Lipid flip-flop in biological membranes: through the lens of TMEM16F

Ke Z Shan; Augustus J Lowry; Maria A González Torres; Megha S Seri; Huanghe Yang

doi:10.1152/physrev.00046.2025

. Author manuscript; available in PMC: 2026 Sep 29.

Published before final editing as: Physiol Rev. 2026 Sep 10:10.1152/physrev.00046.2025. doi: 10.1152/physrev.00046.2025

Lipid flip-flop in biological membranes: through the lens of TMEM16F

Ke Z Shan ¹, Augustus J Lowry ¹, Maria A González Torres ¹, Megha S Seri ¹, Huanghe Yang ^1,^2,^3,^4,^5,^*

PMCID: PMC13619121 NIHMSID: NIHMS2210829 PMID: 42721955

Abstract

The asymmetric distribution of phospholipids between membrane leaflets is a hallmark of all known cells, yet many fundamental questions about membrane lipid asymmetry remain unanswered. Why do cells invest energy to establish and maintain this thermodynamically unstable state? How do cells exploit lipid asymmetry to support essential functions? What are the consequences for health and disease when this organization breaks down? Recent identification of bona fide lipid scramblases, which catalyze rapid phospholipid translocation across the hydrophobic membrane core (lipid flip-flop), has shed new light on these longstanding questions. In particular, studies of the Ca²⁺-activated phospholipid scramblase TMEM16F have revealed how regulated lipid scrambling can couple Ca²⁺ signaling to membrane remodeling, phosphatidylserine (PS) exposure, cellular communication, and disease pathogenesis. In this review, we examine the biology of regulated transmembrane phospholipid flip-flop mediated by lipid scramblases, using TMEM16F as a prototypical model to illustrate core molecular mechanisms, physiological functions, pathological implications, and emerging pharmacological opportunities. By placing TMEM16F within the broader landscape of lipid scramblases and scramblase-like proteins, we highlight how regulated membrane lipid scrambling shapes cell physiology and disease. We also discuss critical knowledge gaps and future directions for understanding this rapidly evolving field.

Graphical Abstract

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1. INTRODUCTION

Self-assembly of amphiphilic molecules into membrane-bound compartments was a critical step in providing physical boundaries for protocells and enabling the evolution of cellular life (1–3). As phospholipids started to dominate the cellular membranes during evolution (4), the hydrophobic core of the phospholipid bilayer created an effective barrier to polar, charged, or bulky solutes and severely limited the spontaneous flip-flop or translocation of phospholipids between leaflets (5–7). Cells thus invest substantial metabolic energy in ATP-dependent phospholipid transporters, flippases and floppases, to establish and regulate an asymmetric distribution of phospholipids across the plasma membrane (8, 9) (Figure 1 and Table 1). Once established, this lipid asymmetry can be rapidly dissipated by passive, energy-independent phospholipid scramblases (PLSases), which catalyze bidirectional, largely nonselective transbilayer movement of multiple phospholipid species (6, 9).

FIGURE 1. — A: Phospholipids are asymmetrically distributed across the plasma membrane bilayer. Illustration showing the asymmetric distribution of major phospholipids in the plasma membrane. Sphingomyelin (SM) and phosphatidylcholine (PC) are enriched in the exofacial/outer leaflet, while phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidic acid (PA) are predominantly localized in the cytosolic/inner leaflet. The numbers were based on the study by Lorent et al (10). B: Phospholipid asymmetry is maintained by ATP-dependent flippases and floppases, which selectively pump phospholipids inward (PS/PE) or outward (PC/SM), respectively. In contrast, ATP-independent scramblases mediate the rapid, non-selective, and bidirectional redistribution of phospholipids, leading to the collapse of phospholipid asymmetry. C: Lipid flippases/floppases and scramblases work in concert to dynamically control membrane lipid asymmetry. Figure schematics were created with BioRender.com.

Table 1.

Major classes of lipid transport systems regulating membrane phospholipid asymmetry

Feature	Flippases	Floppases	Scramblases
Direction	Outer → Inner	Inner → Outer	Bidirectional
Energy	ATP-dependent	ATP-dependent	ATP-independent
Main family	P4-ATPases	ABC transporters	TMEM16/XKR
Representative proteins	ATP11C, ATP8A2	ABCA1, ABCB4	TMEM16F, XKR8
Major substrates	PS, PE	PC, cholesterol	Broad/nonselective
Functional outcome	Maintain asymmetry	Maintain asymmetry	Dissipate asymmetry

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Recognition of membrane lipid asymmetry and the transport mechanisms that dynamically regulate it emerged through a series of landmark discoveries (Figure 2). Following Bretscher’s groundbreaking proposal of membrane phospholipid asymmetry in 1972, experimental studies in the early 1970s established that phospholipids are unequally distributed between the two leaflets of the plasma membrane, with phosphatidylserine (PS) and most phosphatidylethanolamine (PE) confined to the cytoplasmic leaflet (11, 12). The discovery of ATP-dependent aminophospholipid translocation in the 1980s and ABC transporter-mediated phospholipid export in the 1990s subsequently established the opposing flippase and floppase activities that generate and maintain this asymmetry (13–23). In parallel, studies of activated platelets and erythrocytes, apoptotic cells, and patients with Scott syndrome revealed that membrane lipid asymmetry can be rapidly and physiologically disrupted by phospholipid scrambling, although the molecular identity of the responsible Ca²⁺-activated phospholipid scramblase (CaPLSase) remained elusive for decades (24–32).

FIGURE 2. — Figure schematics were created with BioRender.com.

The seminal identification of TMEM16F in 2010 as a bona fide CaPLSase marked a key milestone in understanding regulated membrane lipid flip-flop (33) (Figure 2). Subsequent work has uncovered additional scramblases, including other TMEM16 family members, XKR proteins (34, 35), and other unrelated proteins (36–40) (Table 2), greatly expanding the repertoire of known PLSases and transforming our view of lipid flip-flop-mediated biology. This review focuses on TMEM16F as a prototype PLSase, summarizing advances over the past 15 years in its structure, function, activation mechanisms, and physiological and pathological roles. Rather than providing an exhaustive survey of all phospholipid scramblases, our goal is to use TMEM16F as a lens to illustrate how dynamically regulated loss of lipid asymmetry underlies diverse yet still only partially understood biological processes. Given the rapid expansion of this field and space constraints, we inevitably could not cite all relevant contributions, and any omissions are unintentional. Interested readers are encouraged to refer to recent comprehensive reviews for broader coverage of the PLSase field (6, 9, 41–62).

Table 2.

Established and candidate lipid scramblases

Protein(s)	Primary function/class	Scramblase evidence status	Expression	Subcellular Localization	Activation / regulation	Key physiological functions	Disease relevance	References
TMEM16 family with established Ca ²⁺ -activated scramblases								(46, 51, 52, 79–86)
TMEM16F /ANO6	Ca²⁺-activated phospholipid scramblase (CaPLSase) with Ca²⁺-activated non-selective ion channel activity	Established	Broadly expressed; high functional relevance in platelets, RBCs, immune cells, endothelial cells, osteoblasts, trophoblasts and other cell types	Mainly plasma membrane	Activated by cytosolic Ca²⁺; channel activity is modulated by voltage	PS exposure; procoagulant platelet/RBC activity; microparticle and extracellular vesicle release; membrane repair; cell fusion; immune and vascular signaling	LOF variant: Scott syndrome. GOF variant: Parkinson’s disease. Dysregulated activity implicated in thrombosis, SCD/HX RBC PS exposure, stroke, inflammation, pain, cancer and neurodegeneration	(33, 87–91)
TMEM16A/ANO1	Ca²⁺-activated Cl⁻ channel (CaCC)	Wildtype (WT) is not a PLSase; engineered mutants can confer scrambling	Broadly expressed, including epithelia, smooth muscle, sensory neurons and secretory tissues	Plasma membrane	Activated by cytosolic Ca²⁺; regulated by voltage, lipids, calmodulin	Transepithelial ion transport; smooth muscle contraction; secretion; nociception; sensory processing; membrane excitability	GOF variants reported in moyamoya disease. Aberrant expression/activity implicated in asthma, hypertension, pain, cancer and secretory disorders	(85, 92–107)
TMEM16B/ANO2	CaCC	WT is not a PLSase. Mutations: N.D.	Predominantly neuronal tissue	Plasma membrane	Activated by cytosolic Ca²⁺; regulated by voltage	Olfaction, phototransduction, sensory processing, motor learning; emotional control	Disease causality less developed; deletion involving TMEM16B and VWF in type 3 von Willebrand disease (VWD); autoimmune antibody implicated in multiple sclerosis	(94, 101, 108–122)
TMEM16C/ANO3	SLACK (KNa) -channel regulatory subunit; Putative CaPLSase;	Limited / developing	Predominantly neuronal tissue, including DRG & CNS	Intracellular membranes and plasma membrane depending on isoform/trafficking	Regulates SLACK (KNa) channel in a Ca²⁺-linked manner; putative CaPLSase activated by cytosolic Ca²⁺	Pain processing, neuronal excitability and thermoregulation/seizure susceptibility in rodent models	Autosomal-dominant ANO3 variants cause craniocervical dystonia (DYT24); possible link to AD	(123–128)
TMEM16D/ANO4	non-selective cation channel; Putative CaPLSase	Limited / controversial	Enriched in CNS neurons	Intracellular membranes and/or plasma membrane	Ca²⁺-linked channel activity; scrambling less directly supported	Possible roles in neuronal excitability and Ca²⁺ signaling	Variants reported in neurodevelopmental/epileptic phenotypes with unclear mechanisms	(129–134)
TMEM16E /ANO5	CaPLSase; Ca²⁺-activated non-selective channel	Established	Musculoskeletal-enriched: Skeletal muscle, cardiac muscle, osteoblasts, chondrocytes	Primarily intracellular/ER with regulated sarcolemmal or plasma-membrane presence in some contexts	Cytosolic Ca²⁺ & channel activity is modulated by voltage	Muscle membrane repair; bone homeostasis/ mineralization; musculoskeletal Ca²⁺/lipid signaling; fertilization and coagulation in mice	Bone dysplasia and muscular dystrophy. GOF: gnathodiaphyseal dysplasia (GDD). LOF: Limb-girdle muscular dystrophy type 2L (LGMD2L) and Miyoshi muscular dystrophy type 3 (MMD3)	(135–147)
TMEM16G/ANO7	Prostate-enriched TMEM16 member; putative/controversial scramblase or channel	Limited / controversial	Highly enriched in prostate epithelium and prostate cancer	Plasma membrane and intracellular compartments	Ca²⁺-linked activity proposed; lipid/ion transport mechanisms remain debated	Prostate epithelial function, vesicle biology and cancer-associated remodeling	Prostate cancer biomarker/risk/prognosis associations; no established monogenic scramblase disease	(148–154)
TMEM16H/ ANO8	ER-PM tethering/signal protein; CaPLSase or channel activity not established	Not demonstrated / developing	Broadly expressed in epithelia and secretory tissues	ER-plasma membrane junctions	Ca²⁺ store depletion and PIP₂-dependent tethering; functions in membrane contact sites	Organization of ER–PM Ca²⁺ signaling domains; epithelial/hepatobiliary physiology	Variants associated with intrahepatic cholestasis of pregnancy and hepatobiliary phenotypes; a high-impact rare-variant risk gene	(155–158)
TMEM16J/ ANO9	Controversial cation channel/ signaling protein, putative intracellular CaPLSase	Developing	Broadly expressed; enriched in gastrointestinal tract	Mainly in intracellular compartments	Ca²⁺- and cAMP/PKA-linked regulation	Cytosolic Ca²⁺ regulation; membrane trafficking/signaling and cell growth pathways; may contribute to olfaction	Cancer/gastrointestinal associations reported; scramblase-specific disease links unclear	(159–165)
TMEM16K/ ANO10	ER-resident CaPLSase	Established	Broadly expressed; important in neurons/Purkinje cells	ER and ER-endosome contact sites	Cytosolic Ca²⁺; in vitro activity requires short-chain lipids	ER lipid homeostasis, endosomal sorting, organelle contact-site regulation and cerebellar neuron maintenance	LOF: Spinocerebellar ataxia 10 (SCAR10)	(166–176)
Subdued	Drosophila CaPLSase; Ca²⁺-activated non-selective channel	Established	Drosophila gut epithelium and sensory neurons	Plasma membrane	Activated by cytosolic Ca²⁺; channel activity is modulated by voltage	Heat nociception and gut/immune homeostasis in Drosophila	N/A	(177–180)
nhTMEM16/ afTMEM16	Fungal CaPLSase, Ca²⁺-activated nonselective channel	Established biochemically/ structurally	Nectria haematococca/ Aspergillus fumigatus	Not established; studied in purified/ reconstituted systems	Cytosolic Ca²⁺	N/A	N/A	(83, 181–184)
Ist2	Yeast ER–PM tether and Ca²⁺-independent phospholipid scramblase	Established	Saccharomyces cerevisiae	Cortical ER / ER-plasma membrane contact sites	Constitutive, Ca²⁺-independent activity	Supports Osh6/Osh7-mediated PS transfer and ER–PM lipid homeostasis	N/A	(185–190)
XKR (XK-related) family with established caspase-activated scramblases								(191, 192)
XKR8	Caspase- and kinase-regulated phospholipid scramblase	Established	Broadly expressed	Plasma membrane as complex with basigin or neuroplastin	Caspase-dependent cleavage of C-terminal tail during apoptosis; regulated by phosphorylation/ kinase pathways	Apoptotic PS exposure, efferocytosis, immune tolerance, myoblast differentiation, germ-cell clearance and developmental axon pruning	Xkr8 deficiency in mice causes SLE-like autoimmunity and male infertility; exploited in viral PS exposure/entry contexts	(35, 193–196)
XKR4	Caspase-activated apoptotic scramblase	Strong/ emerging structurally	Highly expressed in the brain	Plasma membrane	Caspase cleavage, dimerization and ligand/cofactor-dependent activation proposed	Apoptotic PS exposure and efferocytosis in neural contexts	Genetic associations include lissencephaly 9/brain malformation; direct scramblase-disease mechanism still developing	(197, 198)
XKR9	Caspase-activated apoptotic scramblase	Strong/ emerging structurally	Intestine, stomach and testis enriched	Plasma membrane	Caspase cleavage activates apoptotic scrambling	Tissue-specific apoptotic PS exposure and efferocytosis	No well-established monogenic human disease	(199)
XKR1 (XK)	Constitutive scramblase-like partner for VPS13A at ER–PM contacts	Strong but still developing	Broadly expressed: RBCs, immune cells, and neurons	Plasma membrane; complex with VPS13A at ER-plasma membrane contact sites	Caspase-independent; functions with VPS13A as lipid-transfer bridge	Bulk lipid homeostasis at contact sites; erythrocyte shape and neurobiology	LOF causes McLeod syndrome/neuroacanthocytosis; functionally linked to VPS13A/chorea-acanthocytosis pathways	(34, 200–202)
XKR2, XKR3, XKR5–7	XKR-family proteins with limited or unclear scramblase evidence	Not demonstrated or limited, depending on paralog	Tissue-specific expression varies by paralog	Predicted membrane proteins; localization varies	N.D.	N.D.	No established scramblase-specific disease links for most paralogs
TMEM63 /OSCA/ TMC mechanosensitive ion channel families; evolutionarily related to the TMEM16 family								(41, 203–206)
TMEM63A	Mechanosensitive ion channel (MSC); possible mechanically activated/cholesterol-regulated lipid scrambling; engineered mutants enable scrambling;	Emerging/ debating	Plasma membrane and lysosomal compartments	Mechanical force/ membrane stretch /osmotic stress	Mechanical force, membrane stretch and osmotic/organellar stress; cholesterol may regulate scrambling	Mechanotransduction, lysosomal mechanosensing/ function and membrane homeostasis	Hypomyelinating leukodystrophy and related neurodevelopmental disease associated with variants	(96, 207–215)
TMEM63B	MSC; possible mechanically activated/cholesterol-regulated lipid scrambling; pathogenic mutants enable scrambling	Emerging/ debating	Broad expression; lung lamellar bodies, nervous system and epithelial systems reported	Plasma membrane and intracellular compartments including lamellar bodies	Mechanical force, membrane stretch and osmotic stress	Mechanotransduction, surfactant secretion in the lung, thirst/ osmosensory pathways and intestinal stem-cell homeostasis	GOF variants linked to developmental and epileptic encephalopathy/neurodevelopmental disease; mutant-acquired scrambling	(96, 207, 212, 215–220)
TMEM63C	Mechanosensitive/ channel and scramblase functions less defined than A/B	Limited/ developing	Broadly expressed, angiotensin II-induced expression in podocytes	ER/mitochondria-associated ER membranes; localization may be context-dependent	Organelle stress/mechanosensitive regulation proposed	Mitochondrial morphology and organelle homeostasis	Biallelic variants associated with hereditary spastic paraplegia/ neurodevelopmental phenotypes;	(221–224)
OSCA 1.1/1.2/2.2	Plant MSC; Scramblase: debating.; Engineered mutants can be scramblases	Emerging/ debating	Arabidopsis roots, shoots, guard cells and pollen tubes depending on paralog	Plant plasma membrane	Osmotic/mechanical stress	Plant osmosensing, mechanotransduction and stress responses	N/A	(225–230)
TMC1/2	MSCs; core components of hair-cell MET complex; claimed as cholesterol-dependent scramblase	Emerging/ debating	Cochlear and vestibular hair cells	Stereocilia tips/MET complex	Mechanical force in MET complex; cholesterol-dependent scrambling reported	Auditory and vestibular mechanotransduction; membrane homeostasis in hair-cell stereocilia	TMC1/TMC2 variants cause hereditary hearing loss and vestibular defects	(231–239)
TMC3–8	Either ion channels or unknown functions	Not demonstrated	Various tissues; TMC6/8 in immune/epithelial contexts	No scramblase activity reported to date	Plasma membrane or intracellular compartments depending on members	TMC4 in salt taste proposed; TMC6/8 in papillomavirus restriction/skin immunity; TMC6 regulates M current in sensory neurons	Biallelic TMC6/TMC8 LOF causes epidermodysplasia verruciformis; other disease links less defined	(240–247)
Other reported scramblases
PLSCR1	Interferon-inducible signaling/scaffold protein historically named phospholipid scramblase	Controversial as standalone CaPLSase	Broad; strongly induced by type I/II interferons	Plasma membrane; intracellular compartments	Interferon-responsive; Ca²⁺-responsive membrane association; reported historically without direct evidence	Antiviral innate immunity, transcription/signaling and cancer-associated signaling	Autoimmune and cancer associations; antiviral restriction factor against SARS-CoV-2 and other viruses	(248–256)
PLSCR2	Immune-associated PLSCR family member	Controversial; unlikely to be CaPLSase	Testis-enriched; low in most other tissues	N.D.	N.D.	Possible immune functions	Disease links unclear	(256, 257)
PLSCR3	Mitochondrial PLSCR-family protein associated with cardiolipin/mitochondrial regulation	Controversial; unlikely to be CaPLSase	Ubiquitous; enriched in mitochondria	Inner mitochondrial membrane	Ca²⁺-linked mitochondrial regulation proposed	Cardiolipin distribution, mitochondrial architecture, mitophagy/metabolism	Metabolic disease/ adiposity/insulin resistance links in model systems	(256, 258–264)
Opsins / Rhodopsin	GPCRs with ATP-independent phospholipid scramblase activity in vitro and specialized photoreceptor-disc context	Strong in reconstitution; in vivo generality debated	Photoreceptor discs for rhodopsin/opsin; other GPCRs vary	Photoreceptor disc membranes and plasma membranes depending on receptor	Constitutive in proteoliposomes; inhibited by cholesterol in some systems; distinct from canonical GPCR signaling	Photoreceptor disc membrane homeostasis; broader GPCR membrane remodeling remains under investigation	RHO mutations cause retinal degeneration, but disease is not generally attributed to scramblase dysfunction	(265–270)
VMP1 (Vacuole membrane protein 1)	ER multi-pass VTT/DedA-domain phospholipid scramblase; autophagy protein	Established ER scramblase	Broad expression	ER, including mitochondria-associated ER membranes/contact sites	Constitutive ATP-independent lipid scrambling; function coordinated with Beclin 1, ATG2 and autophagy machinery	Autophagosome biogenesis, ER lipid equilibration, organelle contact-site biology, Ca²⁺ homeostasis and inflammasome regulation	Pancreatitis, pancreatic cancer, liver steatosis/MASH-like phenotypes and inflammatory/ neurodegenerative contexts	(271–276)
TMEM41B	ER VTT/DedA-domain phospholipid scramblase; autophagy and viral host factor	Established ER scramblase	Broad expression	ER; ER-mitochondria contact-enriched contexts	Constitutive ATP-independent scramblase for PS/PE/PC/cholesterol; function coupled to ATG2-mediated transfer and lipoprotein assembly	Early autophagosome biogenesis; ER lipid homeostasis; VLDL secretion; ER–mitochondria contact maintenance; formation of viral replication organelle; essential for embryonic development and neuronal function	Host-dependency factor for SARS-CoV-2 and other viruses; liver steatosis/MASH-like phenotypes and neurodevelopmental/neuronal dysfunction in models	(37, 272, 275–277)
ATG9 (Autophagy Related 9)	Autophagy transmembrane protein and lipid scramblase in ATG9 vesicles/phagophore membranes	Established biochemically; physiological model strong	Broad expression	Golgi/TGN-derived vesicles, endosomes and phagophore assembly sites	Constitutive scramblase; activity coordinated with ATG2-mediated lipid transfer and membrane curvature/vesicle context	Autophagosome membrane expansion, autophagy initiation and axonal/ neurodevelopmental homeostasis	ATG9A LOF variants cause severe neurodevelopmental/ spastic paraplegia-like disorders; AP-4-related autophagy trafficking links	(36, 275–288)
TMEM170A	ER/nuclear-envelope lipid scramblases associated with BLTP1/Csf1	Emerging / recent	Broad expression	ER and nuclear envelope (NE); ER membrane-contact-site contexts	Constitutive ATP-independent scrambling; physical association with BLTP1/Csf1 reported	Bulk lipid transport at membrane contact sites; ER/nuclear-envelope morphogenesis	N.D.	(289, 290)
VDAC1 / VDAC2 dimer	β-barrel mitochondrial outer-membrane channels that can moonlight as phospholipid scramblases	Strong / emerging	Broadly expressed; abundant mitochondrial outer membrane proteins	Outer mitochondrial membrane	Dimer-dependent ATP-independent scrambling proposed; also canonical metabolite/anion channels	Mitochondrial phospholipid import, metabolite exchange, bioenergetics and apoptosis	VDACs broadly implicated in cancer, neurodegeneration and ischemia-reperfusion; scramblase-specific disease mechanisms remain under investigation	(38, 291)
EMC/Oxa1/YidC-family protein insertases	Protein insertases with hydrophilic grooves that can promote lipid scrambling in vitro/simulations	Developing / mechanistic principle	Broad; depends on insertase complex	ER, inner mitochondrial membrane, bacterial plasma membrane and related systems	Constitutive scrambling via insertase hydrophilic groove proposed; tightly linked to protein insertion machinery	Membrane protein biogenesis; possible leaflet equilibration during membrane protein insertion and organelle biogenesis	Diseases of EMC/Oxa-related insertase dysfunction exist, but scramblase-specific disease mechanisms are not established	(40, 292, 293)

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Note: ER: endoplasmic reticulum; GOF: Gain-of-function; HX: hereditary xerocytosis; LOF: Loss-of-function; N/A: Not applicable; N.D.: Not determined; PM: plasma membrane; RBC: red blood cells; SCD: sickle cell diseases; TGN: trans-Golgi network; VDAC: voltage-dependent anion channel.

1.1. Phospholipid asymmetry: discovery and update

Biological membranes display a striking and highly conserved asymmetry in their phospholipid organization (6, 9, 11, 63). In the plasma membrane, PS and PE are enriched in the cytofacial leaflet, whereas sphingomyelin (SM) and phosphatidylcholine (PC) predominate in the exofacial leaflet (Figure 1A). This now-canonical arrangement was first proposed by Mark Bretscher in 1972, based on biochemical studies demonstrating that membrane-impermeant reagents preferentially labeled proteins rather than aminophospholipids in erythrocytes or red blood cells (RBCs), that phospholipase A-containing venoms selectively hydrolyzed outer-leaflet PC but not PS/PE, and that spontaneous phospholipid “flip-flop” across bilayers occurred at extremely slow rates (11) (Figure 2). Subsequent work using lipase digestion combined with freeze-fracture electron microscopy (EM) provided the first direct visualization of phospholipid sidedness, establishing membrane asymmetry as a fundamental principle of cell biology (64). In the decades since, the concept of lipid asymmetry has been extended from RBCs to platelets (65), nucleated cells (66), and organelles (67). This concept was recently further refined by lipidomics, revealing that headgroup asymmetry is accompanied by asymmetric acyl-chain distributions and non-equimolar leaflet packing (10, 68).

1.2. Molecular players that build and dissipate phospholipid asymmetry

Membrane phospholipid asymmetry is established and dynamically regulated by three major classes of lipid transport proteins: ATP-dependent flippases, ATP-dependent floppases, and ATP-independent phospholipid scramblases (PLSases) (6, 9, 16, 17) (Figure 1B–C and Table 1). Together, these transport systems govern the transbilayer distribution of phospholipids and maintain the distinct lipid composition of the exofacial and cytosolic membrane leaflets.

Phospholipid synthesis occurs predominantly on the cytosolic leaflet of the ER and other organelles (69, 70), requiring newly formed lipids to be translocated across bilayers to achieve proper membrane composition (71). This translocation is mediated by two major classes of active transporters: P4-ATPases or flippases, which pump aminophospholipids, PS and PE, from the exofacial leaflet to the cytosolic leaflet of the plasma membrane (9, 16, 17, 72, 73); and ABC transporter floppases (74), which mediate outward transport of diverse lipid substrates, including PC, SM, and sterols (9, 72, 74) (Figure 1B–C and Table 1). Representative members include ATP11A, ATP11C, and ATP8A2 flippases, as well as ABCA1, ABCB1, and ABCB4 floppases, while many P4-ATPases additionally require CDC50/TMEM30 family cofactors for proper trafficking and activity (9, 16, 17, 72, 73). Together, flippases and floppases invest metabolic energy to maintain lipid asymmetry against entropy and the slow but finite spontaneous flip-flop of lipids.

In contrast, PLSases mediate passive, energy-independent, bidirectional, and largely nonselective lipid movement, rapidly collapsing asymmetry under defined physiological conditions (6, 9) (Figure 1B–C and Table 1). Although a recent study using a cell-based fluorescence polarization assay demonstrated that TMEM16F preferentially scrambles PS and PC over PE on the plasma membrane (75), its permeability to other anionic lipids such as phosphoinositides (PIPs) and PA has not been experimentally examined. Thus, TMEM16F activation likely produces substantial dissipation of pre-existing glycerophospholipid asymmetry rather than complete equilibration of all membrane lipids. Whether TMEM16F similarly redistributes sphingolipids, glycolipids, phosphoinositides, or cholesterol in cells remains less well established.

Early physiological clues for such transporters came from studies of blood coagulation (28, 63, 76) and apoptosis (31, 77, 78) (Figure 2). In particular, PS exposure became recognized as a hallmark of apoptotic cells, suggesting the existence of caspase-dependent phospholipid scramblases involved in apoptotic cell clearance (31, 78). The subsequent identifications of Ca²⁺-activated TMEM16F (33) and caspase-activated XKR8 (35) as bona fide PLSases established direct molecular links between Ca²⁺ signaling, caspase activation, and controlled dissipation of lipid asymmetry. In recent years, additional classes of phospholipid scramblases have continued to emerge across diverse cellular and organellar systems, greatly expanding the landscape of regulated lipid transport (Table 2). Owing to space constraints, this review focuses primarily on TMEM16F as the prototypical PLSase, highlighting how regulated lipid scrambling is integrated with cellular signaling pathways and how it contributes to physiology and disease.

1.3. Conceptual framework of lipid transport mechanisms

The “credit-card slot” or “card-swiping” model (Figure 3A) has emerged as a prevailing conceptual framework for how phospholipids move through scramblases and flippases (7, 294). In this model, the polar or charged headgroup of a phospholipid traverse through the hydrophilic groove or permeation pore within the transporter and interacts with the residues lining the groove. As the lipid molecule moves through the groove, the hydrophilic pathway shields the headgroup from the hydrophobic core of the membrane, avoiding the high energetic cost of extracting the entire lipid from the membrane bilayer. This arrangement dramatically lowers the activation barrier for transbilayer movement from ~20 kcal/mol for non-facilitated, spontaneous flip-flop to ~7 kcal/mol or less for scramblase-mediated translocation (7).

The “credit-card slot” model gained strong support with high-resolution crystal and cryo-EM structures of key lipid transporters, including P4-ATPase flippases (295, 296) and TMEM16 CaPLSases (84, 175, 182, 183, 297, 298) (Figure 3B). These structures revealed membrane-exposed hydrophilic grooves that provide plausible headgroup translocation paths. For TMEM16 CaPLSases, Ca²⁺ binding induces conformational changes that expose or open a membrane-facing hydrophilic groove, allowing phospholipid headgroups to traverse between leaflets while their acyl chains remain largely within the bilayer (Figure 3C). This groove-mediated “credit-card” mechanism is further facilitated by local membrane thinning and deformation, which reduce the energetic barrier and transbilayer distance for phospholipid movement. This structural framework explains numerous functional and computational observations, including the rapid, bidirectional, and largely nonselective scrambling characteristic of TMEM16F (33, 82, 95, 175, 184, 299–301).

Subsequent work has refined this simple model. For instance, an “out-of-the-groove” transport model (Figure 3C) has been proposed to account for scrambling of lipids with very bulky headgroups, such as PEG-conjugated lipids that exceed the width of the groove (302). More recent simulations and structural analyses also highlight a prominent role for local membrane thinning and lipid reorientation adjacent to the permeation pathway (82, 175, 183, 184, 298, 300, 303–305). Rather than acting as a rigid tunnel (Figure 3A), scramblases appear to reshape nearby bilayer architecture, shortening the distance and lowering the energy barrier for headgroup movement while allowing lipids to slide along the groove surface without fully entering its deepest region (Figure 3C).

The most recent structural study of TMEM16F captured a novel Ca²⁺-activated conformation in liposomes in which TM4 detaches from TM3 and slides along the straightened TM6, forming an X-shaped groove architecture (306) (Figure 3C). In this conformation, lipid headgroups are proposed to translocate by skirting the X-shaped groove periphery through a locally thinned membrane region, whereas ions permeate through a structurally coupled but spatially distinct aqueous pathway formed within the groove by TM3-TM6. These findings also suggest that ion conduction and lipid scrambling arise from a shared Ca²⁺-dependent conformational transition rather than independent activation mechanisms. Section 2 provides a more detailed discussion of how these mechanisms are thought to operate in TMEM16 scramblases.

1.4. Dynamic lipid flip-flop delivers nonconventional cell signals

The molecular identification of scramblases and flippases has enabled in-depth investigation of lipid flip-flop in physiology and disease. Over the past decade, accumulating evidence from studying these lipid transporters further strengthened that lipid asymmetry is not static but is dynamically tuned by the opposing activities of flippases/floppases and scramblases (307) (Figure 1C). The discovery of viable cells with constitutively exposed PS has overturned the dogma that loss of lipid asymmetry is synonymous with cell death (95, 308). The observed transient, reversible collapse and restoration of asymmetry (309) suggests that the lipid transporters act in concert to generate lipid flip-flop-mediated spatiotemporal signals that complement, and sometimes replace, more conventional protein-, ion- and lipid metabolism-based signals. In the sections that follow, we first outline the structural and mechanistic basis of TMEM16F-mediated lipid transport, then summarize how its activation is integrated with cellular signaling pathways, and finally explore how TMEM16F-dependent lipid scrambling is deployed in physiological processes and co-opted or disrupted in human disease.

2. STRUCTURE-FUNCTION OF TMEM16F

Over the past 15 years, work from many groups has yielded a rich picture of the structure and function of TMEM16F and its homologs. In this section, we provide a focused overview of key molecular features of TMEM16F, aiming to establish a framework for understanding its roles in physiology and disease. Rather than offering an exhaustive account, we emphasize concepts most relevant to TMEM16F biology. Readers interested in a more in-depth understanding of TMEM16 family structure-function relationships are referred to recent comprehensive reviews (41, 45, 46, 51, 83, 84, 310–313).

2.1. Discovery of TMEM16F CaPLSase

For decades, the molecular basis of phospholipid scrambling remained elusive (Figure 2). A major breakthrough came in 2010, when the Nagata laboratory identified TMEM16F as a key component of the CaPLSase and linked its loss-of-function to Scott syndrome, a rare inherited bleeding disorder (33) (see Section 5.1.1). Using a mouse B-cell line subjected to repetitive fluorescence-activated cell sorting for strong PS exposure, the authors identified a constitutively active TMEM16F mutation, providing the first direct evidence that TMEM16F is an essential component of the CaPLSase. In the same study, sequencing of immortalized B lymphocytes from a Scott syndrome patient (26, 314) revealed a frameshift mutation in TMEM16F that introduces a premature stop codon, further implicating TMEM16F as the defective scramblase in this disorder (33). Shortly thereafter, the Jan laboratory showed that Tmem16f-deficient mice recapitulate the clinical features of Scott syndrome, including severely reduced Ca²⁺-induced PS exposure in platelets and RBCs, impaired thrombin generation, and prolonged tail bleeding time (87). Together, these studies firmly established TMEM16F as the long-sought CaPLSase essential for blood coagulation.

2.3. Experimental measurement of lipid scrambling

Two complementary strategies are widely used to assess TMEM16 scramblase activity (Figure 4). In cell-based assays, elevation of intracellular Ca²⁺ activates TMEM16 scramblases, and the resulting exposure of PS on the cell surface is detected with fluorescent PS-binding probes, most commonly annexin V (AnV) (33, 87, 315, 316). Alternative probes include lactadherin (317, 318), its isolated C2 domain (LactC2) (301), the polarity-sensitive annexin-based probe pSIVA (319, 320), and Zn²⁺–dipicolylamine-based synthetic probes such as PSVue (321, 322). Surface PS exposure can be quantified by fluorescence microscopy, flow cytometry, or real-time fluorescence measurements. By preserving the native cellular environment, these assays enable analysis of scramblase activation, kinetics, and pharmacology.

In reconstitution assays, purified TMEM16 proteins are incorporated into liposomes containing trace amounts of fluorescently labeled phospholipids, most commonly NBD-labeled lipids, and transbilayer movement is monitored fluorometrically following the addition of membrane-impermeant dithionite (323, 324). In protein-free liposomes, in which fluorescent lipids are distributed approximately equally between the two leaflets, dithionite quenches the outer-leaflet fluorophores and rapidly reduces fluorescence by approximately 50%. In proteoliposomes containing an active scramblase, translocation of initially protected inner-leaflet lipids to the outer leaflet renders their fluorophores accessible to dithionite, resulting in substantially greater and, under ideal conditions, near-complete fluorescence quenching.

Together, cell-based assays measure scramblase-dependent PS externalization in a native membrane environment, whereas reconstitution assays can establish the intrinsic lipid-transport activity of purified proteins in a defined membrane system. However, both approaches, are susceptible to artifacts that can produce scramblase-independent PS labeling or fluorescence quenching. In cell-based assays, membrane damage, loss of plasma-membrane integrity, or nonspecific probe binding can mimic PS externalization, whereas in proteoliposome assays, vesicle leakage, membrane disruption, or unintended access of dithionite to the inner leaflet can exaggerate apparent lipid scrambling. Appropriate controls for membrane integrity, protein-free liposomes, inactive mutants, and stimulus dependence are therefore essential. For TMEM16 scramblases with detectable Ca²⁺-activated ion conductance, patch-clamp recording provides a useful orthogonal readout of functional membrane expression, activation, and pharmacological or mutational effects (95, 301, 309, 325–328). Although ionic currents do not directly measure lipid translocation and may not correlate quantitatively with scrambling, concordant changes in channel and scramblase activities can substantially strengthen the conclusion that an observed phenotype reflects TMEM16 activation.

2.3. Intriguing moonlighting function as an ion channel

The identification of TMEM16F as a lipid scramblase was unexpected, because the ten-member mammalian TMEM16 family was initially classified as Ca²⁺-activated chloride channels (CaCCs) based on TMEM16A and TMEM16B, the first members to be identified and functionally characterized (52, 92–94). Electrophysiological characterizations, however, revealed that TMEM16F is a Ca²⁺- and voltage-activated ion channel (87, 329) (Figure 3D). More detailed analyses of ion permeability showed that TMEM16F channel has substantial cation permeability, with reported P_Na/P_Cl ratios of 6.8 (87), 4.8 (177), 1.3 (330), 1.4 (301), 0.5 (331), and 0.3 (332), depending on expression system, ionic conditions, and recording configuration. This relative cation permeability stands in contrast to TMEM16A, which is a classical CaCC with P_Na/P_Cl ~0.1 (87, 333). Given its non-selective ion permeation and small single channel conductance of 0.5 pS, TMEM16F has been described as a Small Conductance, Ca²⁺-Activated, Nonselective (SCAN) channel (87).

2.3. Overall structural features

Structural information is essential to understand the biophysical properties of TMEM16F. Similar to the fungal TMEM16 ortholog NhTMEM16 (182) and TMEM16A (334), TMEM16F is a butterfly-shaped homodimer, with each monomer comprising ten transmembrane (TM) α-helices (297, 298, 304) (Figure 3B). This dimeric arrangement creates a double-barreled architecture, similar to the CLC and Fluc families of anion channels (335, 336), where each subunit contributes its own permeation pathway (297, 304). TMEM16F monomers are thought to function independently, though this has only been experimentally demonstrated through CaCC activity in TMEM16A (337, 338). Notably, TMEM16F substrate permeation pathways are not aligned with the central dimer axis. Instead, each monomer contains a narrow hourglass-shaped groove formed by hydrophilic and nonpolar residues spanning TM helices 3–7, which together define the proteolipid permeation pore (Figure 3B–C). At the protein-lipid interface, TM4 and TM6 line one side of this groove; their extracellular halves come into close apposition at the so-called ‘neck region’ and form a critical gating interface for TMEM16F permeation (see Section 2.5). Within each monomer, three highly conserved Ca²⁺-binding sites are located in the aqueous cavities within the transmembrane region: two adjacent “orthosteric” sites formed mainly by the acidic and polar residues from TM6-TM8 within the permeation pore (45, 297, 304, 339, 340) and a third, allosteric site located between TM2 and TM10 near the dimer interface (175, 297, 341) (see Section 2.4).

2.4. Ca²⁺-dependent activation

Ca²⁺ is the primary driver for TMEM16F activation and gating. Ca²⁺ binding to the orthosteric TM6-TM8 sites of TMEM16F triggers a conformational change where the cytosolic half of TM6 collapses onto TM7 and TM8 (297, 304) (Figure 3B–C). While not structurally resolved, this conformational change is thought to trigger the opening of the permeation pathway, enabling ions and phospholipid headgroups to pass through the hydrophilic groove. Although four Ca²⁺ ions bind per channel at the orthosteric sites, the Hill slope for activation is only approximately 2 (87, 332, 342), indicating a graded concentration-response relationship without a well-defined Ca²⁺ threshold. This implies that significant scramblase activity could occur at sub-micromolar free Ca²⁺ concentrations well below those required for maximal activation.

Similar to TMEM16A CaCC (343), a conserved glycine hinge (G614/615 in the human/mouse TMEM16F orthologs, respectively) in TM6 plays a critical role in the Ca²⁺-induced conformational changes. Unlike TMEM16A, however, TMEM16F lacks an additional residue proximal to the hinge, perhaps explaining why the TM6 helix movement originates from opposing directions in the Ca²⁺-free closed states (Figure 3C). Participation by TM6 in both Ca²⁺ binding and formation of the substrate permeation pathway highlights its key role in activation and gating (344–347).

2.5. Gating and permeation mechanisms

As a passive lipid transporter capable of moving ~4.5 × 10⁴ phospholipids per second (299), TMEM16F can rapidly disrupt membrane lipid asymmetry. Such a high transport rate demands a tight regulation of permeation to prevent uncontrolled scrambling. Structural, functional, and computational studies converge on a central steric gate formed by pore-lining helices TM4, TM5, and TM6 as the key determinant of TMEM16F gating and permeation (95). Three bulky hydrophobic residues, F518 (TM4), Y563 (TM5), and I612 (TM6), constitute the core of this inner gate, guarding lipid and ion permeation. Alanine substitutions at these positions relieve steric hindrance and produce constitutively active scramblases, whereas replacement with larger hydrophobic residues (e.g., leucine, tryptophan) markedly suppresses Ca²⁺-activated scrambling. Introducing polar or charged residues at or near the gate (e.g., F518K, I521K, M522K, Y563K, I612K) further destabilizes this barrier, yielding strong gain-of-function lipid scrambling and ion conductance (95, 96). Notably, some of these mutations (e.g, F518K and Y563K) exhibit constitutive lipid scrambling activity even when primary Ca²⁺-binding sites are disrupted (95), indicating that gate stability itself is a principal determinant of TMEM16F activity.

Analogous inner-gate residues in TMEM16A CaCC also form a hydrophobic barrier for its chloride ion permeation (95, 346). Mutations at or near the inner gate such as L543K, I546K and I547K lower the Ca²⁺ level required for channel opening and, strikingly, confer robust constitutive scramblase activity, highlighting an evolutionary continuum between pure ion channels and scramblases within the TMEM16 family (95, 96). Consistent with this idea, an early study by the Hartzell laboratory showed that TMEM16F ion and lipid permeation require a 15–amino acid segment (residues 525–540) in TM4–TM5 termed the “scrambling domain” (301). Grafting this segment into TMEM16A is sufficient to bestow scramblase activity on the otherwise chloride-selective CaCC. Together, these findings blur the traditional boundary between “channels” and “scramblases,” and support a broader view of the transmembrane channel-scramblase (TCS) superfamily (including TMEM16, OSCA/TMEM63, TMC families) as a continuum in which relatively small changes near the inner gate can tune the balance between ion and lipid permeation (45). Mechanistically, these functional data, together with TMEM16 structures captured in distinct conformations (175), support a “clamshell” gating model in which Ca²⁺ binding remodels interactions between TM4 and TM6 in the neck region, opening a hydrophilic proteolipid pore that permeates phospholipid headgroups and, in some states, ions (Figure 3C).

At the opposite conceptual extreme, work on reconstituted fungal scramblases such as AfTMEM16 in proteoliposomes has shown that they can transport synthetic lipids >5 kDa, challenging a strictly “credit-card slot” view (302, 348, 349). These studies have motivated a “closed-groove” model in which scrambling is driven primarily by conformationally induced membrane thinning near TM4 and TM6, allowing lipids to slip around the protein-lipid interface rather than through a fully open aqueous groove (181, 298, 303, 304, 350) (Figure 3C). This framework helps reconcile robust scrambling activities observed in liposome reconstitutions with the relative paucity of lipid-filled grooves in related structures. A key unresolved issue for the “closed-groove” model, however, is how gating of both ions and lipids is accomplished in TMEM16F and other TMEM16 scramblases.

Recent structures of gate-destabilizing mutations in TMEM16F and TMEM16A offered additional insights. While the TMEM16A I550A structure provided further structural evidence to support the “pore dilation” model of ion permeation (346), the TMEM16F F518H Ca²⁺-bound structure reveals an unexpected rearrangement of TM3 that appears to create partially distinct ion and lipid permeation pathways, with lipid translocation facilitated by local membrane thinning and “closed-groove”-like scrambling (298) (Figure 3C). In summary, structural, functional, and computational findings have greatly advanced the understanding of gating and permeation mechanisms of TMEM16 proteins. It remains unclear which gating mechanism or combination of mechanisms best describes TMEM16F in its native setting: a clamshell-type opening supported by functional dissection and mammalian TMEM16K structures, or a predominantly closed-groove, membrane-thinning mechanism suggested by some fungal orthologs and mutationally open TMEM16F structures. Future studies are required to resolve this question using integrated structural, functional, and computational approaches.

2.6. Regulatory mechanisms

The plasma membrane contains approximately 5 × 10⁶ phospholipids per μm² (351), meaning that complete scrambling of a 1 μm² membrane patch within 10 seconds would require roughly 100 active TMEM16F molecules per μm², based on a transport rate of ~4.5 × 10⁴ phospholipids per second (299). Such a high transport rate thus demands a tight regulation of permeation to prevent uncontrolled scrambling.

In addition to its Ca²⁺-dependent activation, TMEM16F activity is also controlled by various regulatory mechanisms. Alternative splicing generates multiple isoforms differing primarily in their N-terminal and C-terminal cytoplasmic regions. These isoforms exhibit distinct Ca²⁺ sensitivities, kinetics, and trafficking properties, allowing tissue-specific tuning of TMEM16F activity (331). At the plasma membrane, phosphatidylinositol 4,5-bisphosphate (PIP₂) acts as an essential cofactor that stabilizes Ca²⁺-gated opening and prevents TMEM16F channel rundown, effectively restricting robust TMEM16F channel activity to PIP₂-rich cell-surface domains (304, 352). The local membrane environment further modulates function. Changes in lipid packing, thickness, and composition alter how efficiently phospholipids access the hydrophilic “credit-card” groove and thus impact both scrambling and ion permeation (303, 350). Intracellular pH then operates as a rheostat on Ca²⁺ sensitivity by protonating Ca²⁺-binding residues at acidic pH (inhibiting activation) and enhancing Ca²⁺ affinity at alkaline pH (potentiating scrambling and currents), a mechanism likely exploited in pathophysiological settings such as cancer (326). In addition, post-translational modifications, most clearly N-glycosylation, and potentially phosphorylation or ubiquitination, help control TMEM16F folding, trafficking, and stability (299, 353). Together, these diverse regulatory mechanisms fine-tune TMEM16F properties and expression to decode specific combinations of Ca²⁺ signals, lipids, and intracellular conditions to drive lipid flip-flop-mediated cellular events.

3. CELLULAR ACTIVATION MECHANISMS OF TMEM16F

Ca²⁺ is the principal physiological trigger for CaPLSase activation (33, 87, 309, 327–329, 354–356). Compared to TMEM16A CaCC with sub-micromolar Ca²⁺ affinity (52, 87, 93, 94, 329), TMEM16F has markedly low Ca²⁺ sensitivity (EC₅₀ values ranging from 3.4 to 105 μM depending on the measurements) (45, 87, 326, 330–332, 357–359). This relatively low apparent Ca²⁺ sensitivity implies that only large and/or sustained local Ca²⁺ elevations can efficiently activate TMEM16F, consistent with the observations that cells usually do not spontaneously expose PS under basal conditions (33, 87, 90, 95, 308, 309, 327, 360, 361).

Given sustained, global Ca²⁺ elevations (>1 μM) are uncommon in most physiological contexts, especially in non-excitable cells (362), TMEM16F’s graded Ca²⁺ concentration-response relationship may generate scramblase activity at free Ca²⁺ concentrations below 1 μM (87). Importantly, this may be explained in part by the relatively shallow Ca²⁺ dependence of TMEM16F scrambling, which broadens the activation range so that Ca²⁺ levels below the apparent EC₅₀ can still produce measurable lipid scrambling. However, effective TMEM16F activation in vivo generally requires tight spatial coupling to upstream Ca²⁺ sources so that high microdomain Ca²⁺ can be reached near the cytoplasmic leaflet (309, 327, 328, 360).

Growing evidence supports the idea that TMEM16F resides in such Ca²⁺ microdomains formed by specific plasma-membrane Ca²⁺ channels. A well-defined example is the Ca²⁺-permeable TRPV4 channel in human placental trophoblasts (309). Pharmacological activation of TRPV4 with GSK1016790A elicits local Ca²⁺ influx that robustly activates TMEM16F, leading to PS exposure in a trophoblast cell line. Patch-clamp characterizations confirmed that TMEM16F and TRPV4 are co-localized within membrane microdomains, allowing even modest TRPV4 activity to trigger transient PS exposure and reversible lipid scrambling, a requirement for trophoblast fusion (309). TRPV4 inhibition or knockdown suppresses TMEM16F activity and impairs trophoblast fusion, whereas TRPV4 activation drives reversible PS exposure, which is required for syncytialization. These data establish TRPV4-TMEM16F coupling as a physiological Ca²⁺ entry pathway that gates CaPLSase activity during placental development.

The mechanosensitive channel PIEZO1 represents another major upstream Ca²⁺ source for TMEM16F in various cell types. In RBCs, mechanical activation or pharmacologic stimulation of PIEZO1 induces Ca²⁺ influx that directly activates TMEM16F, driving PS exposure (327). PIEZO1-TMEM16F functional coupling is markedly enhanced in hereditary xerocytosis (HX) RBCs carrying PIEZO1 gain-of-function mutations, increasing the propensity for PS exposure and contributing to anemia, splenomegaly, and post-splenectomy thrombosis (327). In sickle cell disease (SCD), deoxygenation-induced sickling similarly activates PIEZO1, leading to enhanced Ca²⁺ entry, TMEM16F activation, PS exposure, PS⁺ microparticle shedding, thrombin generation, and endothelial adhesion (360). In trophoblasts, PIEZO1 is also required for syncytiotrophoblast formation and placental development, acting at least in part via Ca²⁺-dependent activation of TMEM16F (328). In mammalian cell lines, PIEZO1 activation promotes TMEM16F-dependent plasma-membrane expansion, whereas in the absence of TMEM16F the same stimulus triggers massive endocytosis, albeit the evidence of direct PIEZO1-TMEM16F coupling was not provided (363). More recently, a proteomic study reported that TMEM16F is within the PIEZO1 interactome (364), providing biochemical evidence for the PIEZO1-TMEM16F coupling. Together, these studies define PIEZO1-TMEM16F coupling as a central pathway linking mechanical forces to Ca²⁺-driven lipid scrambling in multiple tissues.

In macrophages, purinergic P2X7 channel is a major Ca²⁺ channel that drives TMEM16F activation. Co-immunoprecipitation and functional assays support a close physical and functional association between P2X7 and TMEM16F (365). Upon ATP binding, P2X7-mediated Ca²⁺ elevation activates TMEM16F, promotes PS exposure, macropore formation and dye uptake, membrane blebbing, cell-volume changes, and ultimately ATP-induced cell death (365, 366). TMEM16F has also been reported to support macrophage migration, phagocytosis, and bacterial killing in this model, although whether these effects reflect direct PS-dependent antimicrobial activity or broader roles in membrane repair and immune-cell viability remains unresolved (366).

Collectively, these examples highlight a common design principle: physiological activation of TMEM16F is not driven by diffuse, global Ca²⁺ signals but instead by localized Ca²⁺ microdomains generated by specific plasma membrane channels such as TRPV4, PIEZO1, and P2X7. Although many non-excitable cells exhibit Ca²⁺ oscillations in the low-micromolar range that overlap with the EC₅₀ of TMEM16F, its relatively low Ca²⁺ sensitivity and dependence on spatial proximity suggest that only sustained, locally elevated Ca²⁺ near TMEM16F-enriched microdomains is sufficient for robust activation. This spatial restriction, together with the yet unresolved molecular organization of these microdomains, including contributions from specific lipid species, the spectrin-based cytoskeleton, and potential physical coupling between TMEM16F and upstream channels, ensures that lipid scrambling is triggered only under defined stimuli. This enables precise control of PS exposure, membrane remodeling, and downstream signaling.

The existence of Ca²⁺ microdomains is a cornerstone concept in cellular signaling, enabling highly localized activation of downstream effectors. By analogy, such spatially restricted Ca²⁺ signals raise the possibility that phospholipid scrambling may also occur locally, suggesting that TMEM16F-mediated lipid redistribution could be confined to discrete membrane regions. However, the existence and organization of lipid microdomains are considerably less well established. Lipid microdomains are most convincingly demonstrated for rapidly turning-over species such as PIP₂, where spatial gradients and lateral clustering have been directly observed (367–369). In contrast, for other lipids, including PS, evidence of persistent lateral gradients is more limited, and whether such gradients are generated or maintained by TMEM16F remains unclear. Nevertheless, a study in mast cells undergoing degranulation, showed that Annexin A5 labeling reveals discrete extracellular PS pools that persist for several seconds (370) demonstrating that PS could be locally confined under specific conditions.

4. TMEM16F-MEDIATED CELL SIGNALING AND PHYSIOLOGICAL FUNCTIONS

TMEM16F translocates up to 4.5×10⁴ phospholipids per second across the plasma membrane, orders of magnitude faster than ATP-dependent flippases or floppases, which typically move fewer than 10² lipids per second (9, 299, 371). This striking kinetic disparity indicates that once TMEM16F is activated by elevated intracellular Ca²⁺, it can rapidly collapse membrane lipid asymmetry, triggering acute membrane remodeling and downstream changes of extracellular and intracellular signaling. When cytosolic Ca²⁺ levels return to baseline, TMEM16F activity ceases and ATP-dependent lipid transporters gradually reestablish lipid asymmetry, creating a reversible “on-off” switch for lipid redistribution. This dynamic interplay between Ca²⁺-activated scramblases and energy-dependent lipid pumps positions TMEM16F as a pivotal signaling hub integrating membrane remodeling with key biological processes such as coagulation, immune activation, cell-cell fusion, cell death, and angiogenesis. In this section, we therefore summarize current knowledge of TMEM16F’s physiological functions in three broad categories: extracellular signaling, intracellular signaling, and membrane remodeling (Figure 5). This simplified classification is intended to highlight the often underappreciated signaling role of lipid scramblases. In vivo, however, these signaling modes frequently intersect and act in combination to shape cellular outcomes.

FIGURE 5. — These functions can be broadly grouped into three modes, based on whether TMEM16F-mediated lipid flip-flop primarily affects extracellular signaling, intracellular signaling, or membrane remodeling. TMEM16F may engage multiple modes simultaneously to regulate downstream physiological processes. Figure schematics were created with BioRender.com.

4.1. TMEM16F-mediated extracellular signaling associated with exofacial leaflet lipid remodeling

TMEM16F is best known as a lipid scramblase that externalizes PS to initiate extracellular events (Figure 6). This subsection summarizes its extracellular signaling roles, including facilitating coagulation (87, 88, 135, 372), recruiting phagocytes (373, 374), serving as a membrane fusion signal (91, 375, 376), and modulating ADAM protease activity (377–379).

4.1.1. TMEM16F-mediated PS exposure in blood coagulation

One of the well-established extracellular signaling roles of TMEM16F is its procoagulation function via PS externalization (Figure 6). Surface-exposed PS provides an indispensable platform for the efficient assembly of the intrinsic tenase (Factor IXa/Factor VIIIa) and prothrombinase (Factor Xa/Factor Va) complexes. The prothrombinase complex catalyzes the conversion of prothrombin to thrombin, which cleaves fibrinogen to produce fibrin monomers. These monomers spontaneously polymerize to facilitate the formation of fibrin clot, the structural foundation of hemostasis (6, 380, 381). The physiological necessity of this pathway is illustrated by Scott syndrome, a rare bleeding disorder where deficient TMEM16F-dependent PS exposure disrupts the assembly and activity of these coagulation complexes (30, 33, 382).

Mechanistic studies in mouse models have confirmed TMEM16F’s central role in platelet-dependent coagulation (87, 88, 383). In platelets, PS exposure following thrombin and collagen stimulation, as well as tissue factor-induced thrombin generation, is strictly dependent on TMEM16F (88). Beyond membrane scrambling, TMEM16F mediates microparticle release from activated platelets and platelet ballooning, physical changes that dramatically expand the procoagulant surface area available for clotting (88, 384, 385).

Interestingly, discrepancies in in vivo coagulation phenotypes have been observed between global and platelet-specific TMEM16F KO mice. Global KO mice exhibit prolonged tail bleeding time and an inability to form occlusive thrombi following ferric chloride injury to the carotid artery (87). In contrast, platelet-specific TMEM16F KO mice display no significant difference in tail bleeding time or fibrin clot retraction compared to WT, though laser injury-induced thrombi are smaller and more fragile (88). These findings suggest that TMEM16F in non-platelet cells, such as RBCs and endothelial cells, also contributes to coagulation (135, 327, 360, 372, 386). Indeed, RBCs from Scott syndrome patients show defective PS exposure, impaired prothrombinase activity, reduced factor Va binding, and diminished microparticle release after Ca²⁺ ionophore stimulation (25, 27, 314).

Although blood cells were traditionally considered the primary source of procoagulant PS exposure, mounting evidence shows that endothelial cells also play a significant role, especially under pathological conditions (135, 387, 388). Inflammatory stimuli such as TNF-alpha and lipopolysaccharide induce TMEM16F-dependent PS exposure and enhance endothelial procoagulant activity (135). Knockdown or pharmacological inhibition of TMEM16F markedly reduces this activity (135). Interestingly, TMEM16E, another TMEM16 family member, has also been implicated in promoting procoagulant PS exposure and may function epistatically with TMEM16F (135). Consistently, a recent study showed that endothelial-specific TMEM16F knockout mice exhibit impaired thrombosis, characterized by prolonged tail bleeding time and reduced clot size, further supporting the role of endothelial TMEM16F in promoting blood coagulation (388). These results, alongside the phenotypic differences observed in global versus platelet-specific TMEM16F KO mice (87, 88), highlight the importance of TMEM16F cell types other than platelets in blood coagulation.

Together, these findings position TMEM16F as a central regulator of extracellular procoagulant signaling across multiple cell types. By mediating Ca²⁺-induced PS externalization, TMEM16F enables the rapid assembly of coagulation enzyme complexes on the cell surface. Its functional relevance spans physiological hemostasis and pathological thrombosis, as evidenced by its role in platelets, RBCs, and endothelial cells. The consistent requirement for TMEM16F-dependent PS exposure in promoting coagulation highlights its unique role as a transducer of intracellular Ca²⁺ signals into prothrombotic extracellular cues.

4.1.2. TMEM16F-mediated PS exposure in phagocytosis and immune clearance

Phagocytosis is an essential immune process that maintains tissue homeostasis, and its dysregulation contributes to autoimmune diseases neurodegeneration, and cancer (389, 390). Surface-exposed PS serves as a critical “eat-me” signal on dying cells to attract PS receptors on phagocytes or PS binding proteins that bridge PS and phagocyte surface receptors (48). These interactions facilitate engulfment and subsequent degradation of the dying cells (48). Although PS exposure in phagocytic recognition has traditionally been attributed to caspase-dependent inactivation of flippases and activation of XKR scramblases (48), recent studies have demonstrated that TMEM16F also mediates PS exposure in stressed-but-viable cells, acting as a clearance signal during neurodegenerative conditions (373, 374) (Figure 6).

Ischemia-reperfusion promotes TMEM16F-dependent PS exposure in neurons, and TMEM16F knockdown reduces both motor deficits and microglial phagocytosis in this context (373). Similarly, in a tauopathy model, TMEM16F regulates neuronal PS exposure and microgliosis triggered by phospho-tau (374). Global and neuron-specific TMEM16F knockout mice show reduced hyperphosphorylated tau levels and microgliosis, whereas microglia-specific TMEM16F knockout did not show this phenotype, suggesting a neuron-intrinsic role for TMEM16F in disease pathology (374). These findings suggest that TMEM16F-mediated, reversible PS exposure in stressed-but-viable neurons may contribute to their recognition and removal by microglia.

It is important to note, however, that PS exposure alone may not be sufficient to trigger phagocytosis. In cells overexpressing a constitutively active form of TMEM16F, PS is continuously present on the surface, yet these viable cells are not engulfed by macrophages (308). Similarly, lymphoma cells with constitutive PS exposure are not cleared in vivo, as indicated by comparable tumor weights in mice injected with lymphoma cells with or without TMEM16F gain-of-function mutations (308). The failure of these PS-exposing cells to be removed may stem from several factors. For instance, PS exposure mediated by constitutively activated TMEM16F may be reversed by hyperactive flippases or reduced by shedding of PS positive vesicles (48). Other possibilities include the persistence of inhibitory signals such as “don’t eat me” and “don’t come close to me” cues that prevent phagocyte PS receptors to recognize exposed PS. Phagocytosis can be suppressed by anti-phagocytic molecules and receptors such as SIRPα and Siglec family members (391). Additionally, a dense glycocalyx on the cell surface may obstruct macrophage access to PS, preventing effective recognition and engulfment (392).

Interestingly, TMEM16F likely plays dual functions in phagocytosis: regulating both PS exposure on target cells and functional responses within phagocytes themselves. In macrophages exposed to transient high extracellular ATP, TMEM16F is required for PS exposure, efficient phagocytosis, bacterial killing, cell shrinkage, membrane blebbing, and migration (366). These findings identify TMEM16F as a key mediator of P2X7 receptor-driven immune responses in macrophages (366).

Subsequent studies have extended these findings to microglia, showing that under pathological conditions, TMEM16F promotes microglial motility, phagocytosis, and pro-inflammatory M1 polarization, contributing to neuronal loss and inflammation (393–395). In a peripheral nerve injury model, microglia-specific TMEM16F knockout impairs microglial movement and phagocytosis and prevents the development of mechanical allodynia (394). Similarly, TMEM16F deficiency provides protection in a spinal cord injury model, where global knockout promotes M2 polarization, reduces macrophage infiltration and pro-inflammatory gene expression, and improves motor function (395).

The role of TMEM16F in regulating microglial phenotype is further supported in Alzheimer’s disease models. In APP/PS1 transgenic mice, which exhibit spatial memory deficits and increased hippocampal TMEM16F expression, TMEM16F knockdown enhances M2 polarization, suppresses NLRP3 inflammasome activation, decreases pro-inflammatory cytokine secretion and neuronal apoptosis, and improves memory performance (393). These findings highlight TMEM16F as a critical regulator of phagocyte activation and polarization across diverse disease contexts, with its inhibition offering protective effects against neuroinflammation and tissue damage.

Altogether, TMEM16F serves as a context-specific regulator of phagocytosis by coordinating intercellular signaling between stressed cells and immune effectors. By mediating PS exposure and influencing phagocyte behavior, TMEM16F links membrane remodeling to immune recognition and inflammatory activation. Its dual roles underscore therapeutic potential in diseases driven by aberrant cell clearance or chronic inflammation, particularly in neurodegenerative and autoimmune conditions.

4.1.3. TMEM16F-mediated PS exposure in cell fusion and viral infection

Cell fusion occurs in a wide range of physiological and pathological contexts, including sperm-egg fusion during fertilization, myoblast fusion during myogenesis and muscle repair, trophoblast fusion during placental development, macrophage fusion in osteoclast differentiation and inflammation, tumor-non-tumor cell fusion during cancer progression, and viral-host cell fusion during viral infection (396, 397). Fusion usually proceeds through several key steps: recognition between fusion-competent cells, adhesion, membrane dehydration to allow close apposition, hemifusion (merging of outer leaflets of the plasma membrane), and finally, formation and expansion of the fusion pore (396–398). During this process, two or more cells need to overcome substantial physical barriers that prevent their plasma membranes from merging into a single, multinucleated cell (396, 397). In general, fusogens especially fusion proteins such as syncytin-1/2 in trophoblasts, myomaker and myomerger in myoblasts, and hapless 2 in gametes, change conformations and interact with their receptors in fusing cells to overcome the physical barriers that prevent membrane-membrane fusion in a cell-type specific manner (396, 397).

In addition to fusogens, PS exposure has emerged as a universal “fuse-me” signal in all cell fusion events that have been characterized thus far (Figure 6), including myoblast fusion (399–401), macrophage fusion (402, 403), trophoblast fusion (90, 404, 405), osteoclast fusion (406, 407), viral entry (375, 408), sperm-egg fusion (409), and cancer cell fusion (410). Notably, PS exposure during fusion is transient, reversible, and independent of apoptosis, distinguishing it from cell death-associated PS externalization (90, 399, 400).

Emerging evidence points to TMEM16F as one of the key mediators of the regulated, non-apoptotic PS exposure observed during cell fusion. In placental trophoblasts, Zhang et al. demonstrated that TMEM16F functions as the major Ca²⁺-activated phospholipid scramblase responsible for PS externalization and trophoblast fusion (90). TMEM16F is highly expressed in syncytiotrophoblasts and cytotrophoblasts within human chorionic villi, as well as in primary human trophoblasts and BeWo cells, a human placental cell line (90). In BeWo cells, TMEM16F knockout abolished Ca²⁺-induced PS exposure and significantly impaired forskolin-induced syncytialization. Notably, this PS exposure was caspase-independent and essential for fusion, as treatment with a pan-caspase inhibitor did not affect fusion efficiency, while blocking PS with annexin V, a PS-binding protein, markedly reduced fusion. In vivo, TMEM16F global knockout mice exhibited perinatal lethality, reduced placental and embryonic weights, impaired vascularization in the placenta and extraembryonic membranes, and enlarged maternal blood cavities in the labyrinth layer. While these phenotypes likely reflect a combination of systemic defects, further analysis of syncytiotrophoblast formation revealed a near-complete loss of the SynT-2 layer, as determined by immunostaining for MCT1 and MCT4, which specifically mark the SynT-1 and SynT-2 layers, respectively. Together, these in vitro and in vivo findings support a critical role for TMEM16F in regulating PS exposure and trophoblast fusion (90).

Beyond its role in trophoblasts, TMEM16F also regulates membrane fusion processes in virus-induced cell fusion (91, 375, 376). Post-mortem histological analysis of COVID-19 patients revealed widespread pneumocyte syncytia (411). Mechanistically, overexpression of the spike protein in ACE2-expressing cells upregulates TMEM16F expression and activity, leading to Ca²⁺-dependent and TMEM16F-mediated PS exposure (91, 376). Functionally, TMEM16F overexpression enhances spike-induced cell fusion, while siRNA knockdown or pharmacological inhibition of TMEM16F reduces syncytium formation and suppresses viral replication (91, 376).

TMEM16F-dependent PS exposure also participates directly in viral entry. During HIV-1 infection, engagement of the viral envelope glycoprotein (Env) with host cell coreceptors triggers Ca²⁺ influx, which activates TMEM16F and induces PS externalization to facilitate membrane fusion (375). Blocking exposed PS with PS-binding proteins such as LactC2 or full-length lactadherin inhibits Env-induced fusion, whereas exogenous PS or TMEM16F overexpression promotes fusion. Notably, another negatively charged phospholipid, phosphatidylglycerol (PG), but not the zwitterionic PC, enhances Env-mediated fusion. Temporally, PS exposure occurs after Env-coreceptor binding but before outer-leaflet hemifusion, indicating that TMEM16F-mediated PS externalization primes membranes for merger (375). Similarly, SARS-CoV-2 spike-induced calcium signals activate TMEM16F and PS exposure in host cells, and both genetic silencing and pharmacological inhibition of TMEM16F reduce SARS-CoV-2 infection and viral replication in vitro (91, 376).

TMEM16F also plays an important role in the budding of Ebola virus virions (412). At sites of viral budding, TMEM16F-mediated lipid scrambling allows nascent virions to acquire a PS-rich envelope. This PS decoration supports apoptotic mimicry, enhancing attachment and entry of Ebola virions into target cells via host PS receptors. Genetic or pharmacological inhibition of TMEM16F reduces PS incorporation into budding particles and diminishes their infectivity, indicating that TMEM16F functions as a host factor that couples Ca²⁺-dependent membrane remodeling to efficient Ebola virus spread. Together, these findings identify TMEM16F-mediated PS exposure as a common host mechanism that can be co-opted by diverse enveloped viruses to promote cell-cell fusion, viral entry, and budding, and suggest that targeting TMEM16F may provide a novel antiviral strategy in selected enveloped virus contexts.

Interestingly, TMEM16E, another CaPLSase within the TMEM16 family, also plays an important role in myoblast fusion, a critical step in skeletal muscle repair and regeneration (141, 413). This suggests that different fusogenic cell types may utilize distinct CaPLSases to mediate PS exposure and promote subsequent cell-cell fusion.

Although PS exposure is a conserved feature of diverse fusion events, the mechanisms by which externalized PS promotes membrane merger remain incompletely understood. Its function likely extends beyond simply providing a negatively charged surface and may involve effects on membrane biophysical properties, recruitment of PS-binding proteins, and regulation of fusogen activity. In addition, exposed PS can facilitate ADAM10/17 sheddase activity (377–379), raising the possibility that PS-dependent proteolytic remodeling of membrane proteins may contribute to fusion in some contexts. However, whether ADAM activation represents a general mechanism linking PS exposure to cell fusion remains to be determined.

Taken together, the above studies indicate that externalization of negatively charged phospholipids helps overcome the energetic barriers to membrane-membrane fusion, and that TMEM16 CaPLSase-mediated lipid scrambling serves as an essential driver of this process in both physiological and pathogenic contexts.

4.1.4. TMEM16F-mediated PS exposure in ADAM activation

The ADAM (A Disintegrin and Metalloproteinase) family comprises transmembrane proteases that cleave and shed ectodomains of diverse membrane-associated substrates, such as growth factors, cytokines, receptors, and adhesion molecules (414). Among these proteases, ADAM10 and ADAM17 play central roles in embryonic development, tissue homeostasis, inflammation and disease-associated signaling (414–416). Recent studies have revealed that their sheddase activity is regulated not only by protein-based mechanisms but also by membrane lipid asymmetry, particularly the exposure of PS on cell surface (377–379).

Surface-exposed PS acts as a lipid co-factor for ADAM10 and ADAM17 activation. Both proteases harbor cationic PS-binding motifs within their membrane-proximal domains, and interaction with exposed PS is proposed to position their catalytic domains near membrane-proximal cleavage sites on substrates, thereby enhancing sheddase activity (377–379) (Figure 6). Consistent with this model, ADAM-activating stimuli such as melittin, ionomycin, PMA, and FGF7 induce PS exposure in parallel with substrate shedding. Conversely, ADAM10/17 sheddase activity is suppressed when upstream signaling pathways that drive PS externalization are disrupted or when soluble O-phospho-L-serine, a PS headgroup mimic, is applied to compete with membrane PS binding (377, 379).

TMEM16F-mediated PS exposure provides an important mechanism for ADAM10 and ADAM17 activation (59, 377–379). Overexpression of wild-type or gain-of-function TMEM16F enhances cleavage of ADAM substrates such as amphiregulin, TGF-α, and CD23, and L-selectin, whereas TMEM16F knockdown or loss of Ca²⁺-induced lipid scrambling impairs ADAM10/17-dependent substrate cleavage (377–379). In Scott syndrome cells carrying loss-of-function TMEM16F mutations, ionomycin-induced PS exposure is markedly reduced, accompanied by defective shedding of ADAM17-dependent L-selectin and ADAM10-dependent CD23 (377, 379). These findings support a model in which TMEM16F-mediated lipid scrambling functions upstream of ADAM10/17 activation by supplying the exposed PS required for efficient proteolytic signaling.

This TMEM16F-ADAM axis may have important pathological implications in cancer and viral infection. In cancer-related models, enhanced TMEM16F activity has been linked to increased ADAM10-dependent CD44 cleavage and cell migration (417). This connection is illustrated by studies in naked mole-rat primary skin fibroblasts (NPSFs), which show markedly reduced Ca²⁺-induced PS externalization compared with mouse primary skin fibroblasts. In NPSFs, ADAM10 sheddase activity is strongly suppressed, and CD44 remains largely uncleaved after ionomycin stimulation despite comparable ADAM10 expression, maturation, and membrane localization (417). Introducing hyperactive TMEM16F mutations into NPSFs restores ADAM10-dependent CD44 cleavage and accelerates cell migration, supporting a direct link between TMEM16F-dependent PS exposure, ADAM10 activation, and pro-migratory cellular behavior. TMEM16F-dependent PS exposure and ADAM10/17 activity may also converge in viral infection, as ADAM10 and ADAM17 promote SARS-CoV-2 infection and spike-induced syncytium formation, processes in which TMEM16F-mediated membrane remodeling has also been implicated (91, 376, 418). Together, these studies highlight TMEM16F-mediated PS exposure as a lipid-based mechanism that couples Ca²⁺-dependent membrane remodeling to ADAM protease activation and disease-associated cellular behaviors.

4.2. TMEM16F-mediated intracellular signaling associated with cytosolic leaflet lipid remodeling

While TMEM16F is best known as a lipid scramblase that externalizes PS to initiate extracellular events, such as facilitating coagulation (87, 88, 135, 372), recruiting phagocytes (373, 374), serving as a membrane fusion signal (90, 91, 375, 376), and modulating ADAM protease activity (377–379), its ability to reshape intracellular signaling networks has been comparatively underappreciated. In particular, the consequences of depleting PS and other anionic phospholipids from the cytosolic leaflet, and the resulting effects on membrane-associated signaling proteins, have only recently begun to attract attention.

Anionic phospholipids in the cytosolic leaflet provide a key electrostatic platform that recruits cytoplasmic proteins through polybasic regions or specialized lipid-binding domains such as pleckstrin homology (PH) and C2 domains (Figure 7) (419). Proteins with polybasic domains that bind PS through largely electrostatic interactions include Src family kinases, KRAS, PIP5K, Rac1, and Rho GTPases (420–424). Proteins carrying PH or C2 domains include PKC isoforms, synaptotagmins, Raf, sphingosine kinase, Akt, and phospholipase C (419, 425, 426). These proteins regulate processes ranging from cell proliferation and neurotransmitter release to cytoskeletal remodeling, immune activation, and oncogenesis (419, 424).

FIGURE 7. — Membrane-associated proteins with polycationic domains (+++) or PS-binding domains normally interact with negatively-charged PS on the inner leaflet of the plasma membrane. TMEM16F activation leads to PS externalization and disrupts these interactions, thereby regulating intracellular signaling cascades that are important for amplifying T cell receptor signaling, dampening natural killer (NK) cell responses, regulating angiogenesis, membrane expansion, and facilitating regulated cell death, such as apoptosis, pyroptosis, and ferroptosis. Figure schematics were created with BioRender.com.

Upon TMEM16F activation, redistribution of inner-leaflet anionic phospholipids disrupts this membrane-binding platform and drives the dissociation of lipid-dependent proteins from the plasma membrane (Figure 7). Although TMEM16F exhibits measurable headgroup preference among glycerophospholipids, likely favoring PS and PC over PE (56), its broad substrate range makes scrambling of additional anionic lipids such as PIP₂ and PA plausible, though direct biochemical evidence for these specific substrates remains limited (268, 297, 299). This combined depletion of PS, PIP₂, and PA weakens both electrostatic and lipid-specific interactions, leading to the relocalization of membrane-associated signaling proteins and reorganization of signaling complexes (Figure 7) (422). Given that anionic lipid-binding proteins govern diverse cellular processes (419, 424), TMEM16F-dependent lipid redistribution can profoundly reshape intracellular signaling dynamics and cellular behavior.

In this section, we focus on emerging observations that TMEM16F-mediated lipid scrambling not only generates extracellular PS cues, but also remodels the inner leaflet electrostatic landscape, thereby regulating membrane-proximal kinases, junctional complexes, and signaling pathways that control T cell activation, natural killer (NK) cell function, angiogenesis, membrane expansion, and cell death.

4.2.1. TMEM16F regulates intracellular signaling in immune cells

TMEM16F can modulate immune-cell signaling by altering lipid–protein interactions at the plasma membrane. In T cells, the positively charged cytoplasmic domain of CD3ε (CD3εCD) associates electrostatically with anionic lipids in the inner leaflet (427). Upon TCR engagement, Ca²⁺ influx activates TMEM16F and promotes PS externalization, weakening this membrane interaction and releasing the cytoplasmic domains of bystander TCR-CD3 complexes from the membrane. This release enhances TCR signaling and T cell activation, whereas TMEM16F depletion suppresses and gain-of-function TMEM16F enhances these responses (428).

However, the underlying electrostatic mechanism remains incompletely resolved. TCR stimulation and experimental elevation of Ca²⁺ can also activate PLCγ and deplete PI(4,5)P₂, which, despite its lower abundance than PS, contributes strongly to inner-leaflet charge because of its high charge density. Moreover, Ca²⁺-independent activation of the gain-of-function TMEM16F (95) produced robust PS externalization without a detectable change in phosphoinositide distribution or global inner-leaflet charge (429). TMEM16F-mediated PS scrambling should therefore not be assumed to uniformly reduce the electrostatic potential of the entire inner leaflet. Instead, the T-cell findings may reflect local or context-dependent remodeling of anionic lipid-protein interactions at the immunological synapse, potentially together with PLC-dependent phosphoinositide hydrolysis.

In NK cells, TMEM16F-mediated reduction of inner-leaflet PS destabilizes Src family kinase membrane association, leading to decreased NK cell activation. TMEM16F was activated in NK cell lines or primary NK cells via Ca²⁺ ionophores, target cell co-culture, or PMA, with effects confirmed by TMEM16F overexpression (WT or activated D408G mutant) or knockdown. TMEM16F activation in NK cells promotes the dissociation of the Src family kinases Fyn and Lck from the plasma membrane, resulting in reduced NK cell activation. Fyn and Lck contain polybasic regions that mediate electrostatic interactions with anionic phospholipids, including PS, to support their stable membrane localization and signaling competence. TMEM16F-mediated scrambling decreases inner-leaflet PS abundance and weakens these electrostatic interactions, leading to kinase redistribution away from the plasma membrane and attenuation of downstream activating signals (430). Of note, these approaches did not explicitly exclude lipase-mediated PIP₂ hydrolysis during Ca²⁺ rises, which could similarly disrupt polybasic region interactions; hence, PIP₂ changes represent one possible alternative or complementary mechanism. Thus, similar to observations in other cell types, Src family kinases in NK cells behave as PS-dependent signaling proteins whose membrane association is sensitive to TMEM16F-driven changes in the inner-leaflet electrostatic landscape.

These findings demonstrated that TMEM16F-mediated lipid redistribution acts as an intracellular signaling modulator, dynamically tuning the localization and activity of PS-binding proteins. By controlling the electrostatic landscape of the inner leaflet, TMEM16F extends its functional repertoire beyond extracellular signaling and fine-tunes immune cell activation in a context-dependent manner.

4.2.2. TMEM16F regulates intracellular signaling in endothelial cells

Beyond immune cells, TMEM16F also plays a critical role in regulating intracellular signaling in endothelial cells, with direct consequences for angiogenesis (Figure 7). The first indication in mammals came from Tmem16f-deficient mouse placentas, which exhibited impaired fetal blood vessel development (90). To disentangle endothelial-intrinsic effects from defective trophoblast fusion, Shan et al. combined in vitro tube formation assays using HUVECs with in vivo analyses of retinal vascular development in neonatal mice (361). Both systems demonstrated that endothelial TMEM16F is essential for angiogenesis in mammals, consistent with an earlier observation of angiogenic defects in a morpholino-based zebrafish model (431).

Mechanistically, TMEM16F regulates angiogenesis through intracellular signaling, particularly the Src signaling pathway (361). In TMEM16F-deficient endothelial cells, retention of anionic phospholipids at the inner leaflet enhances Src kinase recruitment to the plasma membrane, leading to increased Src activation, excessive VE-cadherin phosphorylation, and subsequent VE-cadherin internalization and degradation. Proximity ligation assays further confirm increased phosphorylated Src kinase at the membrane in TMEM16F knockdown cells, consistent with prior work in macrophages and NK cells showing that inner-leaflet lipid composition regulates Src family kinase localization (422, 430, 432). This Src-VE-cadherin signaling axis destabilizes adherens junctions, impairs endothelial barrier integrity, disrupts sprouting behavior, and ultimately compromises angiogenic capacity.

Together with studies in immune cells (428, 430), these findings support a dual-function model for TMEM16F. While PS externalization is well recognized for mediating extracellular signaling events, the same scramblase also modulates intracellular pathways by tuning inner-leaflet lipid composition and the localization of membrane-associated signaling proteins. Given the large repertoire of PS-binding proteins, TMEM16F and other PLSases likely coordinate additional intracellular signaling networks that remain to be defined in future studies.

4.2.3. TMEM16F regulates membrane expansion through inner leaflet lipid remodeling

The asymmetric enrichment of anionic phospholipids in the cytoplasmic leaflet contributes to membrane organization by regulating the association of membrane-binding proteins involved in membrane constriction and trafficking. Recent studies suggest that TMEM16F-mediated scrambling of cytoplasmic anionic phospholipids can alter these protein-lipid interactions and thereby promote plasma membrane expansion (363) (Figure 7). Membrane remodeling often requires local membrane expansion to provide additional surface area for biological processes such as blebbing, vesicle release, phagocytic engulfment, enzymatic reactions, and cytokinesis (433–436). A well-established upstream trigger for these rapid membrane expansion events is an elevation of intracellular Ca²⁺, which drives membrane repair, cell spreading, and immune cell activation (363, 434, 437–439).

In Jurkat T cells, ionomycin-induced Ca²⁺ influx leads to massive membrane expansion, which coincides with PS exposure, followed by gradual membrane area reduction and concurrent vesicle shedding (434). In contrast, TMEM16F knockout cells exhibit a 50% reduction in membrane area after ionomycin treatment. This reduction is accompanied by robust endocytosis rather than vesicle release. Mechanistically, Ca²⁺-induced membrane expansion may occur through several overlapping pathways, including the release of membrane reservoirs, fusion of intracellular vesicles with the plasma membrane, and lipid transfer from internal organelles (437–439). One proposed mechanism is that Ca²⁺-dependent activation of TMEM16F relaxes dynamin-constricted plasma membrane invaginations, thereby releasing pre-existing membrane reservoirs (363). Dynamins are large GTPases that mediate membrane constriction during fission, endocytosis, and membrane invagination formation (440, 441). As dynamin preferentially binds to negatively charged phospholipids (442, 443), depletion of cytoplasmic anionic phospholipids by TMEM16F-mediated scrambling may weaken dynamin-dependent constriction of PM invaginations, thereby releasing membrane reservoirs and enabling rapid surface expansion (363). Importantly, unlike dynamin’s GTPase-dependent role in endocytosis, GTPase activity is not required for dynamin-mediated closure of these PM reservoirs or for TMEM16F-driven expansion (363). Consistent with this model, sequestration of cytoplasmic anionic phospholipids triggered moderate PM expansion, mimicking TMEM16F activation (363). Nonetheless, alternative mechanisms, such as enhanced vesicle-plasma membrane fusion or lipid transfer from organelles, have not been excluded. A deeper understanding of how TMEM16F regulates plasma membrane expansion may provide insight into the general mechanisms of membrane remodeling, as well as intracellular processes involving membrane expansion, including those mediated by intracellular scramblases during autophagosome formation and lipid droplet biogenesis (36, 277, 282).

Physiologically, TMEM16F-mediated membrane expansion and subsequent vesicle release may modulate immune signaling through the regulation of PD-1, an immune checkpoint receptor that inhibits T cell activation. In wild-type cells, both surface and total PD-1 levels significantly decrease after ionomycin treatment, likely due to vesicle shedding. In contrast, TMEM16F KO cells show a decrease in surface PD-1 while maintaining the total PD-1 levels, suggesting that PD-1 is internalized into endosomes rather than released from the cell surface (434). This impaired vesicle shedding and failure to downregulate PD-1 expression may explain why TMEM16F-deficient T cells retain higher PD-1 levels and exhibit features of functional exhaustion (444). However, it remains unclear whether and how PD-1-containing endosomes reintegrate into the plasma membrane to mediate inhibitory signaling during T cell activation. Furthermore, reports on TMEM16F’s role in endocytosis are inconsistent: Bricogne et al. observed that TMEM16F activation by ionomycin suppresses endocytosis, whereas Hu et al. reported TMEM16F localization to late endosomes following T cell receptor stimulation. These discrepancies may reflect differences in cellular context or stimulation mode and warrant further investigation.

4.2.4. TMEM16F modulates multiple regulated cell death pathways in a context-dependent manner.

While TMEM16F contributes to various aspects of cellular homeostasis, it has also been increasingly associated with the regulation of multiple forms of regulated cell death (RCD) (445). RCD is a highly orchestrated process governed by intracellular signaling networks and is essential for physiological events such as tissue homeostasis and remodeling, as well as pathological conditions including neurodegeneration, autoimmune disorders, and tumorigenesis (446). Importantly, although XKR-dependent PS exposure frequently accompanies cell death, PS externalization is not universally coupled to apoptotic execution and can occur independently in diverse physiological and pathological contexts. Moreover, while TMEM16F contributes to membrane remodeling and signaling events associated with several forms of RCD, its role is often indirect, context-dependent, and distinct from the primary execution machinery of these pathways. TMEM16F has been linked to several distinct RCD pathways, including apoptosis, pyroptosis, necroptosis, and ferroptosis (445, 447), though the precise molecular mechanisms remain incompletely understood. Nonetheless, its involvement in these pathways positions TMEM16F as a context-dependent modulator of intracellular signaling events that influence cell fate.

Apoptosis is a non-lytic and non-inflammatory form of cell death (448). During apoptosis, PS exposure is primarily mediated by the XKR caspase-activated scramblases, rather than by TMEM16F (35, 383). Nonetheless, several studies suggest that TMEM16F contributes to apoptotic progression through different mechanisms. Specifically, TMEM16F has been shown to promote cell shrinkage and facilitate apoptotic signaling (329, 449, 450). Apoptosis induced by Fas ligand or staurosporine activates TMEM16F, and TMEM16F knockdown suppresses apoptosis-associated cell shrinkage and caspase-3 activation (329, 449). Conversely, TMEM16F overexpression enhances apoptosis and modulates gene expression by increasing Bax and decreasing Bcl-2 levels (329, 450). Collectively, while TMEM16F is not the major contributor to apoptotic PS exposure, it facilitates apoptosis by influencing cellular morphology and downstream caspase-dependent pathways.

In contrast to apoptosis, pyroptosis is a lytic and pro-inflammatory form of regulated cell death. It is typically triggered by microbial infection or inflammatory stimuli, which activate caspase-1 or caspase-11/4/5 and lead to gasdermin D cleavage (451). The cleaved N-terminal fragment of gasdermin D oligomerizes to form membrane pores, resulting in Ca²⁺ influx, cell swelling, membrane rupture, and the release of pathogen- and damage-associated molecular patterns (451, 452). Overexpression of gasdermin D induces a marked rise in intracellular Ca²⁺ and subsequent activation of TMEM16F (453). Notably, TMEM16F knockdown reduces gasdermin D-mediated cell death and suppresses lactate dehydrogenase (LDH) release (453), indicating a functional contribution to pyroptosis. In contrast, TMEM16F does not appear to be involved in necroptosis, another form of lytic cell death. Although necroptosis induced by TNFα, SMAC mimetic, and Z-VAD (TSZ) is associated with Ca²⁺ influx and TMEM16F activation, TMEM16F knockdown fails to attenuate necroptotic cell death (454). These findings suggest that TMEM16F selectively amplifies pyroptotic, but not necroptotic, responses, reflecting its context-dependent involvement in inflammatory cell death.

Ferroptosis is a distinct form of regulated, non-apoptotic cell death driven by iron-dependent phospholipid peroxidation (455). It has been implicated in diverse pathological conditions, including neurodegeneration, cancer, and immune dysregulation (456, 457). TMEM16F has recently emerged as a modulator of ferroptotic cell death, though its precise role is still under debate.

Early studies reported that TMEM16F promotes ferroptosis. In TMEM16F-deficient cells, ferroptotic death was significantly attenuated across multiple cell types, including peritoneal macrophages, Jurkat T cells, and cancer lines such as pulmonary adenocarcinoma and osteosarcoma (447). In an Alzheimer’s disease cell model, β-amyloid was shown to increase TMEM16F expression and elevate reactive oxygen species (ROS) levels, accompanied by upregulation of ferroptosis-associated proteins in a TMEM16F-dependent manner (458). Similarly, TMEM16F overexpression in gastrointestinal stromal tumors has also been associated with increased levels of ferroptosis-related markers, such as ROS accumulation, elevated intracellular iron, and changes in lipid metabolism gene expression, although these findings were not confirmed using ferroptosis-specific inducers or inhibitors (450). In endothelial cells, erythrophagocytosis-induced ferroptosis triggered by uremia or hypoxia in pulmonary arterial hypertension models is correlated with TMEM16F upregulation and increased PS exposure (459, 460). These findings underscore a potential link between ferroptotic signaling and TMEM16F-dependent membrane remodeling in pathological contexts such as thrombosis and pulmonary vascular remodeling. In addition to Ca²⁺-dependent activation, TMEM16F has been shown to be responsive to ROS (461, 462), which may represent an alternative activation mechanism during ferroptosis. Beyond ferroptosis, TMEM16F is known to regulate membrane remodeling processes such as bleb formation in response to diverse stimuli across multiple cell types (447, 450, 463, 464). While membrane blebbing has been observed during ferroptosis in certain cell types, such as placental trophoblast (465), the molecular mechanisms driving this phenotype remain poorly defined. Together, these observations suggest that TMEM16F contributes to ferroptotic membrane disruption and cell death, although the precise links between its scramblase activity, lipid peroxidation, and plasma membrane destabilization remain to be fully elucidated.

In contrast, a recent 2025 study proposed that TMEM16F functions as a suppressor of ferroptosis, protecting cells by mediating Ca²⁺-dependent phospholipid scrambling at discrete plasma membrane lesion sites (466). According to their model, TMEM16F redistributes phospholipids locally to reduce membrane tension and delay rupture, without influencing lipid peroxidation. TMEM16F-deficient cells were reported to be hypersensitive to ferroptosis, while gain-of-function mutants protect against it. Notably, the study also reported that Ca²⁺ chelation by EGTA addition increased ferroptotic death. This model was further linked to tumor immunology, where TMEM16F loss promoted lytic ferroptosis, DAMP release, and improved response to anti-PD-1 therapy.

Although compelling, these conclusions are at odds with multiple lines of evidence. A substantial body of literature indicates that Ca²⁺ influx promotes ferroptosis rather than preventing it. Ca²⁺ entry precedes membrane rupture and is required for ferroptotic execution (467–469). Ca²⁺ chelators, including BAPTA-AM and EGTA, have been shown to suppress ferroptosis in multiple systems (470–472), and disruption of intracellular Ca²⁺ release similarly mitigates ferroptotic death (473). These results directly oppose the idea that Ca²⁺-dependent TMEM16F activation is broadly protective. Mechanistically, Ca²⁺ influx activates downstream pathways required for ROS production and lipid peroxidation amplification (474, 475), suggesting that sustained Ca²⁺ elevations should potentiate, not limit, ferroptotic injury.

Moreover, TMEM16F-mediated ion conductance has been linked to changes in membrane integrity under high Ca²⁺ conditions, but its direct role in ferroptotic membrane destabilization remains unresolved, and strong causal evidence is limited (447, 464). While the 2025 study emphasizes spatially restricted scrambling, it is unclear whether this mechanism is sufficient to counteract the high oxidative burden characteristic of ferroptosis. Importantly, the protective effect of EGTA observed in multiple earlier studies contrasts with the increased ferroptosis sensitivity reported in the 2025 paper, raising the possibility that their observations reflect cell-type–specific Ca²⁺ handling, distinct stages of ferroptotic progression, or assay-dependent variability.

Taken together, while the 2025 study (466) introduces a novel and potentially context-specific model of TMEM16F as a ferroptosis suppressor, most prior evidence, including work on Ca²⁺ dependence, ROS responsiveness, and TMEM16F-associated membrane remodeling, supports a pro-ferroptotic function. TMEM16F’s role may indeed vary with cell identity, Ca²⁺ dynamics, or the temporal sequence of membrane injury. Nevertheless, the broad interpretation that TMEM16F-mediated scrambling universally protects against ferroptosis is difficult to reconcile with extensive data showing that Ca²⁺ influx and TMEM16F activity tend to facilitate, rather than prevent, ferroptotic membrane failure.

More generally, across multiple forms of regulated cell death, TMEM16F functions as a context-dependent modulator of cell fate rather than a pathway-specific effector. In this broader framework, disruption of phospholipid asymmetry may contribute to cell death regulation through coordinated changes in both inner and outer membrane leaflets, linking intracellular signaling with membrane remodeling processes. These membrane remodeling aspects are discussed in the following section.

4.3. TMEM16F-mediated membrane remodeling associated with integrated cellular signaling responses

TMEM16F-mediated lipid scrambling not only generates extracellular and intracellular lipid signals but also remodels the biophysical properties of the plasma membrane. In resting mammalian cells, the two membrane leaflets differ in lipid composition and biophysical properties: the exoplasmic leaflet is relatively enriched in more saturated, tightly packed lipids, whereas the cytoplasmic leaflet contains higher levels of anionic phospholipids, such as PS and phosphoinositides, as well as phospholipids with more unsaturated acyl chains (10, 307, 476, 477). Upon activation, lipid scrambling can dissipate selected transbilayer phospholipid gradients and expose normally inner-leaflet lipids, such as PS and PE, on the cell surface. Rather than simply converting an asymmetric membrane into a fully symmetric one, scrambling likely remodels the bilayer in a context-dependent manner by altering leaflet composition, surface charge, lipid packing, curvature stress, and the recruitment of membrane-associated proteins. These changes can facilitate membrane deformation, vesiculation, membrane expansion, repair, phagocytic recognition, and other downstream cellular responses (307, 478) (Figure 8). In this section, we summarize how TMEM16F-dependent lipid scrambling contributes to membrane blebbing, vesiculation, and repair.

FIGURE 8. — TMEM16F-mediated lipid flip-flop alters the biophysical properties of the plasma membrane, enabling membrane remodeling manifested as bleb formation, vesicle shedding, and lesion removal during membrane repair. Figure schematics were created with BioRender.com.

4.3.1. Membrane blebbing

Membrane blebbing is a dynamic membrane remodeling process in which the plasma membrane forms temporary, spherical bulges or protrusions (479). Membrane blebbing is commonly observed during cell death such as apoptosis (480, 481) and ferroptosis (465), cytokinesis (482), cell migration (433), cell spreading (483), cancer invasion (484), membrane repair (485), and cell fusion (486). Membrane blebs are typically initiated by localized weakening of the cortical actin network and its detachment from the plasma membrane, followed by expansion mediated by membrane-actin linker proteins such as ezrin, radixin, and moesin (ERM). Subsequent F-actin re-assembly at the bleb cortex drives contraction and retraction (433, 479). While the cytoskeletal basis of blebbing is relatively well characterized, how the plasma membrane environment is remodeled to enable bleb initiation is less well understood.

Recent studies suggest that TMEM16F activity can contribute to bleb formation in specific contexts through membrane remodeling (Figure 8). In chemically induced blebbing models, cells generate Ca²⁺- and TMEM16F-dependent giant plasma membrane vesicles accompanied by extensive phospholipid scrambling (320, 478). Beyond chemical stimuli, other triggers of blebbing, including nanosecond pulsed electric field (464), pore-forming toxins (463), Ca²⁺ ionophores (478), and ATP stimulation of P2X7 receptors (366), also require TMEM16F activity.

Mechanistically, TMEM16F-dependent lipid scrambling appears to facilitate bleb formation by reducing lipid packing and thereby increasing membrane deformability (478). Loss of lipid asymmetry has long been associated with mechanical instability of the plasma membrane (487). Consistently, TMEM16F activation results in a more loosely packed plasma membrane, and blebbing phenotypes in TMEM16F-deficient cells can be rescued by interventions that similarly decrease lipid packing, such as cholesterol extraction, elevated temperature, or sodium deoxycholate treatment (478).

In addition to altering lipid packing, TMEM16F-mediated phospholipid redistribution may facilitate blebbing through effects on membrane-cytoskeleton interactions and membrane curvature (488). ERM proteins, which link F-actin to the plasma membrane, preferentially bind to negatively charged phospholipids such as PIP₂ (489–491). It has been proposed that TMEM16F activation exposes negatively charged phospholipids, destabilizing ERM-membrane interactions and thereby facilitating bleb initiation (488). However, ERM proteins display much stronger affinity for PIP₂ compared with PS (489–491), and loss of PIP₂ can also occur in TMEM16F-deficient cells, likely as a result of Ca²⁺-induced activation of phospholipase C (478). Further studies will be required to determine whether TMEM16F directly scrambles PIP₂ or whether PS exposure alone is sufficient to disrupt ERM anchoring at the plasma membrane.

The morphology and dynamics of blebs vary across cell types, underscoring the context-dependent nature of bleb formation. While TMEM16F expression and Ca²⁺ influx are required in many systems, the relative contributions of lipid scrambling, Ca²⁺ signaling, and altered lipid packing to bleb initiation remain unresolved. In some contexts, additional pathways may contribute to bleb formation, raising the possibility that multiple molecular mechanisms can converge on similar morphological outcomes. Clarifying how TMEM16F is activated in different settings, and how it coordinates with other processes, will be key to understand how membrane remodeling drives cell type-specific responses.

4.3.2. Vesicle release

4.3.2.1. TMEM16F-mediated vesicle release in blood coagulation

Vesicle release from the plasma membrane is a major form of membrane remodeling in which microvesicles bud directly from the cell surface and undergo fission to release portions of the plasma membrane and cytosol (492–495). This process enables activated or stressed cells to export bioactive cargos, including lipids, proteins, and nucleic acids, and to communicate with neighboring cells. Distinct from exosome release, which relies on fusion of internal endosomal vesicles with the plasma membrane, vesicle release occurs via outward budding, a process facilitated by phospholipid scrambling and cytoskeletal rearrangements (492, 494). Central to this process is TMEM16F-dependent lipid scrambling, which has emerged as an important mechanism facilitating plasma membrane vesiculation (Figure 8). Activation of TMEM16F leads to phospholipid redistribution and outward budding from the cell surface, resulting in vesicle shedding across multiple cell types (24, 88, 320, 360, 372, 463). These vesicles, in turn, contribute to diverse physiological functions, including hemostasis, immunity, and intercellular communication.

In coagulation, TMEM16F-mediated PS exposure on the plasma membrane generates platforms for assembling coagulation factors and initiating thrombin generation (see section 4.1.1). Beyond surface PS exposure, PS-positive microparticles released from activated cells serve as additional procoagulant surfaces for coagulation (24, 495). TMEM16F plays an indispensable role in generating these procoagulant microvesicles from platelets, RBCs, and ECs (24, 88, 320, 360, 372, 463). Upon platelet activation by Ca²⁺ ionophore, thrombin, or collagen, TMEM16F mediates Ca²⁺-dependent PS scrambling and membrane budding, driving the release of PS-exposed vesicles. In Scott syndrome, caused by loss-of-function mutations in TMEM16F, platelet-derived vesicle release and associated procoagulant activity are markedly reduced (24). Consistently, platelet-specific Tmem16f knockout mice exhibit drastically impaired microparticle release and diminished thrombin generation in response to activation (88). TMEM16F also governs platelet membrane morphology during activation. Wild-type platelets undergo dynamic shape transitions from rod-like structures to flattened remnants, whereas TMEM16F-deficient platelets remain arrested in the rod-like state, underscoring TMEM16F’s role in membrane shedding and microparticle release (88).

Defective vesiculation extends beyond platelets. RBCs from Scott syndrome patients show diminished vesicle release and reduced factor Va binding (25). Recent characterization of RBCs from Tmem16f-deficient mice further demonstrated that TMEM16F is a major driver of Ca²⁺-induced microparticle release (360). Conversely, sickled RBCs from patients with SCD show enhanced TMEM16F scrambling activity, which correlates with increased release of PS-positive microparticles. Pharmacological inhibition of TMEM16F activation effectively suppresses microparticle release from sickle RBCs. These findings indicate that increased TMEM16F activity is a major contributor to the release of thrombogenic microparticles in SCD and suggest that TMEM16F inhibition may reduce SCD-associated thrombotic risk, in part by limiting microparticle generation. In endothelial cells, TMEM16F activation similarly promotes microparticle release under pathological conditions. For instance, in hyperuricemia, uric acid induces TMEM16F-dependent PS exposure and microparticle release, thereby augmenting endothelial procoagulant activity (372).

4.3.2.2. TMEM16F-mediated vesicle release beyond coagulation

An emerging question is whether TMEM16F simply enables membrane budding or also influences cargo selection. Although direct evidence for a dedicated TMEM16F-dependent cargo-sorting machinery remains limited, TMEM16F-mediated PS exposure, membrane curvature changes, and cytoskeletal remodeling may help define budding sites that preferentially incorporate specific lipid, protein, or protein-aggregate cargo in a cell type- and stimulus-dependent manner. In this context, TMEM16F-dependent vesicle release has been implicated in biological processes beyond hemostasis, including bone mineralization (89), immune regulation (496), and neurodegenerative disease (497).

In skeletal development, TMEM16F-mediated membrane remodeling may contribute to the formation or function of osteoblast-derived matrix vesicles. During embryogenesis, TMEM16F is strongly expressed in differentiating and mature osteoblasts and is required for normal skeletal morphology and mineral deposition (89). Tmem16f deficiency does not appear to impair lineage differentiation of chondrocytes, osteoblasts, or osteoclasts, but delays and reduces osteoblast mineralization, resulting in smaller and deformed skeletal structures during embryonic development. Although many skeletal abnormalities largely resolve in adulthood, adult Tmem16f knockout mice retain a mild mineralization defect characterized by accumulation of unmineralized osteoid (89). Because bone mineralization depends on the release of matrix vesicles that concentrate Ca²⁺, phosphate, and mineralization-associated lipids to initiate hydroxyapatite nucleation (498), it is plausible that TMEM16F facilitates mineral deposition by promoting vesicle budding from osteoblast membranes or by influencing the lipid composition of matrix vesicles (6). The contribution of PS to mineralization remains incompletely defined. PS has been proposed to stabilize Ca²⁺-phosphate complexes and promote mineral nucleation (499, 500), whereas TMEM16F activation induces PS externalization in osteoblasts (89). How TMEM16F-dependent scrambling controls PS distribution, matrix vesicle cargo, and subsequent mineral deposition remains an important open question.

TMEM16F-dependent vesicle release also contributes to immune regulation in inflammatory disease. In rheumatoid arthritis, an autoimmune disease that leads to joint inflammation and cartilage degradation, neutrophil-derived microparticles are enriched in the pro-resolving and anti-inflammatory protein annexin A1, and are elevated in synovial fluid compared with plasma (496). In a murine model of inflammatory arthritis, Tmem16f-deficient mice exhibited reduced shedding of neutrophil-derived microparticles, more severe cartilage destruction, and greater loss of sulfated glycosaminoglycans, despite comparable inflammation and synovitis (496). These findings suggest that TMEM16F-dependent vesiculation supports cartilage protection at least in part by enabling the release of AnxA1-enriched microparticles. Whether TMEM16F directly promotes AnxA1 sorting into budding vesicles or instead licenses vesicle release from pre-existing AnxA1-enriched membrane domains remains to be determined. Future studies using cell type-specific TMEM16F deletion and proteomic/lipidomic profiling of released vesicles could clarify how TMEM16F-dependent microparticles acquire protective cargo during inflammatory challenge.

In the nervous system, TMEM16F-dependent vesicle release has been linked to the intercellular spread of pathogenic protein aggregates. Synucleinopathies, including Parkinson’s disease, are characterized by abnormal aggregation and deposition of α-synuclein, and extracellular vesicle-mediated release of α-synuclein is thought to contribute to disease propagation (501). Consistent with this model, TMEM16F-dependent membrane remodeling and vesicle release facilitate the extracellular release and intercellular transmission of pathogenic α-synuclein species, whereas TMEM16F loss reduces aggregate propagation in vivo (497). These findings suggest that TMEM16F-mediated vesiculation can influence not only the quantity of vesicles released but also the export of disease-relevant protein cargo.

Taken together, these examples indicate that TMEM16F-dependent vesicle release is not limited to generating procoagulant surfaces. Instead, TMEM16F may shape intercellular communication by coupling Ca²⁺-dependent lipid scrambling to the release of vesicles carrying context-specific cargo, such as mineralization-associated lipids and ions, pro-resolving proteins, or pathogenic protein aggregates. Defining how TMEM16F-dependent budding sites are selected and how cargo is incorporated into these vesicles remains a key challenge for future studies.

4.3.3. Membrane repair

Membrane repair is a cellular process that rapidly reseals sites of plasma membrane injury caused by mechanical stress, pore-forming toxins, or electric pulses, thereby preventing cytolysis and maintaining homeostasis (438). Failure to repair damaged membranes can lead to Ca²⁺ overload, mitochondrial dysfunction, ROS accumulation, and cell death, whereas dysregulated repair underlies a range of human pathologies, including muscular dystrophies and cardiomyopathies (438, 502). Repair can be achieved through multiple mechanisms, including exocytotic patching of damaged sites, blebbing and vesicle shedding, constriction and pinching off of lesions, or endocytic internalization, with the choice of mechanism often depending on cellular context (503). Ca²⁺ influx is a near-universal trigger for these processes, coordinating rapid recruitment of repair machinery and facilitating phospholipid rearrangements at the injury site (438, 503).

Indeed, Ca²⁺ influx at the site of damage, induced by pore-forming toxins, detergents, complements, or nanosecond pulsed electric fields, activates TMEM16F, triggering lipid scrambling and vesicle shedding, which facilitates lesion removal and plasma membrane resealing (463, 464). Cells lacking TMEM16F display increased membrane damage and reduced survival under these conditions, highlighting the functional importance of TMEM16F-mediated vesicle shedding in repair (463). When TMEM16F-deficient cells are exposed to pore-forming toxins such as listeriolysin O produced by Listeria monocytogenes, they fail to turn on an effective repair response, exhibiting rapid and uncontrolled membrane damage. Tmem16f-deficient mice exhibit compromised control of Listeria infection and show greater neutrophil sensitivity to the toxin, directly linking TMEM16F function to survival against membrane-damaging pathogens (463) (Figure 8).

Recent work further supports a broader role for Ca²⁺-activated lipid scrambling in membrane repair beyond the plasma membrane. In the context of lysosomal membrane damage, Niekamp et al. identified a Ca²⁺-dependent, endosomal sorting complexes required for transport (ESCRT)-independent repair pathway driven by sphingomyelin scrambling and turnover. Using a fluorescent cytosolic sphingomyelin probe, the authors showed that treatment with the Ca²⁺ ionophore ionomycin induces TMEM16F-dependent sphingomyelin scrambling at the plasma membrane. In contrast, when lysosomal damage is induced by L-leucyl-L-leucine O-methyl ester (LLOMe) in the presence of Ca²⁺, sphingomyelin is rapidly exposed on the cytosolic surface of lysosomes in a manner independent of TMEM16F expression. These findings imply the existence of a TMEM16F-like lysosomal scramblase that mediates sphingomyelin translocation across the lysosomal membrane. Importantly, cytosolic sphingomyelin exposed during lysosomal damage is converted to ceramide by neutral sphingomyelinase 2, which promotes inward membrane budding and facilitates lysosomal membrane repair (504). However, the molecular identity of the lysosomal scramblase remains unknown.

Together, these studies suggest that Ca²⁺-activated phospholipid scrambling constitutes a conserved membrane repair strategy that operates at distinct cellular membranes through context-specific scramblases. TMEM16F mediates plasma membrane repair primarily through lipid scrambling and vesicle shedding, whereas lysosomal repair relies on a distinct, yet unidentified, scramblase that promotes sphingomyelin exposure and ceramide-driven membrane remodeling. How TMEM16F-mediated plasma membrane repair interfaces with other repair pathways, such as ESCRT-mediated constriction or annexin-dependent membrane stabilization, remains unclear. Additionally, whether TMEM16F’s sphingomyelin scrambling activity contributes directly to plasma membrane repair, analogous to the sphingomyelin-dependent mechanism described at lysosomes, warrants further investigation.

5. TMEM16F IN DISEASE

TMEM16F has emerged as a key scramblase at the crossroads of physiology and pathology. Dysregulated TMEM16F activity, through germline mutations, polymorphisms, or altered expression, has been implicated in a growing spectrum of human diseases (Figure 9). These range from rare Mendelian scramblaseopathies (diseases caused by genetic mutations of lipid scramblases), to complex disorders including HX, SCD, neurodegeneration, viral infection, vascular disease, autoimmune disorders, and cancer. In this section, we summarize current evidence connecting TMEM16F to human pathology, distinguishing settings where it acts as a primary disease driver from those in which it functions as a critical modifier of disease progression.

FIGURE 9. — Figure schematics were created with BioRender.com.

5.1. TMEM16F scramblaseopathy and polymorphism

5.1.1. Scott syndrome

Loss-of-function TMEM16F scramblaseopathy leads to Scott syndrome, a rare, autosomal recessive thrombopathy characterized by defective Ca²⁺-induced PS exposure on blood cells (Figure 9) (30, 33, 382). This mild bleeding disorder was first described by Weiss et al., reporting a patient with impaired platelet procoagulant activity and defective thrombin generation despite normal platelet count, adhesion, aggregation, and secretion (28). Subsequent studies revealed that factor Va activity was normal, but factor Xa binding to the patient’s platelets was markedly reduced (76), implicating a deficiency in platelet surface sites required for factor Va-factor Xa assembly. Consistently, reduced thrombin generation was accompanied by a decrease in factor Xa production (30). Although the bulk phospholipid composition of Scott syndrome platelets is normal (28), PS exposure on the outer leaflet is defective (30). In addition to abnormal procoagulant activity, Scott syndrome platelets exhibit markedly reduced microparticle release upon stimulation (24). It is worth noting that the defect extends beyond platelets. RBCs from Scott patients show impaired procoagulant activity, reduced microvesicle release, and defective phospholipid scrambling (25, 505). Similarly, the immortalized B lymphocytes from the patient display diminished PS exposure (26, 314). Intriguingly, recent work in the endothelial cell-dependent coagulation suggests that the related scramblase TMEM16E may act epistatically with TMEM16F, raising the possibility that other scramblases could mitigate the consequences of TMEM16F loss in Scott syndrome (135).

Scott syndrome has also been identified in canines (506, 507). An inbred colony of affected dogs exhibits clinical and cellular features resembling human patients, including diminished platelet procoagulant activity and impaired PS scrambling despite normal platelet count, morphology, and phospholipid composition (506, 507). Pedigree analyses in both humans and dogs indicate that Scott syndrome follows an autosomal recessive inheritance pattern (27, 506, 507). Tmem16f-deficient mice also recapitulate Scott syndrome, with markedly reduced Ca²⁺-induced PS exposure in platelets, RBCs, and splenic B cells and severely impaired platelet thrombin generation (87). With the advent of genetic testing, additional Scott patients carrying different TMEM16F loss-of-function variants have been identified in humans (508–512), and canines (507, 513). Most recently, a homozygous frameshift variant, c.1943del (p.Arg648Hisfs23), was reported in a 12-year-old girl with excessive bleeding, prolonged menorrhagia, and iron deficiency (510). In both global and platelet-specific TMEM16F knockout mice, PS exposure in response to thrombin/collagen and tissue factor-induced thrombin generation is TMEM16F-dependent, and TMEM16F is required for microparticle release from activated platelets, reinforcing its central role in procoagulant activity (88).

To date, only a handful of TMEM16F/ANO6 variants have been reported in unrelated Scott syndrome families, underscoring the extreme rarity of this condition despite its relatively mild bleeding phenotype (33, 382, 508–512). This apparent discrepancy may have several, non-mutually exclusive explanations. First, Scott syndrome is likely underdiagnosed because bleeding manifestations are often mild and conventional hemostasis tests can be normal or only subtly abnormal (514). Second, TMEM16F-independent scramblases or compensatory pathways may partially preserve PS exposure in selected cell types or physiological contexts. Third, severe TMEM16F deficiency may have developmental or extra-hematopoietic consequences that reduce the likelihood of identifying affected individuals. In support of this possibility, TMEM16F has important developmental functions beyond hemostasis, including roles in angiogenesis, trophoblast fusion, and placental development. Homozygous Tmem16f knockout in mice causes partial prenatal lethality associated with placental and fetal vascular defects (90, 361), raising the possibility that severe TMEM16F loss-of-function in humans could contribute to pregnancy loss or other developmental manifestations. Together, these considerations suggest that Scott syndrome may be underrecognized, biologically broader than a platelet procoagulant defect alone, and potentially shaped by both compensatory mechanisms and developmental selection. Inclusion of TMEM16F in genetic screening panels for unexplained bleeding disorders may therefore improve diagnosis and help define the full clinical spectrum of TMEM16F deficiency (510, 511).

5.1.2. Synucleinopathy

Beyond its role in hematological disorders, TMEM16F mutations have recently been linked to synucleinopathies (Figure 9), which currently represent the only other TMEM16F mutation-associated diseases described in humans aside from Scott syndrome (497). Synucleinopathies, including Parkinson’s disease (PD), dementia with Lewy bodies, and multiple system atrophy, are characterized by intracellular aggregation and intercellular propagation of α-synuclein.

TMEM16F variant pAla703Ser was recently identified in PD patients from an Ashkenazi Jewish population (497). This substitution lies near the primary Ca²⁺ binding sites within the pore, and ionomycin stimulation of Ala703Ser-expressing HEK293T cells revealed mildly enhanced lipid scrambling, consistent with a gain-of-function phenotype. Complementary in vitro studies using iPSC-derived dopaminergic neurons showed that Ala703Ser enhances scramblase activity and promotes vesicular α-synuclein release (497).

Functional studies in murine models overexpressing the pathogenic α-synuclein A53T variant further support a role for TMEM16F in α-synuclein spread. Genetic ablation of TMEM16F markedly reduced the intercellular transfer of misfolded α-synuclein and attenuated the accumulation of pathological inclusions in distant brain regions (497). Notably, TMEM16F loss did not alter intracellular α-synuclein abundance, indicating that TMEM16F primarily modulates extracellular secretion and aggregate transmissibility rather than α-synuclein synthesis or degradation.

Together, these findings suggest that aberrant TMEM16F-mediated lipid scrambling promotes the extracellular spreading of neurotoxic α-synuclein, potentially amplifying neuroinflammatory signaling and driving disease progression. They highlight TMEM16F as a candidate therapeutic target for limiting aggregate propagation and neurodegeneration in synucleinopathies.

5.1.3. Ankylosing spondylitis

Genome-wide association studies (GWAS) in Han Chinese have identified a susceptibility locus for ankylosing spondylitis (AS) within the TMEM16F region at chromosome 12q12, implicating the intronic SNP rs17095830 in disease risk (515) (Figure 9). Subsequent work studying a Taiwanese cohort, however, did not confirm an association of rs17095830 (or rs4552569) with overall AS susceptibility or standard clinical indices such as BASDAI, BASFI, and BAS-G, but did show a significant association between rs17095830 and inflammatory bowel disease (IBD) among patients with AS (516) (Figure 9). These findings suggest that TMEM16F variants may act as phenotype-modifying loci (for example, influencing IBD comorbidity) rather than universal risk factors across all populations, and they underscore possible ethnic differences in ANO6-related AS genetics. Cell and animal models demonstrate that TMEM16F is essential for proper bone mineralization by osteoblasts (89), providing a mechanistic rationale that TMEM16F polymorphisms could contribute to aberrant new bone formation in AS, although a direct causal pathway from specific human SNPs to fibrosis and ankylosis has not yet been established.

5.2. Diseases derived from dysregulation of TMEM16F

5.2.1. Hematologic disorders

Aberrant PS exposure on RBCs is associated with hypercoagulation, increased adhesion to the endothelium, clearance by phagocytosis, and hemolysis (25, 517–521). Utilizing Tmem16f-deficient mice that recapitulate the phenotype of Scott syndrome, TMEM16F was established as the principal Ca²⁺-activated phospholipid scramblase that mediates PS exposure in human and murine RBCs (87, 327). This places dysregulated TMEM16F activity at the center of PS-related hematologic abnormalities. Furthermore, the mechanosensitive Ca²⁺ channel PIEZO1 was identified as a key upstream activator of TMEM16F in RBCs (327, 360), linking mechanical stress and Ca²⁺ influx to TMEM16F-dependent scrambling. Together, these findings underscore a TMEM16F-centered signaling pathway that contributes to hematologic disorders like hereditary xerocytosis (HX) and sickle cell disease (SCD).

5.2.1.1. TMEM16F in hereditary xerocytosis (HX)

Gain-of-function mutations in the mechanosensitive cation channel PIEZO1 cause HX or dehydrated hereditary stomatocytosis (DHS) (522–524). HX is an autosomal dominant hemolytic anemia characterized by red cell dehydration, fatigue, jaundice, pallor, and iron overload (523, 525, 526). These mutations slow channel inactivation and/or increase mechanosensitivity, leading to excessive Ca²⁺ influx through PIEZO1 (527, 528). In RBCs, this Ca²⁺ elevation activates the Gardos (IK, KCa3.1) K⁺ channel to drive KCl loss and cellular dehydration, a hallmark of HX phenotype (524).

With the identification of TMEM16F as a CaPLSase in RBCs (87) and its direct activation by Ca²⁺ entry through PIEZO1, Liang et al further demonstrated that HX RBCs have an increased propensity to expose PS upon activation of PIEZO1 (327) (Figure 9). Excessive Ca²⁺ entry through gain-of-function PIEZO1 mutant channels enhances PIEZO1-TMEM16F functional coupling, leading to abnormal scrambling that disrupts membrane lipid asymmetry and drives pathological PS externalization on HX RBCs. The resulting surface PS has broad pathogenic consequences. As an “eat-me” signal for splenic macrophages (518, 521, 529), PS accelerates clearance of HX RBCs, contributing to anemia and splenomegaly. In parallel, PS-positive RBCs and PS-rich microparticles provide procoagulant membrane surfaces for tenase and prothrombinase complex assembly (25, 519), aligning with the heightened thrombotic risk observed in HX, particularly after splenectomy when PS-exposed cells and microparticles are no longer efficiently removed (327, 522, 525). In addition, PS exposure also promotes the adherence of HX RBCs to endothelial cells (530, 531).

These mechanistic insights suggest that targeting PIEZO1-TMEM16F coupling may be therapeutically beneficial. Liang et al. showed that partial pharmacological inhibition of PIEZO1 suppresses TMEM16F-mediated PS exposure, Ca²⁺ overload, echinocytosis, and hemolysis in HX RBCs, while preserving essential baseline PIEZO1 functions (327). Notably, decoupling PIEZO1 from TMEM16F requires lower inhibitor concentrations than those needed to fully block PIEZO1 conductance, suggesting a therapeutic window.

Collectively, these findings position aberrant PIEZO1-TMEM16F signaling as a core pathogenic mechanism in HX and highlight selective modulation of PIEZO1 activity, and that its coupling to TMEM16F, could serve as a rational therapeutic strategy. More broadly, they raise the possibility that similar channel-scramblase coupling may underlie PS dysregulation and prothrombotic phenotypes in other hemolytic disorders.

5.2.1.2. TMEM16F in sickle cell disease (SCD)

SCD is one of the most prevalent inherited blood disorders that affects 7.7 million people worldwide, including ~100,000 individuals in the United States and 1 in 500 African Americans (532). A hallmark of SCD is chronically elevated PS exposure on sickle RBCs, which contributes to anemia, hypercoagulability, and vaso-occlusion (533–535). Although this abnormal PS exposure was recognized as early as the late 1970s (535–538), its molecular basis remained elusive for decades.

Recent work by Liang et al. revealed that TMEM16F is the key CaPLSase that mediates the excessive PS exposure in sickle RBCs downstream of PIEZO1 activation and Ca²⁺ influx (360) (Figure 9). Deoxygenation-induced sickling alters the mechanical properties of sickle RBCs and promotes PIEZO1 activation, leading to exaggerated Ca²⁺ entry and enhanced TMEM16F activation. TMEM16F-mediated lipid scrambling also drives shedding of PS⁺ microvesicles, dramatically expanding the available procoagulant membrane surfaces, promoting thrombin generation, and increasing thrombotic risk. In addition, PS externalization increases the adhesion of sickled RBCs to the endothelium, a critical trigger for vaso-occlusion events (360). These mechanistic findings are consistent with clinical observations that PS-positive RBCs and microvesicles contribute to thrombotic complications and vaso-occlusive crises in SCD (539, 540).

Partial PIEZO1 inhibition with GsMTx-4 or benzbromarone attenuates Ca²⁺ overload, PS scrambling, microparticle shedding, thrombin formation, and endothelial adhesion in SCD RBCs (360). Benzbromarone, most often used in the treatment of gout, thus emerges as a candidate for repurposing, although its off-target effects and safety profile underscore the need for next-generation PIEZO1 inhibitors with improved selectivity. These results demonstrate that modulating PIEZO1-dependent TMEM16F activation provides a tangible strategy to reduce prothrombotic PS exposure in SCD (360).

Across HX and SCD, a unifying theme emerges: pathological activation of the PIEZO1-TMEM16F axis is a central driver of PS dysregulation, hemolysis, and thrombotic complications. TMEM16F serves as the final executor of Ca²⁺-dependent PS externalization, acting as a convergence point linking mechanosensation, membrane remodeling, and procoagulant signaling in RBC disorders. Future priorities include developing highly selective PIEZO1 inhibitors, mapping context-specific regulators of TMEM16F, and defining how mechanical forces, oxygen tension, and redox stress dynamically tune channel-scramblase coupling in human disease. Together, these efforts may enable mechanism-based interventions that restore membrane homeostasis and improve outcomes across multiple hemolytic disorders.

5.2.2. Endothelial and vascular dysfunction

TMEM16F contributes to several vascular pathologies that exhibit increased TMEM16F expression and heightened endothelial procoagulant activity (Figure 9). In vascular disorders such as hyperlipidemia and hyperuricemia, pathological stimuli including oxidized low-density lipoprotein (oxLDL) and uric acid drive robust upregulation of TMEM16F, which increases endothelial prothrombotic responses (372, 386). Beyond promoting PS externalization, these stimuli also trigger TMEM16F-dependent microparticle release, membrane remodeling, and increased sensitivity to circulating stress cues, reflecting a broader disruption of endothelial homeostasis (372, 386). In vivo, high-fat diet rodent models further support this association by showing elevated TMEM16F levels in the vascular wall together with systemic hypercoagulability (386).

Endothelial erythrophagocytosis in uremia and hypoxic pulmonary arterial hypertension also elevate TMEM16F expression, linking TMEM16F activation to thrombosis in settings of vascular injury, hemolysis, and hypoxia (459, 460) (Figure 9). While these studies primarily define TMEM16F as a mediator of endothelial procoagulant activation, they collectively suggest that TMEM16F is upregulated across diverse metabolic, inflammatory, and hypoxic vascular insults. These observations provide important context for the broader roles of TMEM16F in endothelial dysfunction (361).

5.2.3. Neurological disorders and stroke

Neurological disorders such as ischemic stroke, Alzheimer’s disease (AD), tauopathies, spinal cord injury, and neuropathic pain represent rising global health challenges. Despite their distinct clinical manifestations, these neurological disorders share common mechanisms including progressive neuronal loss, chronic glial activation, and maladaptive neuroinflammatory signaling.

Recent studies converge on a central role for TMEM16F in brain pathologies, including neurodegeneration, neuropathic pain, motor dysfunction, and injury-induced neuronal loss. Across studies, inhibition or deletion of TMEM16F in neurons or microglia frequently mitigates neuronal damage, neuroinflammation, pathological protein spread, and functional deficits in animal models. Therefore, uncovering the pathophysiological roles of TMEM16F will facilitate developing novel therapeutic approaches to limit progression across a spectrum of neurological disorders.

5.2.3.1. TMEM16F in ischemic stroke

Ischemic stroke, caused by thrombotic obstruction of cerebral blood flow, leads to acute neuronal death, neuroinflammation, and long-term neurological deficits, with microglia acting as a central regulator of these processes (541). Microglia, the resident macrophages of the central nervous system, are rapidly activated after ischemic onset (542, 543). Immediately following stroke, microglia adopt an anti-inflammatory M2 phenotype that supports debris clearance and tissue repair. However, this response progressively shifts toward a pro-inflammatory M1 state, characterized by cytokine release, phagocytosis of stressed neurons, and secondary neuronal loss (543, 544). A key mechanism enabling this detrimental phagocytosis is the exposure of PS, an “eat-me” signal, on the stressed-but-viable neurons (543).

To determine whether TMEM16F contributes to ischemia-induced neuronal loss, Zhang et al. employed a middle cerebral artery occlusion/reperfusion (MCAO/R) model combined with lentiviral TMEM16F knockdown in rats (373). Interestingly, after ischemic stroke induction, TMEM16F expression levels increase significantly in the brain tissue and primary neuron culture, and peak 12 hours after stroke onset (373). This rise was accompanied by elevated neuronal PS exposure at the same time point, which subsequently declined at 24 hours, suggesting that PS externalization is reversible during early neuronal stress. TMEM16F knockdown reduced microglial phagocytosis in the ischemic penumbra, preserved neuronal survival, and improved motor outcomes after stroke, indicating that TMEM16F promotes the removal of stressed-but-salvageable neurons (373). These findings suggest that TMEM16F may drive reversible PS exposure on neurons, marking them for microglial engulfment and thereby exacerbating post-ischemic neurodegeneration. Targeting TMEM16F may provide an alternative therapeutic avenue to limit post-ischemic neurodegeneration and maladaptive inflammation (Figure 9).

Notably, ischemia-induced upregulation of TMEM16F parallels earlier findings in endothelial cells, where hypoxia similarly increased TMEM16F expression (431). This points to a broader, tissue-independent mechanism through which hypoxia elevates TMEM16F, raising the possibility that TMEM16F contributes to pathology across diverse hypoxia-associated diseases, including cardiovascular, ocular, metabolic, and cancer-related disorders. Elucidating the regulatory pathways governing hypoxia-driven TMEM16F expression may uncover conserved mechanisms relevant to multiple disease contexts.

5.2.3.2. TMEM16F in neurodegenerative diseases

Tauopathies, characterized by the accumulation of pathological hyperphosphorylated tau, are a defining feature of several neurodegenerative diseases, including Alzheimer’s disease (AD) and multiple forms of dementia (545). Using a P301S tauopathy mouse model, Zubia et al. demonstrated that global or neuron-specific TMEM16F knockout reduced tau accumulation and microglial inflammatory activation, whereas microglia-specific TMEM16F deletion did not produce significant changes at the 6–7 month timepoint (374). In primary embryonic hippocampal cultures, TMEM16F-deficient neurons exhibited hyperphosphorylated tau levels comparable to wildtype controls, suggesting that neuronal TMEM16F-mediated PS exposure, rather than tau phosphorylation per se, represents a key driver of microglial activation and tau-related neuroinflammation (374). The divergence between in vivo and in vitro findings likely reflects developmental and environmental differences: embryonic neurons in culture lack the complex glial interactions, extracellular matrix, and aged inflammatory milieu present in 6–7-month-old mouse brains.

Mechanistically, how TMEM16F-derived PS exposure exacerbates tau pathology remains unresolved. TMEM16F may promote the release of tau-containing microvesicles, a proposed mechanism for tau spreading supported by broader extracellular vesicle literature (546). Alternatively, PS exposure on phospho-tau–burdened neurons could serve as an “eat-me” signal marking the cells for microglial phagocytosis, potentially influencing whether microglia clear or instead propagate tau pathology. Detailed mechanisms require further in vivo validation in tauopathy models.

Beyond tauopathy, TMEM16F also contributes to AD-related neuroinflammation. In APP/PS1 AD mice, TMEM16F expression is increased in the hippocampus, accompanied by elevated microglial NLRP3 inflammasome activation, pro-inflammatory cytokine production, and neuronal injury. TMEM16F knockdown in APP/PS1 mice shifted microglia toward an anti-inflammatory M2 phenotype, reduced neuronal apoptosis, and improved cognitive performance (393). These findings suggest that TMEM16F influences PS exposure on tau-burdened neurons and microglial inflammatory states in AD-related neurodegenerative contexts (Figure 9).

5.2.3.3. TMEM16F in pain

Apart from regulating microglia in neurodegeneration, TMEM16F also shapes microglial responses in neuropathic pain (394, 395) (Figure 9). Neuropathic pain arises from injury to the somatosensory system and is sustained by central immune alterations including microglial activation and cytokine release (547, 548). By first establishing that TMEM16F is expressed in microglia, Batti et al. used a microglia-specific TMEM16F knockout combined with partial nerve ligation to probe its functional contribution to pain. This targeted approach revealed that TMEM16F is required for the development of mechanical allodynia and for microglial-mediated synaptic remodeling, as TMEM16F-deficient microglia showed impaired phagocytic activity and reduced loss of inhibitory GABAergic terminals. A later study in spinal cord injury expanded this view, showing that TMEM16F expression increases in activated microglia and macrophages and that global TMEM16F deficiency protects against tissue damage, aberrant inflammation, and cutaneous hypersensitivity. TMEM16F loss shifted microglia toward an anti-inflammatory M2 phenotype, suggesting that TMEM16F influences not only phagocytic remodeling but also the broader immunological state (395).

Together, these studies indicate that TMEM16F promotes pro-nociceptive microglial functions through multiple mechanisms, including synaptic pruning and inflammatory activation. Given that TMEM16F drives microvesicle shedding in other immune and non-immune contexts, impaired vesicle production in TMEM16F-deficient microglia could dampen neuron-glia signaling loops relevant to pain, although this remains untested. Future work should determine whether TMEM16F regulates microglial Ca²⁺-dependent signaling and extracellular vesicle cargo, and whether pharmacological TMEM16F inhibition could serve as an analgesic strategy.

5.2.4. TMEM16F in immune and infectious diseases

TMEM16F regulates immune homeostasis and host-pathogen interactions through several related processes, including Ca²⁺-dependent PS exposure, membrane repair, extracellular vesicle release, and modulation of immune-cell signaling (Figure 9). These functions are highly cell type- and context-dependent. In some settings, TMEM16F supports host defense by preserving membrane integrity or enabling protective immune-cell responses; in others, its lipid-scrambling activity is exploited by pathogens to promote entry, budding, or cell-cell fusion.

In adaptive immunity, TMEM16F functions as a regulator of T cell activation. During TCR stimulation, TMEM16F-mediated lipid scrambling alters plasma membrane lipid organization and promotes dissociation of signaling components from the membrane, thereby limiting excessive T cell activation (444). Consistent with this checkpoint function, TMEM16F-deficient T cells can become hyperactivated during infection but subsequently develop severe exhaustion and fail to control chronic viral infection (444). These findings suggest that TMEM16F can potentiate early TCR signaling while also limiting excessive or prolonged activation during chronic stimulation, thereby helping tune both the amplitude and duration of T cell responses.

TMEM16F also contributes to innate immune-cell function, although the underlying mechanisms remain incompletely defined. In macrophages and other phagocytes, TMEM16F has been implicated in membrane repair after pore-forming damage, a process that may be especially relevant during bacterial infection or inflammasome activation (463). In liver-resident Kupffer cells, TMEM16F supports host defense against Listeria monocytogenes; TMEM16F deficiency leads to increased bacterial burden, excessive liver inflammation, and metabolic dysfunction (549). These findings suggest that TMEM16F helps preserve macrophage/Kupffer-cell integrity and antimicrobial function during infection. However, whether TMEM16F directly enhances bacterial phagocytosis and killing through PS exposure on pathogen-containing vesicles, or instead acts more broadly by supporting membrane repair and immune-cell viability, remains to be determined. It is also not yet clear how much TMEM16F-dependent PS exposure during infection exceeds PS externalization driven by ATP depletion or nonspecific cell stress.

TMEM16F-dependent vesicle release can further shape immune responses. In inflammatory arthritis, neutrophil-derived microparticles are enriched in annexin A1 and protect cartilage from inflammatory damage (496). Tmem16f-deficient mice release fewer neutrophil-derived microparticles and exhibit worsened cartilage destruction despite comparable synovitis, suggesting that TMEM16F-dependent vesiculation can generate anti-inflammatory or pro-resolving extracellular vesicles in specific disease contexts. Thus, TMEM16F-mediated PS exposure and vesicle release may influence immunity not only by changing the plasma membrane of immune cells, but also by controlling the release of vesicles carrying immunomodulatory cargo.

In viral infections, TMEM16F can be co-opted by enveloped viruses. Several studies suggest that virus-induced Ca²⁺ influx and TMEM16F-dependent PS exposure facilitate membrane fusion, syncytium formation, or virion infectivity. For example, TMEM16F activity contributes to SARS-CoV-2 spike-induced syncytia, and pharmacological inhibition or genetic knockdown of TMEM16F reduces spike-mediated cell-cell fusion (91, 376). TMEM16F has also been implicated in Ebola virus budding and incorporation of PS into virions, which may promote apoptotic mimicry and enhance viral infectivity (412, 550). More generally, lipid scrambling may support the shedding or infectivity of enveloped viruses by enriching viral membranes with exposed PS, although the relative contributions of TMEM16F, other scramblases, and nonspecific cell injury likely vary among viruses and host-cell types.

These diverse observations emphasize that TMEM16F has dual roles in infection and immunity. In immune cells, TMEM16F can support membrane repair, vesicle-mediated resolution, and controlled signaling. In virus-infected or virus-targeted cells, the same lipid-scrambling machinery may be exploited to promote viral spread. This context dependence is also important for interpreting macrophage and microglial polarization. TMEM16F-dependent PS exposure in tumors may contribute to immunosuppressive, M2-like macrophage programs, whereas TMEM16F loss or inhibition in CNS injury models has been reported to shift microglia/macrophages toward an anti-inflammatory M2-like state. These apparently divergent findings likely reflect differences in cell type, disease setting, source of PS exposure, and local inflammatory cues rather than a universal effect of TMEM16F on macrophage polarization. Therapeutic targeting of TMEM16F in immune and infectious diseases will therefore require careful attention to the specific cell type, pathogen, and disease stage.

5.2.5. TMEM16F in cancer

Unlike its homologous family member TMEM16A/ANO1 CaCC, which generally promotes cancer growth (107), TMEM16F exhibits diverse, tissue- and context-dependent roles across different malignancies (55, 551) (Figure 9). On the pro-tumor side, TMEM16F is overexpressed in glioma/glioblastoma, where high TMEM16F levels correlate with poor prognosis, and knockdown suppresses proliferation and invasion in part by dampening ERK activation and nuclear translocation. Similar associations are reported in gastric adenocarcinoma, pancreatic cancer, and melanoma, where TMEM16F overexpression links to advanced clinicopathological features, including higher tumor stage and lymph node metastasis, enhanced cell proliferation, and worse overall survival, and mechanistic studies implicate ERK-pathway activation and pro-migratory signaling (552–554). Pan-cancer analyses further support a poor-prognosis signature of high TMEM16F in several cancer types (552).

However, in some cancers TMEM16F also shows tumor-suppressive features. In gastrointestinal stromal tumors (GIST), TMEM16F expression is significantly lower than adjacent normal tissues, and TMEM16F overexpression inhibits tumor cell growth and xenograft progression, accompanied by increased markers of apoptosis, pyroptosis, and ferroptosis (450). Public data sets and integrative analyses also suggest that in breast cancer TMEM16F behaves as a ferroptosis-related gene where lower expression associates with poorer prognosis, and that higher TMEM16F expression can be a favorable prognostic marker in breast and cervical cancers in some cohorts (555, 556). Together, these studies illustrate that TMEM16F can act as either an oncogenic driver or a tumor suppressor depending on tissue of origin, oncogenic wiring, and the dominant cell-intrinsic death pathways engaged.

Beyond effects on cancer cells themselves, TMEM16F appears to shape the tumor immune microenvironment. Pan-cancer transcriptomic analyses show that high TMEM16F expression correlates with increased immune and stromal scores and altered immune cell composition, including higher tumor-associated macrophage and T-cell infiltration (551, 557). In multiple cancer types, TMEM16F expression and PS exposure associate with macrophage polarization toward an M2-like phenotype and with altered immune checkpoint landscapes, including negative correlations with CTLA4 and PD-1 and a weak positive correlation with PD-L1, suggesting a complex and context-dependent relationship with immunosuppressive signaling (55, 551, 557–559). More recent functional studies demonstrate that TMEM16F knockout or pharmacological inhibition can limit tumor growth by reprogramming the tumor immune microenvironment and reducing immune evasion, providing evidence that TMEM16F contributes to tumor-associated immune suppression (558, 559).

Taken together, current evidence portrays TMEM16F as a highly context-dependent cancer regulator rather than a simple oncogene or tumor suppressor. In some settings it supports proliferation, invasion, and metastasis; in others it sensitizes cells to regulated cell death (apoptosis, pyroptosis, ferroptosis) and associates with better outcomes. At the same time, TMEM16F-dependent PS exposure and membrane remodeling may recalibrate macrophage polarization, T-cell infiltration, and immune checkpoint landscapes in the tumor microenvironment. These complexities argue strongly against a “one-size-fits-all” TMEM16F-targeted therapy. Future work needs to (i) mechanistically validate bioinformatic predictions in defined cancer models, (ii) dissect how TMEM16F intersects with ferroptosis and other death pathways in a tumor-type-specific manner, and (iii) clarify how TMEM16F-driven PS externalization shapes local immune responses.

6. TMEM16F PHARMACOLOGY

Targeting TMEM16F has substantial therapeutic potential for diseases in which pathological PS exposure, membrane remodeling, or cell fusion contributes to disease progression, including thrombosis, sickle cell disease, viral infection, cancer, and potentially neurodegenerative disorders. However, the development of potent, selective, and well-validated TMEM16F modulators remains a major unmet need. Most reported TMEM16F inhibitors were empirically repurposed from TMEM16A CaCC pharmacology (Table 3), despite early electrophysiological evidence that TMEM16F and TMEM16A have distinct pharmacological profiles (87). Moreover, many putative TMEM16F inhibitors have substantial off-target liabilities, including disruption of intracellular Ca²⁺ signaling (91, 560–562), interference with fluorescent PS probes or fluorescence quenching (325), and indirect effects on intracellular signaling pathways, pH homeostasis, or the membrane environment (561, 562). Thus, inhibition of annexin V labeling or cell-based PS exposure does not necessarily establish direct scramblase inhibition.

Table 3.

Summary of TMEM16F pharmacological inhibitors

Inhibitor	Chemical Class/ Rationale	Conclusion	Supporting Evidence	Limitations / Caveats
Niflumic acid (NFA)	Fenamate NSAID; non-selective CaCC blocker repurposed to TMEM16F	Controversial	Inhibits P2X7-evoked TMEM16F-like whole-cell currents (366)	Broad spectrum Cl⁻ channel inhibitor; no evidence on inhibiting lipid scrambling (366); reduces Ca²⁺ elevation through off-target effects on Ca²⁺ handling (560); no effect on TMEM16F current in excised patches (87)
NPPB (5-nitro-2-(3-phenylpropylamino) benzoic acid)	Classical non-selective CaCC blocker repurposed to TMEM16F	Controversial	Inhibits P2X7-evoked TMEM16F-like whole-cell currents (366); used together with NFA as a pharmacological TMEM16F blocker in innate immunity studies (366)	Broad spectrum Cl⁻ channel inhibitor; no evidence on inhibiting lipid scrambling (366); no effect on TMEM16F current in excised patches (87)
Tannic acid (TA) and epigallocatechin gallate (EGCG)	Natural polyphenols, TMEM16A inhibitors repurposed to TMEM16F	No direct effect	Inhibit TMEM16F scramblase in single-molecule and cell-based fluorescence assays (299, 353)	Act as fluorescence quenchers of fluorescently tagged annexin V instead of inhibiting TMEM16F (325); has no obvious effect on TMEM16F current (325) and scrambling in proteoliposomes (564)
Benzbromarone	Uricosuric benzofuran derivative; anti-gout drug; TMEM16A CaCC inhibitor repurposed to TMEM16F	No direct effect	Inhibits Ca²⁺-induced PS exposure in multiple cells (endothelial cells (135), platelets (566)); inhibits TMEM16F whole-cell current (447); prevents ferroptosis models (447)	Primary target is URAT1 (uric acid transporter); no effect on ionomycin-induced lipid scrambling (447); no effect on TMEM16F current using whole-cell or excised patch clamp (327)
CaCCinh-A01	Synthetic CaCC small molecule inhibitor repurposed to TMEM16F	Controversial	Reduces ferroptosis in a TMEM16F-dependent manner (447); prevents TMEM16F-mediated HIV infection (375); inhibits Ca²⁺-induced PS exposure in HUVECs (135); inhibits TMEM16F whole cell current from HEL and megakaryocytes (567)	Reduces Ca²⁺ elevation by off-targeting Ca²⁺ handling (560)
Niclosamide	FDA-approved antihelminthic; differentially potentiates or inhibits TMEM16A depending on Ca²⁺ and voltage (562, 568); repurposed to TMEM16F (91)	Controversial	Blocks SARS-CoV-2 syncytia (91); CryoEM structure solved, revealing a binding pocket on the extracellular side (563); reduces PS exposure (563); Inhibits TMEM16F whole-cell current (91, 561, 569)	Reduces intracellular Ca²⁺ through acidification (561, 562); lack of excised patch clamp validation (91, 563); no effect on TMEM16F scrambling in proteoliposomes (564)
1PBC (1-(2-(trifluoromethyl)phenyl)-benzamide)	Synthetic TMEM16A inhibitor (565, 570); repurposed to TMEM16F (563)	Controversial	CryoEM structure solved, binds to the same pocket as niclosamide (563); delays PS exposure ~2-fold (563); some selectivity for scramblase over ion conduction pathway at specific mutations (563).	Reduces Ca²⁺ influx (563); mutational effects not clearly defined(563); at similar concentrations that inhibit TMEM16A does not block TMEM16F current (565).
Salinomycin	Antiparasitic polyether ionophore antibiotics repurposed for TMEM16F (91)	Controversial	Anti-syncytia activity in SARS-CoV-2 context; anti-TMEM16F effect proposed (91)	No direct inhibitory effect on TMEM16F current (91); no other evidence to further support.
Clofazimine	FDA-approved antibiotic/antileprosy repurposed for TMEM16F (91)	Controversial	Inhibits PS exposure and syncytia formation in spike-expressing cells (91); used as TMEM16F inhibitor in tumor models (trogocytosis) (571).	Reduces Ca²⁺ oscillations in spike-expressing cells (91); no inhibitory effect on TMEM16F current directly (91); does not inhibit scrambling in proteoliposomes (564).
Ivermectin	Antiparasitic macrolide; proposed to be a TMEM16A inhibitor (572); repurposed for TMEM16F (91)	Controversial	Inhibits spike-mediated syncytia formation(91); inhibits TMEM16F-mediated scrambling in a cancer/ferroptosis context (466).	Does not inhibit TMEM16A or TMEM16F currents or scrambling (561).
TMEM16F affibodies	Engineered affibody proteins, biologics (573)	Controversial	Binding to TMEM16F confirmed; reduces microglial phagocytosis and modulates neuronal cell death (573).	No direct effect on purified TMEM16F scrambling in liposomes observed (573); likely a binder instead of an inhibitor.
A6–001	Novel synthetic compound derived from high-throughput screening of TMEM16F Cl− channel activity (376)	Most promising	Inhibits SARS-CoV-2-induced PS scrambling and viral replication in multiple cell types (376); inhibits TMEM16F also inhibits TMEM16A at higher concentrations; no effect on Ca²⁺ elevation (376).	Mechanism of binding to TMEM16F not structurally validated (376).

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Among currently available compounds, A6–001 appears to have the strongest functional validation (376). Identified through a high-throughput screen based on TMEM16F-mediated I⁻ conductance, A6–001 inhibits both TMEM16F-dependent lipid scrambling and ionic current without detectably perturbing intracellular Ca²⁺ signaling. It also suppresses SARS-CoV-2-induced PS exposure and viral replication in multiple cellular models. These features make A6–001 an important lead compound for TMEM16F pharmacology. However, its binding site, structural mechanism, selectivity against TMEM16A/TMEM16B and other ion channels, and broader in vivo utility remain to be defined.

Phenotypic screens for inhibitors of SARS-CoV-2-induced syncytia formation also identified niclosamide, clofazimine, and salinomycin as candidate TMEM16F inhibitors (91). Subsequent structural studies further advanced the field by showing that niclosamide and the TMEM16A blocker 1PBC can bind a groove-pocket near the extracellular TM1/TM6 interface of TMEM16F, linking ligand binding to inhibition of scrambling and channel activity (563). Nevertheless, these compounds remain questionable as bona fide TMEM16F inhibitors. Niclosamide attenuates intracellular Ca²⁺ elevation (561, 562) and has been reported to lack inhibitory activity against TMEM16F-mediated scrambling in proteoliposomes (564). Similarly, 1PBC can perturb Ca²⁺ homeostasis (563) and has been reported not to inhibit TMEM16F current (565). These discrepancies highlight the strong context dependence of current TMEM16F pharmacological reagents.

Moving forward, the field would benefit from a standardized, multi-readout validation pipeline. Candidate compounds should first be assessed in cell-based lipid-scrambling assays, paired with whole-cell and excised-patch electrophysiology, and then advanced to purified-protein systems such as proteoliposomes. Cross-paralog counterscreens against TMEM16A and TMEM16B, together with broader ion-channel and Ca²⁺-signaling panels, will be critical to distinguish direct TMEM16F inhibition from indirect effects on upstream activation pathways or assay readouts. Finally, mutational mapping and cryo-EM should be used to define whether inhibitory activity reflects bona fide TMEM16F engagement or assay-context-dependent artifacts. Such efforts are needed to transform TMEM16F pharmacology from a collection of useful but imperfect probes into a rigorous therapeutic and mechanistic toolkit.

7. CLOSING REMARKS

Over the past 15 years, we have witnessed an explosion of work on the biology of lipid flip-flop. The molecular identification of multiple phospholipid scramblases, most notably TMEM16 and XKR family members, has transformed a phenomenological concept into a mechanistically grounded field. These discoveries not only explain decades of observations on Ca²⁺- and caspase-dependent scrambling in health and disease, but also provide molecular handles to uncover new mechanisms and functions of lipid flip-flop. Although we still lack a definitive answer to why plasma membranes universally maintain phospholipid asymmetry (beyond the intuitive notion of PS sequestration on the cytosolic leaflet), it is now clear that cells actively exploit this asymmetry and its regulated dissipation to drive rapid, reversible membrane remodeling. Mounting evidence has demonstrated that dynamic flip-flop transiently remodels bilayer properties and phospholipid-protein interactions, creating a non-conventional signaling modality that complements classical protein-, ion-, and metabolism-based pathways (Figure 5).

At the molecular level, structural, functional, and computational studies have converged on a “credit-card slot” model for lipid translocation by scramblases and flippases. Additional mechanisms, including membrane thinning and out-of-groove translocation, further refine this model. The identification of the activation gate in TMEM16F has added a gating control component to the “credit-card slot” model, analogous to ion transport through channels. In this way, a conformational change of the lipid gates within the hydrophilic groove or proteolipid pore, regardless of lipid in- or out-of-groove models, is required to tightly regulate lipid permeation (Figure 3). Mutations that destabilize this gate not only alter TMEM16F scrambling, but can convert pure ion channels, such as TMEM16A CaCC and the evolutionarily related OSCA/TMEM63 mechanosensitive channels, into lipid scramblases (95, 96). The recent identification of disease-causing missense mutations (218, 574) that convert TMEM63B from an ion channel into a scramblase underscores the pathophysiological importance of this channel-to-scramblase switch (215). By dissolving the conceptual boundary between “channel” and “scramblase,” these findings advance our understanding of the broader TCS superfamily (TMEM16, OSCA/TMEM63, and TMC) (45) and reveal shared principles governing ion and lipid permeation. A key future priority is to rigorously examine whether and how lipids and ions transport through wildtype OSCA/TMEM63 and TMC proteins, several of which have been proposed to possess scramblase activity (Table 2) (225, 231, 575–577).

At the cellular level, genetic and acute manipulation of scramblases has revealed unexpected breadth in lipid flip-flop function, encompassing membrane expansion and repair, regulated cell death, cell-cell fusion, and virus-host fusion. The long-standing dogma that PS exposure marks irreversible apoptosis has been replaced by a more nuanced view in which PS externalization can be transient, reversible, and tightly localized, serving as a spatiotemporal biomarker and effector of physiological events in viable cells. The largely unexplored intracellular roles of lipid transporters are an important frontier: inner-leaflet lipid composition and dynamics almost certainly influence protein targeting, signal transduction, cytoskeletal remodeling, and organelle-organelle communication. Future work should dissect scramblase-mediated signaling within the cytosolic leaflet and its crosstalk with canonical kinase and second-messenger cascades. Moreover, most studies have focused on PS, even though many scramblases are broadly nonselective. Understanding how redistribution of other lipids, such as PE, SM, PI and its phosphorylated derivatives (Figure 1A) contribute to membrane curvature, microdomain organization, and effector recruitment is likely to uncover additional, unanticipated dimensions of lipid flip-flop biology. Finally, despite clear genetic links to human disease, many TMEM16 CaPLSases are predominantly localized to intracellular organelles, and their physiological functions remain largely undefined (57, 578). Therefore, elucidating these “hidden” scramblaseopathies is a pressing task.

From a disease perspective, aberrant TMEM16F activity has been implicated in thrombotic disorders, hereditary red cell diseases such as SCD and HX, vascular and inflammatory complications, neurodegeneration, pain, viral infection, and cancer (Figure 9). The relatively mild bleeding phenotype in Scott syndrome suggests that partial pharmacological inhibition of TMEM16F could be tolerated and may offer therapeutic benefit in conditions driven by pathological PS exposure and membrane remodeling. However, accurately measuring scramblase activity is technically challenging, and many reported TMEM16F inhibitors lack specificity or may not act directly on the scramblase (Table 3) (91, 325, 568). A major priority is therefore to develop bona fide TMEM16F-specific modulators by integrating structure-based design, high-throughput and in silico screening, and, where feasible, antibody- or biologic-based approaches. In parallel, indirect strategies that target upstream Ca²⁺ channels may be therapeutically useful in settings such as red blood cell disorders, including SCD and HX, where disease-relevant Ca²⁺ influx provides the key trigger for TMEM16F activation. Exploiting the “therapeutic window” created by TMEM16F’s relatively low Ca²⁺ sensitivity, partial inhibition of the upstream Ca²⁺ channel such as PIEZO1 (327, 360) or directly targeting amplifier proteins such as KCNN4/Gardos Ca²⁺-activated K⁺ channel (579) for the Ca²⁺ channel-TMEM16F coupling axis can selectively suppress pathological scrambling without abolishing the essential function of the Ca²⁺ channel. Future therapeutic efforts should pursue these upstream and downstream approaches in tandem.

Progress in the field will also depend critically on methodological advances. In vivo monitoring of lipid flip-flop remains difficult; current probes and assays are largely limited to PS and often provide population-averaged or end-point measurements. Developing sensitive tools to track multiple lipid species in live cells, in real time and at subcellular resolution, will be essential. Equally important is the ability to control scramblase activity with high spatial and temporal precision. Most existing chemical and physical stimuli produce global, sustained activation that does not recapitulate the localized, transient flip-flop events likely used for signaling. New approaches, such as optogenetic, chemogenetic, or mechanogenetic actuators tailored to scramblases and their upstream Ca²⁺ channels, will be crucial for dissecting causality and mapping the signaling logic of lipid flip-flop.

In conclusion, the field is transitioning from descriptive phenomena toward a mechanistic and integrative understanding of lipid flip-flop in physiology and disease. The convergence of structural biology, biophysics, cell biology, genetics, and chemical biology has begun to reveal how scramblases dynamically remodel membranes to control cell signaling, behaviors, and fate (Table 2). Continued progress promises not only deeper insight into fundamental membrane biology, but also new opportunities to therapeutically target lipid transporter function to prevent or treat thrombosis, hematologic disease, neurodegeneration, infectious diseases, and cancer.

CLINICAL HIGHLIGHTS.

Regulated lipid scrambling is increasingly recognized as a clinically important membrane process that connects Ca²⁺ signaling, phosphatidylserine (PS) exposure, membrane remodeling, extracellular vesicle release, and disease. TMEM16F, a prototypical Ca²⁺-activated phospholipid scramblase, illustrates how disruption of membrane lipid asymmetry contributes to human pathology. Loss-of-function TMEM16F “scramblaseopathy” variants cause Scott syndrome, a bleeding disorder characterized by defective Ca²⁺-dependent PS exposure in platelets and other blood cells. Conversely, excessive or dysregulated TMEM16F activity has been implicated in blood disorders such as hereditary xerocytosis and sickle cell disease, as well as in neurodegenerative and inflammatory disorders. TMEM16F-dependent membrane remodeling also influences enveloped virus infection, pain, and cancer by regulating viral fusion and budding, microglial activation, and immune remodeling. The clinical relevance of lipid scrambling extends beyond TMEM16F. ANO5/TMEM16E mutations cause muscular dystrophies and gnathodiaphyseal dysplasia; ANO10/TMEM16K mutations cause cerebellar ataxia; XKR8 regulates apoptotic-cell clearance and immune tolerance; and emerging scramblases such as TMEM41B, VMP1, ATG9A, TMEM63, and TMC proteins connect lipid scrambling to autophagy, lipid metabolism, sensory function, and neurological diseases. These findings reveal unexpected consequences of disrupted membrane lipid asymmetry and highlight lipid scrambling as a broad therapeutic frontier.

ACKNOWLEDGEMENT

We thank Dr. Pengfei Liang for assistance with this review. We appreciate Dr. Yuanyuan Zhao for critical comments. Figure schematics, including the Graphical Abstract, were created with BioRender.com. The authors used AI-assisted writing tools (ChatGPT) to help edit language and improve clarity. All scientific content, data interpretation, and conclusions were developed and verified by the authors.

GRANTS

This work was supported by National Institutes of Health Grants NIH-R35GM153196 and NIH-R21OD037849 to H.Y.

Footnotes

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

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PERMALINK

Lipid flip-flop in biological membranes: through the lens of TMEM16F

Ke Z Shan

Augustus J Lowry

Maria A González Torres

Megha S Seri

Huanghe Yang

Abstract

Graphical Abstract

1. INTRODUCTION

FIGURE 1. Membrane phospholipid asymmetry and major lipid transporters that maintain or disrupt this asymmetry.

Table 1.

FIGURE 2. Milestones in the discovery of membrane lipid asymmetry and lipid transporters.

Table 2.

1.1. Phospholipid asymmetry: discovery and update

1.2. Molecular players that build and dissipate phospholipid asymmetry

1.3. Conceptual framework of lipid transport mechanisms

FIGURE 3. Structure and Function of TMEM16F.

1.4. Dynamic lipid flip-flop delivers nonconventional cell signals

2. STRUCTURE-FUNCTION OF TMEM16F

2.1. Discovery of TMEM16F CaPLSase

2.3. Experimental measurement of lipid scrambling

FIGURE 4. Experimental strategies for measuring lipid scramblase activity.

2.3. Intriguing moonlighting function as an ion channel

2.3. Overall structural features

2.4. Ca2+-dependent activation

2.5. Gating and permeation mechanisms

2.6. Regulatory mechanisms

3. CELLULAR ACTIVATION MECHANISMS OF TMEM16F

4. TMEM16F-MEDIATED CELL SIGNALING AND PHYSIOLOGICAL FUNCTIONS

FIGURE 5. An overview of known physiological functions of TMEM16F.

4.1. TMEM16F-mediated extracellular signaling associated with exofacial leaflet lipid remodeling

FIGURE 6. Extracellular signaling events regulated by TMEM16F-mediated lipid flip-flop.

4.1.1. TMEM16F-mediated PS exposure in blood coagulation

4.1.2. TMEM16F-mediated PS exposure in phagocytosis and immune clearance

4.1.3. TMEM16F-mediated PS exposure in cell fusion and viral infection

4.1.4. TMEM16F-mediated PS exposure in ADAM activation

4.2. TMEM16F-mediated intracellular signaling associated with cytosolic leaflet lipid remodeling

FIGURE 7. Intracellular signaling regulated by TMEM16F-mediated lipid flip-flop.

4.2.1. TMEM16F regulates intracellular signaling in immune cells

4.2.2. TMEM16F regulates intracellular signaling in endothelial cells

4.2.3. TMEM16F regulates membrane expansion through inner leaflet lipid remodeling

4.2.4. TMEM16F modulates multiple regulated cell death pathways in a context-dependent manner.

4.3. TMEM16F-mediated membrane remodeling associated with integrated cellular signaling responses

FIGURE 8. Membrane remodeling regulated by TMEM16F-mediated lipid flip-flop.

4.3.1. Membrane blebbing

4.3.2. Vesicle release

4.3.2.1. TMEM16F-mediated vesicle release in blood coagulation

4.3.2.2. TMEM16F-mediated vesicle release beyond coagulation

4.3.3. Membrane repair

5. TMEM16F IN DISEASE

FIGURE 9. Overview of the involvement of TMEM16F in human diseases.

5.1. TMEM16F scramblaseopathy and polymorphism

5.1.1. Scott syndrome

5.1.2. Synucleinopathy

5.1.3. Ankylosing spondylitis

5.2. Diseases derived from dysregulation of TMEM16F

5.2.1. Hematologic disorders

5.2.1.1. TMEM16F in hereditary xerocytosis (HX)

5.2.1.2. TMEM16F in sickle cell disease (SCD)

5.2.2. Endothelial and vascular dysfunction

5.2.3. Neurological disorders and stroke

5.2.3.1. TMEM16F in ischemic stroke

5.2.3.2. TMEM16F in neurodegenerative diseases

5.2.3.3. TMEM16F in pain

5.2.4. TMEM16F in immune and infectious diseases

5.2.5. TMEM16F in cancer

6. TMEM16F PHARMACOLOGY

Table 3.

7. CLOSING REMARKS

CLINICAL HIGHLIGHTS.

ACKNOWLEDGEMENT

GRANTS

Footnotes

REFERENCES

ACTIONS

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