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TMEM16F is a surface membrane protein critical for platelet procoagulant activity, which exhibits both phospholipid scramblase and ion channel activities following sustained elevation of cytosolic Ca2+. The extent to which the ionic permeability of TMEM16F is important for platelet scramblase responses remains controversial. To date, only one study has reported the electrophysiological properties of TMEM16F in cells of platelet/megakaryocyte lineage, which observed cation-selectivity within excised patch recordings from murine marrow-derived megakaryocytes. This contrasts with reports using whole-cell recordings that describe this channel as displaying either selectivity for anions or being relatively non-selective amongst the major physiological monovalent ions. We have studied TMEM16F expression and channel activity in primary rat and mouse megakaryocytes and the human erythroleukemic (HEL) cell line that exhibits megakaryocytic surface markers. Immunocytochemical analysis was consistent with surface TMEM16F expression in cells from all three species. Whole-cell recordings in the absence of K+-selective currents revealed an outwardly rectifying conductance activated by a high intracellular Ca2+ concentration in all three species. These currents appeared after 5–6 minutes and were blocked by CaCCinh-A01, properties typical of TMEM16F. Ion substitution experiments showed that the underlying conductance was predominantly Cl–-permeable in rat megakaryocytes and HEL cells, yet non-selective between monovalent anions and cations in mouse megakaryocytes. In conclusion, the present study further highlights the difference in ionic selectivity of TMEM16F in platelet lineage cells of the mouse compared to other mammalian species. This provides additional support for the ionic "leak" hypothesis that the scramblase activity of TMEM16F does not rely upon its ability to conduct ions of a specific type. [ABSTRACT FROM AUTHOR]

The cystic fibrosis transmembrane conductance regulator (CFTR) is the secretory chloride channel in epithelial tissues that has a central role in cystic fibrosis (CF) lung and gastrointestinal disease. A recent publication demonstrates a close association between CFTR and TMEM16A, the calcium-activated chloride channel. Thus, no CFTR chloride currents could be detected in airways and large intestine from mice lacking epithelial expression of TMEM16A. Here, we demonstrate that another plasma membrane–localized TMEM16 paralogue, TMEM16F, can compensate for the lack of TMEM16A. Using TMEM16 knockout mice, human lymphocytes, and a number of human cell lines with endogenous protein expression or heterologous expression, we demonstrate that CFTR can only function in the presence of either TMEM16A or TMEM16F. Double knockout of intestinal epithelial TMEM16A/F expression did not produce offsprings, suggesting a lethal phenotype in utero. Plasma membrane–localized TMEM16A or TMEM16F is required for exocytosis and expression of CFTR in the plasma membrane. TMEM16A/F proteins may therefore have an impact on disease severity in CF. Key messages: • Cystic fibrosis is caused by the defective Cl− channel cystic fibrosis transmembrane conductance regulator (CFTR). • A close relationship exists between CFTR and the calcium-activated chloride channels TMEM16A/TMEM16F. • In conditional airway and intestinal knockout mice, lymphocytes from Scott disease patients and in overexpressing cells, CFTR is not functional in the absence of TMEM16A and TMEM16F. • TMEM16A and TMEM16F support membrane exocytosis and are essential for plasma membrane insertion of CFTR. [ABSTRACT FROM AUTHOR]

Key points: TMEM16 proteins can operate as Ca2+‐activated Cl− channels or scramble membrane phospholipids, which are both highly relevant mechanisms during disease. Overexpression of TMEM16A and TMEM16F were found to be partially active at 37°C and at resting intracellular Ca2+ concentrations. We show that TMEM16 Cl− currents and phospholipid scrambling can be activated by modification of plasma membrane phospholipids, through reactive oxygen species and phospholipase A2. While phospholipids and Cl− ions are likely to use the same pore within TMEM16F, TMEM16A only conducts Cl− ions. Lipid regulation of TMEM16 proteins is highly relevant during inflammation and regulated cell death such as apoptosis and ferroptosis. Abstract: TMEM16/anoctamin (ANO) proteins form Ca2+‐activated ion channels or phospholipid scramblases. We found that both TMEM16A/ANO1 and TMEM16F/ANO6 produced Cl− currents when activated by intracellular Ca2+, but only TMEM16F was able to expose phosphatidylserine to the outer leaflet of the plasma membrane. Mutations within TMEM16F or TMEM16A/F chimeras similarly changed Cl− currents and phospholipid scrambling, suggesting the same intramolecular pathway for Cl− and phospholipids. When overexpressed, TMEM16A and TMEM16F produced spontaneous Cl− currents at 37°C even at resting intracellular Ca2+ levels, which was abolished by inhibition of phospholipase A2 (PLA2). Connversely, activation of PLA2 or application of active PLA2, as well as lipid peroxidation induced by reactive oxygen species (ROS) using staurosporine or tert‐butyl hydroperoxide, enhanced ion currents by TMEM16A/F and in addition activated phospholipid scrambling by TMEM16F. Thus, TMEM16 proteins are activated by an increase in intracellular Ca2+, or independent of intracellular Ca2+, by modifications occurring in plasma and intracellular membrane phospholipids. These results may help to explain why regions distant to the TMEM16 pore and the Ca2+ binding sites control Cl− currents and phospholipid scrambling. Regulation of TMEM16 proteins through modification of membrane phospholipids occurs during regulated cell death such as apoptosis and ferroptosis. It contributes to inflammatory and nerve injury‐induced hypersensitivity and generation of pain and therefore provides a regulatory mechanism that is particularly relevant during disease. [ABSTRACT FROM AUTHOR]

Activated receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain like (MLKL) are essential components of the necroptotic pathway. Phosphorylated MLKL (pMLKL) is thought to induce membrane leakage, leading to cell swelling and disintegration of the cell membrane. However, the molecular identity of the necroptotic membrane pore remains unclear, and the role of pMLKL for membrane permeabilization is currently disputed. We observed earlier that the phospholipid scramblase and ion channel TMEM16F/anoctamin 6 cause large membrane currents, cell swelling, and cell death when activated by a strong increase in intracellular Ca. We, therefore, asked whether TMEM16F is also central to necroptotic cell death and other cellular events during necroptosis. Necroptosis was induced by TNFα, smac mimetic, and Z-VAD (TSZ) in NIH3T3 fibroblasts and the four additional cell lines HT, 16HBE, H441, and L929. Time-dependent changes in intracellular Ca, cell morphology, and membrane currents were recorded. TSZ induced a small and only transient oscillatory rise in intracellular Ca, which was paralleled by the activation of outwardly rectifying Cl currents, which were typical for TMEM16F/ANO6. Ca oscillations were due to Ca release from endoplasmic reticulum, and were independent of extracellular Ca. The initial TSZ-induced cell swelling was followed by cell shrinkage. Using typical channel blockers and siRNA-knockdown, the Cl currents were shown to be due to the activation of ANO6. However, the knockdown of ANO6 or inhibitors of ANO6 did not inhibit necroptotic cell death. The present data demonstrate the activation of ANO6 during necroptosis, which, however, is not essential for cell death. [ABSTRACT FROM AUTHOR]

The anoctamin (ANO, TMEM16) family of Ca-activated Cl channels consists of ten members with different cellular functions (ANO1-10). ANO1 is a Ca-activated Cl channel in secretory epithelial cells of exocrine pancreas, salivary glands, or enterocytes. Expression of ANO1 also promotes cell proliferation and migration of tumor cells. ANO6 is essential for Ca-dependent scrambling of membrane phospholipids in platelets, red blood cells, and lymphocytes. ANO10 modulates Ca signals in macrophages and has a role in cerebellar ataxia and other neurological disorders. All three anoctamins have been proposed to control intracellular Ca signals. Anoctamins may also form the basolateral Ca-activated Cl channel in the retinal pigment epithelium (RPE). We show that native human, bovine, porcine, and mouse RPEs express ANO1, ANO6, and ANO10. Growth arrested and confluent RPR cells expressed ANO1 in the plasma membrane, whereas ANO6 and ANO10 were found in the primary cilium. Ussing chamber experiments showed that the application of ATP to the apical (retinal) side of porcine RPE induced a Ca-activated Cl secretion. Activation was inhibited by basolateral (choroidal) administration of the ANO inhibitors AO1, niflumic acid (NFA), and 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS). The results suggest that ANO1 is responsible for basolateral Ca-dependent Cl secretion in RPE, whereas ANO6 and ANO10 may have different functions, such as modulating Ca signals. [ABSTRACT FROM AUTHOR]

A remarkable feature of apoptosis is the initial massive cell shrinkage, which requires opening of ion channels to allow release of K, Cl, and organic osmolytes to drive osmotic water movement and cell shrinkage. This article focuses on the role of the Cl channels LRRC8, TMEM16/anoctamin, and cystic fibrosis transmembrane conductance regulator (CFTR) in cellular apoptosis. LRRC8A-E has been identified as a volume-regulated anion channel expressed in many cell types. It was shown to be required for regulatory and apoptotic volume decrease (RVD, AVD) in cultured cell lines. Its presence also determines sensitivity towards cytostatic drugs such as cisplatin. Recent data point to a molecular and functional relationship of LRRC8A and anoctamins (ANOs). ANO6, 9, and 10 (TMEM16F, J, and K) augment apoptotic Cl currents and AVD, but it remains unclear whether these anoctamins operate as Cl channels or as regulators of other apoptotic Cl channels, such as LRRC8. CFTR has been known for its proapoptotic effects for some time, and this effect may be based on glutathione release from the cell and increase in cytosolic reactive oxygen species (ROS). Although we find that CFTR is activated by cell swelling, it is possible that CFTR serves RVD/AVD through accumulation of ROS and activation of independent membrane channels such as ANO6. Thus activation of ANO6 will support cell shrinkage and induce additional apoptotic events, such as membrane phospholipid scrambling. [ABSTRACT FROM AUTHOR]

Background / Aims: TMEM16F is a transmembrane protein from a conserved family of Ca2+-activated proteins, which is highly expressed in several tissues. TMEM16F confers phospholipid scramblase activity and Ca2+-activated electrolyte channel activity. Potentially thereby, TMEM16F is involved in cell cycle control and apoptotic signaling. The present study evaluated the role of TMEM16F on cell proliferation and viability in Human Embryonic Kidney cells. Methods: An inducible knockdown of TMEM16F was generated and markers of apoptosis and proliferation were assessed via flow cytometry, western blotting and MTT uptake assay under different conditions. Results: TMEM16F knockdown resulted in attenuated growth of HEK293 cells. This observation correlated with an increased phosphatidylserine exposure and a decreased fraction of viable cells. Interestingly, the cells were not sensitized to Staurosporine-induced cell death. Western blot analyses displayed a parallel activation of pro- and antiapoptotic signaling pathways: Caspase 3 cleavage and Cyclin D1 abundance were simultaneously increased. Furthermore, knockdown of TMEM16F led to activation of AKT signaling. Conclusion: TMEM16F modifies viability of Human Embryonic Kidney cells via its function as a phospholipid scramblase and activation of AKT signaling pathways. [ABSTRACT FROM AUTHOR]

Activation of ion channels and pores are essential steps during regulated cell death. Channels and pores participate in execution of apoptosis, necroptosis and other forms of caspase-independent cell death. Within the program of regulated cell death, these channels are strategically located. Ion channels can shrink cells and drive them towards apoptosis, resulting in silent, i.e. immunologically unrecognized cell death. Alternatively, activation of channels can induce cell swelling, disintegration of the cell membrane, and highly immunogenic necrotic cell death. The underlying cell death pathways are not strictly separated as identical stimuli may induce cell shrinkage and apoptosis when applied at low strength, but may also cause cell swelling at pronounced stimulation, resulting in regulated necrosis. Nevertheless, the precise role of ion channels during regulated cell death is far from being understood, as identical channels may support regulated death in some cell types, but may cause cell proliferation, cancer development, and metastasis in others. Along this line, the phospholipid scramblase and Cl/nonselective channel anoctamin 6 (ANO6) shows interesting features, as it participates in apoptotic cell death during lower levels of activation, thereby inducing cell shrinkage. At strong activation, e.g. by stimulation of purinergic P2Y receptors, it participates in pore formation, causes massive membrane blebbing, cell swelling, and membrane disintegration. The LRRC8 proteins deserve much attention as they were found to have a major role in volume regulation, apoptotic cell shrinkage and resistance towards anticancer drugs. [ABSTRACT FROM AUTHOR]

Since the discovery of TMEM16A (anoctamin 1, ANO1) as Ca-activated Cl channel, the protein was found to serve different physiological functions, depending on the type of tissue. Subsequent reports on other members of the anoctamin family demonstrated a broad range of yet poorly understood properties. Compromised anoctamin function is causing a wide range of diseases, such as hearing loss (ANO2), bleeding disorder (ANO6), ataxia and dystonia (ANO3, 10), persistent borrelia and mycobacteria infection (ANO10), skeletal syndromes like gnathodiaphyseal dysplasia and limb girdle muscle dystrophy (ANO5), and cancer (ANO1, 6, 7). Animal models demonstrate CF-like airway disease, asthma, and intestinal hyposecretion (ANO1). Although present data indicate that ANO1 is a Ca-activated Cl channel, it remains unclear whether all anoctamins form plasma membrane-localized or intracellular chloride channels. We find Ca-activated Cl currents appearing by expression of most anoctamin paralogs, including the Nectria haematococca homologue nhTMEM16 and the yeast homologue Ist2. As recent studies show a role of anoctamins, Ist2, and the related transmembrane channel-like (TMC) proteins for intracellular Ca signaling, we will discuss the role of these proteins in generating compartmentalized Ca signals, which may give a hint as to the broad range of cellular functions of anoctamins. [ABSTRACT FROM AUTHOR]

During cell swelling, Cl channels are activated to lower intracellular Cl concentrations and to reduce cell volume, a process termed regulatory volume decrease (RVD). We show that anoctamin 6 (ANO6; TMEM16F) produces volume-regulated anion currents and controls cell volume in four unrelated cell types. Volume regulation is compromised in freshly isolated intestinal epithelial cells from Ano6−/− mice and also in lymphocytes from a patient lacking expression of ANO6. Ca influx is activated and thus ANO6 is stimulated during cell swelling by local Ca increase probably in functional nanodomains near the plasma membrane. This leads to stimulation of phospholipase A (PLA) and generation of plasma membrane lysophospholipids, which activates ANO6. Direct application of lysophospholipids also activates an anion current that is inhibited by typical ANO6 blocker. An increase in intracellular Ca supports activation of ANO6, but is not required when PLA is fully activated, while re-addition of arachidonic acid completely blocked ANO6. Moreover, ANO6 is activated by low intracellular Cl concentrations and may therefore operate as a cellular osmosensor. High intracellular Cl concentration inhibits ANO6 and activation by PLA. Taken together, ANO6 supports volume regulation and volume activation of anion currents by action as a Cl channel or by scrambling membrane phospholipids. Thereby, it may support the function of LRRC8 proteins. [ABSTRACT FROM AUTHOR]

Intestinal epithelial electrolyte secretion is activated by increase in intracellular cAMP or Ca and opening of apical Cl channels. In infants and young animals, but not in adults, Ca-activated chloride channels may cause secretory diarrhea during rotavirus infection. While detailed knowledge exists concerning the contribution of cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) channels, analysis of the role of Ca-dependent Cl channels became possible through identification of the anoctamin (TMEM16) family of proteins. We demonstrate expression of several anoctamin paralogues in mouse small and large intestines. Using intestinal-specific mouse knockout models for anoctamin 1 (Ano1) and anoctamin 10 (Ano10) and a conventional knockout model for anoctamin 6 (Ano6), we demonstrate the role of anoctamins for Ca-dependent Cl secretion induced by the muscarinic agonist carbachol (CCH). Ano1 is preferentially expressed in the ileum and large intestine, where it supports Ca-activated Cl secretion. In contrast, Ano10 is essential for Ca-dependent Cl secretion in jejunum, where expression of Ano1 was not detected. Although broadly expressed, Ano6 has no role in intestinal cholinergic Cl secretion. Ano1 is located in a basolateral compartment/membrane rather than in the apical membrane, where it supports CCH-induced Ca increase, while the essential and possibly only apical Cl channel is CFTR. These results define a new role of Ano1 for intestinal Ca-dependent Cl secretion and demonstrate for the first time a contribution of Ano10 to intestinal transport. [ABSTRACT FROM AUTHOR]

Loss of plasma membrane asymmetry is a hallmark of apoptosis, but lipid bilayer asymmetry and loss of asymmetry can contribute to numerous cellular functions and responses that are independent of programmed cell death. Exofacial exposure of phosphatidylserine occurs in lymphocytes and mast cells after antigenic stimulation and in the absence of apoptosis, suggesting that there is a functional requirement for phosphatidylserine exposure in immunocytes. In this review we examine current ideas as to the nature of this functional role in mast cell activation. Mechanistically, there is controversy as to the candidate proteins responsible for phosphatidylserine translocation from the internal to external leaflet, and here we review the candidacies of mast cell PLSCR1 and TMEM16F. Finally we examine the potential relationship between functionally important mast cell membrane perturbations and phosphatidylserine exposure during activation. [ABSTRACT FROM PUBLISHER]

Anoctamin 1 (TMEM16A, Ano1) is a recently identified Ca2+-activated chloride channel and a member of a large protein family comprising 10 paralogues. Before Ano1 was identified as a chloride channel protein, it was known as the cancer marker DOG1. DOG1/Ano1 is expressed in gastrointestinal stromal tumours (GIST) and particularly in head and neck squamous cell carcinoma, at very high levels never detected in other tissues. It is now emerging that Ano1 is part of the 11q13 locus, amplified in several types of tumour, where it is thought to augment cell proliferation, cell migration andmetastasis. Notably, Ano1 is upregulated through histone deacetylase (HDAC), corresponding to the known role of HDAC in HNSCC. As Ano1 does not enhance proliferation in every cell type, its function is perhaps modulated by cell-specific factors, or by the abundance of other anoctamins. Thus Ano6, by regulating Ca2+-induced membrane phospholipid scrambling and annexin V binding, supports cellular apoptosis rather than proliferation. Current findings implicate other cellular functions of anoctamins, apart from their role as Ca2+-activated Cl– channels. [ABSTRACT FROM AUTHOR]

Anoctamin 6 (Ano6; TMEM16F gene) is a ubiquitous protein; the expression of which is defective in patients with Scott syndrome, an inherited bleeding disorder based on defective scrambling of plasma membrane phospholipids. For Ano6, quite diverse functions have been described: (1) it can form an outwardly rectifying, Ca-dependent and a volume-regulated Cl channel; (2) it was claimed to be a Ca-regulated nonselective cation channel permeable for Ca; (3) it was shown to be essential for Ca-mediated scrambling of membrane phospholipids; and (4) it can regulate cell blebbing and microparticle shedding. Deficiency of Ano6 in blood cells from Scott patients or Ano6 null mice appears to affect all of these cell responses. Furthermore, Ano6 deficiency in mice impairs the mineralization of osteoblasts, resulting in reduced skeletal development. These diverse results have been obtained under different experimental conditions, which may explain some of the contradictions. This review therefore aims to summarize the currently available information on the diverse roles of Ano6 and tries to clear up some of the existing controversies. [ABSTRACT FROM AUTHOR]