actin cytoskeleton [#11, #30]. As a single-molecule scramblase the purified protein moves ~4.5×10^4 lipids/second by channel-like facilitated diffusion with headgroup preference for PS and PC over PE [#10, #43]. Cryo-EM and mutagenesis define an inner hydrophobic gate (F518/Y563/I612) and an active conformation in which upward rotation of the cytosolic domain creates an X-shaped groove that thins the membrane, establishing that lipids skirt this groove while ions pass through an adjacent protein-delimited pore as distinct but overlapping permeation pathways [#9, #14, #15]. Upstream Ca2+-influx channels including PIEZO1 and TRPV4 are functionally coupled to TMEM16F within Ca2+ microdomains to gate its activation [#25, #26]. Through Ca2+-dependent PS exposure and membrane expansion TMEM16F mediates platelet procoagulant activity and blood coagulation [#1, #2], osteoblast bone mineralization [#6], trophoblast syncytialization [#16, #25], plasma membrane repair after pore-forming toxin injury [#17], and ectosome/extracellular-vesicle release [#46]; in T cells it terminates TCR signaling via late-endosomal multivesicular bodies while at the synapse it can also tune the membrane electrostatic potential that controls TCR-CD3 engagement [#18, #21]. Loss-of-function mutation of TMEM16F underlies the human bleeding disorder Scott syndrome through defective phospholipid scrambling [#0].\",\n  \"teleology\": [\n    {\n      \"year\": 2010,\n      \"claim\": \"Establishing the molecular identity of the long-sought Ca2+-dependent phospholipid scramblase resolved which protein exposes PS on activated cells and tied it to human disease.\",\n      \"evidence\": \"expression cloning in Ba/F3 cells, FACS PS-exposure assay, and Scott syndrome patient mutation sequencing\",\n      \"pmids\": [\"21107324\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"mechanism of lipid translocation undefined\", \"relationship to ion conduction unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defining TMEM16F as a Ca2+-activated nonselective cation channel and demonstrating a platelet bleeding/thrombosis phenotype in knockout mice linked the protein's electrical activity to procoagulant PS exposure in vivo.\",\n      \"evidence\": \"knockout mouse, patch clamp in megakaryocytes and heterologous cells, pore mutagenesis, platelet PS assays, arterial thrombosis model\",\n      \"pmids\": [\"23021219\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"whether channel and scramblase share one pathway unresolved\", \"direct Ca2+-binding site not yet mapped\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Platelet-specific and human Scott-syndrome studies confirmed TMEM16F is the dominant platelet scramblase required for procoagulant PS exposure and microparticle shedding while separating Ca2+-dependent from apoptotic PS scrambling.\",\n      \"evidence\": \"platelet-conditional knockout mice with thrombin generation and intravital imaging; Scott syndrome B-lymphocyte patch clamp with FasL/ABT-737 stimuli\",\n      \"pmids\": [\"26417084\", \"23618909\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"does not define the structural basis of scrambling\", \"Ca2+-independent apoptotic scramblase identity unaddressed\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Heterologous electrophysiology characterized TMEM16F's outwardly rectifying Ca2+-activated Cl- conductance, its low Ca2+ sensitivity relative to TMEM16A/B, and the pore residues governing ion selectivity.\",\n      \"evidence\": \"whole-cell patch clamp in HEK cells, ion substitution, pharmacology, pore-region mutagenesis (R592E, K616E, R636E)\",\n      \"pmids\": [\"23426967\", \"23630341\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"overexpression may not reflect native conductance\", \"structural pore not resolved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Domain-swap and mutagenesis work localized a discrete scramblase domain and the shared pore region, and demonstrated that channel and scramblase activities are coupled and co-regulated by a single activating mutation.\",\n      \"evidence\": \"ANO1-ANO6 chimeras, pore-region point mutagenesis, dimerization cross-linking, splice-isoform and D409G activating-mutant analysis with dual scrambling/channel readouts\",\n      \"pmids\": [\"26057829\", \"24478309\", \"26108457\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"whether ions and lipids use identical or merely overlapping paths unresolved\", \"no atomic structure yet\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identifying the conserved acidic Ca2+-binding residues established how Ca2+ directly stabilizes and activates the scramblase.\",\n      \"evidence\": \"comprehensive acidic-residue mutagenesis, blue-native PAGE for Ca2+-dependent complex, scrambling assay\",\n      \"pmids\": [\"27227820\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"conformational consequences of Ca2+ binding not visualized\", \"stoichiometry of Ca2+ binding not defined here\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Single-molecule reconstitution with purified protein and the PIP2-regulation study quantified scrambling as channel-like facilitated lipid diffusion and identified an inner-leaflet lipid cofactor controlling Ca2+ gating.\",\n      \"evidence\": \"purified TMEM16F single-molecule microarray scramblase assay with thermodynamic analysis; patch clamp with PIP2 depletion/reconstitution and N-terminal basic-residue mutagenesis\",\n      \"pmids\": [\"29507235\", \"29382763\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"physiological PIP2 dynamics during native activation unclear\", \"lipid selectivity not addressed at single-molecule level\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Cryo-EM of closed and Ca2+-bound states plus gate and lipid-binding mutagenesis defined the activation conformations and provided structural evidence that lipid scrambling and ion conduction proceed through separate but adjacent pathways.\",\n      \"evidence\": \"cryo-EM of two conformational states, structure-based mutagenesis of an inner gate (F518/Y563/I612) and lipid/membrane-distortion residues, paired ion and scrambling assays\",\n      \"pmids\": [\"30785399\", \"31291589\", \"31015464\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"fully active scrambling-competent conformation not yet captured\", \"dynamics of gating not directly observed\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Cryo-EM of activating mutants captured the rearrangement exposing hydrophilic membrane patches and concomitant pore opening, defining a TMEM16F-specific activation mechanism.\",\n      \"evidence\": \"activating-mutant screen, cryo-EM, functional ion and scrambling assays\",\n      \"pmids\": [\"36335104\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"mutant conformation may differ from wild-type active state\", \"lipid trajectory not directly imaged\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Drug-bound cryo-EM structures localized a scrambling groove with a lipid trail outside the ion pore, giving structural proof of separable lipid and ion permeation pathways and a druggable pocket.\",\n      \"evidence\": \"cryo-EM with niclosamide/1PBC, groove and pocket mutagenesis, electrophysiology and PS exposure assays\",\n      \"pmids\": [\"37573365\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"inhibitor selectivity across paralogues not fully defined\", \"lipid occupancy in the groove inferred not directly resolved during transport\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"High-resolution cryo-EM of active TMEM16F in liposomes resolved an X-shaped groove that forms a transmembrane pore and thins the membrane, demonstrating ions pass through the protein pore while lipids skirt the groove.\",\n      \"evidence\": \"cryo-EM in native-like liposomes, mutagenesis, ion/scrambling assays, molecular dynamics simulations\",\n      \"pmids\": [\"41998358\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"temporal order of pore opening versus membrane thinning not established\", \"lipid headgroup preference mechanism not structurally explained\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identifying upstream Ca2+ sources (PIEZO1 in red cells, TRPV4 in trophoblasts, Orai in fibroblasts) and biophysical regulators (pH competition, ion-selectivity shifts, actin) showed how TMEM16F is gated within physiological Ca2+ microdomains.\",\n      \"evidence\": \"RBC and trophoblast patch clamp with channel inhibition/knockdown, Ca2+-binding-site pH-competition mutagenesis (E667Q), inside-out actin pharmacology, dynamic-selectivity recordings\",\n      \"pmids\": [\"38033286\", \"35670667\", \"33346788\", \"31318330\", \"29964013\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"molecular tethering of TMEM16F to upstream channels unknown\", \"spatial organization of microdomains not resolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Cell-type-specific knockouts mapped TMEM16F PS-scrambling and membrane-expansion functions onto diverse physiological and pathological processes spanning development, immunity, repair, vesicle release, and disease.\",\n      \"evidence\": \"conditional/tissue-specific knockouts and CRISPR ablation across osteoblasts, trophoblasts, T cells, microglia, motoneurons, endothelium, tumor cells, and neurons; membrane-repair, ectosome, EV-release, and SARS-CoV-2 entry assays\",\n      \"pmids\": [\"22936354\", \"32494719\", \"31995754\", \"27810927\", \"33758060\", \"30679690\", \"34404808\", \"35839776\", \"32101737\", \"27332874\", \"39874534\", \"38941274\", \"41198619\", \"41100671\", \"39487963\", \"46\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"many roles share the single PS-scrambling mechanism but downstream effectors differ by cell type\", \"non-canonical signaling roles (e.g. endothelial Src/VE-cadherin) lack defined molecular linkage\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How TMEM16F achieves headgroup preference for PS and PC and how a single conformational change orchestrates the temporal coordination of ion conduction, lipid scrambling, and membrane curvature in living cells remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"structural basis of PS/PC over PE selectivity not defined\", \"in vivo coupling of scrambling to vesicle scission and curvature generation incompletely mapped\", \"proposed non-scramblase signaling functions mechanistically unexplained\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 3, 10, 43]},\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [1, 32, 33]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [10, 11, 46]},\n      {\"term_id\": \"GO:0140299\", \"supporting_discovery_ids\": [12, 27]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 4, 29]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [18]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-109582\", \"supporting_discovery_ids\": [1, 2, 40]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [18, 21, 44]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [35, 36, 37]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [22, 46]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [21, 42]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"PIEZO1\", \"TRPV4\", \"DNM2\", \"CFTR\", \"ORAI1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}