Control of Ion Transport by Tmem16a Expressed in Murine Intestine

Cl- secretion by the human and murine intestinal epithelium occurs through the cystic fibrosis transmembrane conductance regulator (cftr). However, the Ca2+ activated Cl- channel Tmem16a was shown to contribute to Cl- secretion, mainly, but not exclusively, as a basolaterally located Cl- channel that controls basolateral Ca2+ signaling, and thus activation of basolateral Ca2+ dependent Sk4 K+ channels. In intestinal goblet cells, Tmem16a was shown to regulated Ca2+ signals required for exocytosis of mucus. Because a recent report denied the existence and functional role of Tmem16a in murine intestine, we reexamined in detail expression of mRNA and protein for Tmem16a in mouse colon. In experiments using short-circuited Ussing chamber and whole cell patch-clamp techniques, we further compared ion transport in wild type (WT) colon with that in mice with intestinal epithelial specific knockout of Tmem16a. As reported earlier we fully confirm expression of Tmem16a in colonic epithelial cells and the role of Tmem16a for both Ca2+-dependent and cAMP-regulated ion secretion

Anoctamins in Volume Regulation, Proliferation and Apoptotic Cell Death

The family of anoctamins consists of ten different proteins (called ANO1-10 or TMEM16A-K). A variety of functions have been attributed to anoctamins such as that of Ca2+ activated Cl- channels controlling Cl- secretion, cell volume, cell migration and proliferation, and that of phospholipid scramblases contributing to apoptotic cell death. Some anoctamin members are overexpressed in tumors and have been linked to developmental defects. Mutations in anoctamins can cause genetic diseases and cancer. However, it is unknown how anoctamins fulfill these various functions. Before ANO1 was identified as a protein forming a chloride channel, it was known as the cancer marker DOG1. DOG1/ANO1 is expressed in gastrointestinal stromal tumors (GIST) and particularly in head and neck squamous cell carcinoma (HNSCC), and is not detected at very high levels in other tissues. We found that ANO1 strongly augments cell proliferation, cell migration and metastasis. We also found that ANO1 expression is controlled by histone deacetylases (HDAC), corresponding well to the known role of HDAC in HNSCC. As ANO1 did not enhance proliferation in every cell type, its function is perhaps modulated by cell-specific factors. In contrast to ANO1, we found that ANO6, by operating as a Ca2+ activated membrane phospholipid scramblase, supports cellular apoptosis and necroptosis rather than promoting proliferation. Earlier findings from our laboratory and other groups indicated that ANO6 is a Cl- channel, a phospholipid scramblase and a nonselective cation channel activated by intracellular Ca2+ and pro-apoptotic stimuli. Moreover, previous studies characterized ANO6 as an outwardly rectifying Cl- channel (ORCC) and volume-regulated anion channel (VRAC) that is activated during apoptotic cell death. In the experiments presented here I found a major role of ANO6 for both regulatory volume decrease (RVD) as well as apoptotic volume decrease (AVD). RVD and AVD were examined in four different cell types under physiological conditions, i.e. in the presence of physiological intracellular and extracellular bath solutions and at 37 °C. Moreover swelling activated whole cell currents and volume regulation was assessed in freshly isolated intestinal epithelial cells from wild type and anoctamin 6 knockout mice. The data indicate that ANO6 generates a VRAC and is activated during RVD by membrane stretch, Ca2+ influx through TRP channels and activation of phospholipase A2 (PLA2), which generates membrane lysophospholipids (LPL). Accumulation of LPL in the plasma membrane enhances membrane tension, which possibly induces a conformational change of ANO6, thereby opening the Cl- conductive pore and causing RVD. Remarkably, RVD was largely reduced in intestinal epithelial cells from ANO6 knockout mice. The results also demonstrate that ANO6 is activated by low intracellular Cl-, which suggests that ANO6 operates as an osmosensor. ANO6 was further demonstrated to participate in the innate immune response in macrophages. We could demonstrate that in macrophages Ca2+ influx through P2X7 channels leads to initial cell shrinkage, phospholipid scrambling, cell membrane blebbing and ultimately cell death. Cell migration and phagocytic activity was found to be largely reduced in Ano6-/- macrophages. We proposed that ANO6 plays a central role for the immune defense realized by macrophages. The data also indicate that P2X7-induced cell death in macrophages is due to activation of apoptosis as well as necrosis. Another member of the anoctamin family, ANO10, was also demonstrated to be expressed in macrophages and to participate in innate immune response. In collaboration with the Max Planck institute of experimental Medicine in Göttingen we showed that the genomic ANO10 mutation R263H increases serum titers of antibodies directed against Lyme disease causing Borrelia. We suggest that ANO10-R263H enhances susceptibility towards Borrelia infection and attenuates clearance of spirochetes after tick bites. ANO10-R263H caused a reduced VRAC activity and RVD. Loss of ANO10 expression in macrophages compromised phagocytosis and clearance of Borrelia, attenuated volume regulation and reduced apoptotic cell death. Taken together the results demonstrate that anoctamins participate in the regulation of proliferation and cell death and play important roles in cancer and innate immune defense

Contribution of TMEM16F to pyroptotic cell death

Abstract Pyroptosis is a highly inflammatory form of programmed cell death that is caused by infection with intracellular pathogens and activation of canonical or noncanonical inflammasomes. The purinergic receptor P2X7 is activated by the noncanonical inflammasome and contributes essentially to pyroptotic cell death. The Ca2+ activated phospholipid scramblase and ion channel TMEM16F has been shown earlier to control cellular effects downstream of purinergic P2X7 receptors that ultimately lead to cell death. As pyroptotic cell death is accompanied by an increases in intracellular Ca2+, we asked whether TMEM16F is activated during pyroptosis. The N-terminal cleavage product of gasdermin D (GD-N) is an executioner of pyroptosis by forming large plasma membrane pores. Expression of GD-N enhanced basal Ca2+ levels and induced cell death. We observed that GD-N induced cell death in HEK293 and HAP1 cells, which was depending on expression of endogenous TMEM16F. GD-N activated large whole cell currents that were suppressed by knockdown or inhibition of TMEM16F. The results suggest that whole cell currents induced by the pore forming domain of gasdermin-D, are at least in part due to activation of TMEM16F. Knockdown of other TMEM16 paralogues expressed in HAP1 cells suggest TMEM16F as a crucial element during pyroptosis and excluded a role of other TMEM16 proteins. Thus TMEM16F supports pyroptosis and other forms of inflammatory cell death such as ferroptosis. Its potent inhibition by tannic acid may be part of the anti-inflammatory effects of flavonoids

Cellular volume regulation by anoctamin 6: Ca2+, phospholipase A2 and osmosensing

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. Ca2+ influx is activated and thus ANO6 is stimulated during cell swelling by local Ca2+ increase probably in functional nanodomains near the plasma membrane. This leads to stimulation of phospholipase A(2) (PLA(2)) 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 Ca2+ supports activation of ANO6, but is not required when PLA(2) 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(2). 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

Cl− channels in apoptosis

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

Cellular defects by deletion of ANO10 are due to deregulated local calcium signaling

TMEM16K (ANO10) belongs to a family of ion channels and phospholipid scramblases. Mutations in ANO10 cause neurological and immunological defects, and abrogated ion transport. Here we show that Ano10 knockout in epithelial cells leads to defective ion transport, attenuated volume regulation and deranged Ca2+ signaling. Intestinal epithelial cells from Ano10 null mice are reduced in size and demonstrate an almost abolished spontaneous and TNF alpha-induced apoptosis. Similar defects were found in mouse peritoneal Ano10 null macrophages and in human THP1 macrophages with reduced ANO10 expression. A cell cycle dependent colocalization of Ano10 with acetylated tubulin, centrioles, and a submembranous tubulin containing compartment was observed in Fisher rat thyroid cells. Axs, the Drosophila ortholog of ANO10 is known for its role in mitotic spindle formation and association with the endoplasmic reticulum and Ca2+ signaling. We therefore propose that mutations in ANO10 cause cellular defects and genetic disorders through deranged local Ca2+ signaling. (C) 2016 Elsevier Inc. All rights reserved

TMEM16A Mediates Mucus Production in Human Airway Epithelial Cells

TMEM16A is a Ca2+-activated chloride channel that was shown to enhance production and secretion of mucus in inflamed airways. It is, however, not clear whether TMEM16A directly supports mucus production, or whether mucin and TMEM16A are upregulated independently during inflammatory airway diseases such as asthma and cystic fibrosis (CF). We examined this question using BCi-NS1 cells, a human airway basal cell line that maintains multipotent differentiation capacity, and the two human airway epithelial cell lines, Calu-3 and CFBE. The data demonstrate that exposure of airway epithelial cells to IL-8 and IL-13, two cytokines known to be enhanced in CF and asthma, respectively, leads to an increase in mucus production. Expression of MUC5AC was fully dependent on expression of TMEM16A, as shown by siRNA knockdown of TMEM16A. In addition, different inhibitors of TMEM16A attenuated IL-13-induced mucus production. Interestingly, in CFBE cells expressing F508 delCFTR, IL-13 was unable to upregulate membrane expression of TMEM16A or Ca2+-activated whole cell currents. The regulator of TMEM16A, CLCA1, strongly augmented both Ca2+- and cAMP-activated Cl2 currents in cells expressing wtCFTR but failed to augment membrane expression of TMEM16A in F508 delCFTR-expressing CFBE cells. The data confirm the functional relationship between CFTR and TMEM16A and suggest an impaired upregulation of TMEM16A by IL-13 or CLCA1 in cells expressing the most frequent CF-causing mutation F508 delCFTR

CFTR supports cell death through ROS-dependent activation of TMEM16F (anoctamin 6)

Cystic fibrosis transmembrane conductance regulator (CFTR) is the essential chloride and bicarbonate channel in the apical membrane of epithelial cells. CFTR was also proposed earlier to conduct glutathione (GSH) out of airway epithelial cells to be enriched in the apical airway surface liquid to neutralize reactive oxygen species (ROS). Although earlier studies suggested that release of GSH by wild type (wt) CFTR may lead to an increase in cytosolic ROS, we did not detect different ROS levels in cells expressing wt-CFTR and mutant F508del-CFTR, independent of CFTR-activation or exposure to the ROS donor tert-butyl hydroperoxide. The Ca2+-activated phospholipid scramblase and ion channel TMEM16F (anoctamin 6, ANO6) is also expressed in airway cells. ANO6 produced outwardly rectifying Cl- currents (ORCC) and scrambled plasma membrane phospholipids when activated by increase in cytosolic ROS and consecutive peroxidation of plasma membrane lipids. ANO6 activity is enhanced by CFTR, probably through translocation of signaling proteins to the plasma membrane. The present data suggest that enhanced cell death in CFTR-expressing cells is due to upregulation of ANO6-activity. In ANO6 knockout mice, the number of apoptotic cells in the intestinal epithelium was strongly reduced, supporting the role of ANO6 for cell death. Thus, ANO6 and CFTR act cooperatively on ROS-mediated cell death, which is not further augmented by cAMP-dependent stimulation. We propose that ANO6 supports cell death correlated with expression of CFTR, possibly by inducing ferroptosis

Ca2+ signals, cell membrane disintegration, and activation of TMEM16F during necroptosis

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 Ca2+. We, therefore, asked whether TMEM16F is also central to necroptotic cell death and other cellular events during necroptosis. Necroptosis was induced by TNF alpha, smac mimetic, and Z-VAD (TSZ) in NIH3T3 fibroblasts and the four additional cell lines HT29, 16HBE, H441, and L929. Time-dependent changes in intracellular Ca2+, cell morphology, and membrane currents were recorded. TSZ induced a small and only transient oscillatory rise in intracellular Ca2+, which was paralleled by the activation of outwardly rectifying Cl- currents, which were typical for TMEM16F/ANO6. Ca2+ oscillations were due to Ca2+ release from endoplasmic reticulum, and were independent of extracellular Ca2+. 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

Relationship between TMEM16A/anoctamin 1 and LRRC8A

TMEM16A/anoctamin 1/ANO1 and VRAC/LRRC8 are independent chloride channels activated either by increase in intracellular Ca2+ or cell swelling, respectively. In previous studies, we observed overlapping properties for both types of channels. (i) TMEM16A/ANO1 and LRRC8 are inhibited by identical compounds, (ii) the volume-regulated anion channel VRAC requires compartmentalized Ca2+ increase to be fully activated, (iii) anoctamins are activated by cell swelling, (iv) both channels have a role for apoptotic cell death, (v) both channels are possibly located in lipid rafts/caveolae like structures, and (vi) VRAC and anoctamin 1 currents are not additive when each are fully activated. In the present study, we demonstrate in different cell types that loss of LRRC8A expression not only inhibited VRAC, but also attenuated Ca2+ activated Cl- currents. Moreover, expression of LRRC8A enhanced Ca2+ activated Cl- currents, and both LRRC8A and ANO1 could be coimmunoprecipitated. We found that LRRC8A becomes accessible to biotinylation upon exposure to hypotonic bath solution, while membrane capacitance was not enhanced. When intracellular Ca2+ was increased in ANO1-expressing cells, the membrane capacitance was enhanced and increased binding of FM4-64 to the membrane was observed. As this was not seen in cells lacking ANO1 expression, a role of ANO1 for exocytosis was suggested. We propose that ANO1 and LRRC8A are activated in parallel. Thus, ionomycin or purinergic stimulation will not only activate ANO1 but also LRRC8 currents. Cell swelling will not only activate LRRC8/VRAC, but also stimulate ANO1 currents by enhancing compartmentalized Ca2+ increase and/or through swelling induced autocrine release of ATP