RESUMEN
Inflammatory airway diseases like cystic fibrosis, asthma and COVID-19 are characterized by high levels of pulmonary cytokines. Two well-established antiparasitic drugs, niclosamide and ivermectin, are intensively discussed for the treatment of viral inflammatory airway infections. Here, we examined these repurposed drugs with respect to their anti-inflammatory effects in airways in vivo and in vitro. Niclosamide reduced mucus content, eosinophilic infiltration and cell death in asthmatic mouse lungs in vivo and inhibited release of interleukins in the two differentiated airway epithelial cell lines CFBE and BCi-NS1.1 in vitro. Cytokine release was also inhibited by the knockdown of the Ca2+-activated Cl- channel anoctamin 1 (ANO1, TMEM16A) and the phospholipid scramblase anoctamin 6 (ANO6, TMEM16F), which have previously been shown to affect intracellular Ca2+ levels near the plasma membrane and to facilitate exocytosis. At concentrations around 200 nM, niclosamide inhibited inflammation, lowered intracellular Ca2+, acidified cytosolic pH and blocked activation of ANO1 and ANO6. It is suggested that niclosamide brings about its anti-inflammatory effects at least in part by inhibiting ANO1 and ANO6, and by lowering intracellular Ca2+ levels. In contrast to niclosamide, 1 µM ivermectin did not exert any of the effects described for niclosamide. The present data suggest niclosamide as an effective anti-inflammatory treatment in CF, asthma, and COVID-19, in addition to its previously reported antiviral effects. It has an advantageous concentration-response relationship and is known to be well tolerated.
Asunto(s)
Asma , COVID-19 , Ratones , Animales , Anoctamina-1/metabolismo , Ivermectina/farmacología , Ivermectina/uso terapéutico , Niclosamida/farmacología , Niclosamida/uso terapéutico , Anoctaminas/metabolismo , Pulmón/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Calcio/metabolismo , Inflamación/tratamiento farmacológico , Antiinflamatorios , Canales de Cloruro/metabolismoRESUMEN
Background and Aims: Paneth cells play a central role in intestinal innate immune response. These cells are localized at the base of small intestinal crypts of Lieberkuhn. The calcium-activated chloride channel TMEM16A and the phospholipid scramblase TMEM16F control intracellular Ca2+ signaling and exocytosis. We analyzed the role of TMEM16A and TMEM16F for Paneth cells secretion. Methods: Mice with intestinal epithelial knockout of Tmem16a (Tmem16a-/-) and Tmem16f (Tmem16f-/-) were generated. Tissue structures and Paneth cells were analyzed, and Paneth cell exocytosis was examined in small intestinal organoids in vitro. Intracellular Ca2+ signals were measured and were compared between wild-type and Tmem16 knockout mice. Bacterial colonization and intestinal apoptosis were analyzed. Results: Paneth cells in the crypts of Lieberkuhn from Tmem16a-/- and Tmem16f-/- mice demonstrated accumulation of lysozyme. Tmem16a and Tmem16f were localized in wild-type Paneth cells but were absent in cells from knockout animals. Paneth cell number and size were enhanced in the crypt base and mucus accumulated in intestinal goblet cells of knockout animals. Granule fusion and exocytosis on cholinergic and purinergic stimulation were examined online. Both were strongly compromised in the absence of Tmem16a or Tmem16f and were also blocked by inhibition of Tmem16a/f. Purinergic Ca2+ signaling was largely inhibited in Tmem16a knockout mice. Jejunal bacterial content was enhanced in knockout mice, whereas cellular apoptosis was inhibited. Conclusion: The present data demonstrate the role of Tmem16 for exocytosis in Paneth cells. Inhibition or activation of Tmem16a/f is likely to affect microbial content and immune functions present in the small intestine.
RESUMEN
Human CD137 (4-1BB), a member of the TNF receptor family, and its ligand CD137L (4-1BBL), are expressed on immune cells and tumor cells. CD137/CD137L interaction mediates bidirectional cellular responses of potential relevance in inflammatory diseases, autoimmunity and oncology. A soluble form of CD137 exists, elevated levels of which have been reported in patients with rheumatoid arthritis and various malignancies. Soluble CD137 (sCD137) is considered to represent a splice variant of CD137. In this report, however, evidence is presented that A Disintegrin and Metalloproteinase (ADAM)10 and potentially also ADAM17 are centrally involved in its generation. Release of sCD137 by transfected cell lines and primary T cells was uniformly inhibitable by ADAM10 inhibition. The shedding function of ADAM10 can be blocked through inhibition of its interaction with surface exposed phosphatidylserine (PS), and this effectively inhibited sCD137 generation. The phospholipid scramblase Anoctamin-6 (ANO6) traffics PS to the outer membrane and thus modifies ADAM10 function. Overexpression of ANO6 increased stimulated shedding, and hyperactive ANO6 led to maximal constitutive shedding of CD137. sCD137 was functionally active and augmented T cell proliferation. Our findings shed new light on the regulation of CD137/CD137L immune responses with potential impact on immunotherapeutic approaches targeting CD137.
Asunto(s)
Proteína ADAM10/metabolismo , Proteína ADAM17/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Artritis Reumatoide/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Anoctaminas/metabolismo , Artritis Reumatoide/patología , Membrana Celular/metabolismo , Células HEK293 , Células HT29 , Humanos , Neoplasias/patología , Proteínas de Transferencia de Fosfolípidos/metabolismo , Linfocitos T/metabolismo , Linfocitos T/patologíaRESUMEN
Anoctamin 6/TMEM16F (ANO6) is a dual-function protein with Ca2+-activated ion channel and Ca2+-activated phospholipid scramblase activities, requiring a high intracellular Ca2+ concentration (e.g., half-maximal effective Ca2+ concentration [EC50] of [Ca2+]i > 10 µM), and strong and sustained depolarization above 0 mV. Structural comparison with Anoctamin 1/TMEM16A (ANO1), a canonical Ca2+- activated chloride channel exhibiting higher Ca2+ sensitivity (EC50 of 1 µM) than ANO6, suggested that a homologous Ca2+-transferring site in the N-terminal domain (Nt) might be responsible for the differential Ca2+ sensitivity and kinetics of activation between ANO6 and ANO1. To elucidate the role of the putative Ca2+-transferring reservoir in the Nt (Nt-CaRes), we constructed an ANO6-1-6 chimera in which Nt-CaRes was replaced with the corresponding domain of ANO1. ANO6- 1-6 showed higher sensitivity to Ca2+ than ANO6. However, neither the speed of activation nor the voltage-dependence differed between ANO6 and ANO6-1-6. Molecular dynamics simulation revealed a reduced Ca2+ interaction with Nt- CaRes in ANO6 than ANO6-1-6. Moreover, mutations on potentially Ca2+-interacting acidic amino acids in ANO6 Nt- CaRes resulted in reduced Ca2+ sensitivity, implying direct interactions of Ca2+ with these residues. Based on these results, we cautiously suggest that the net charge of Nt- CaRes is responsible for the difference in Ca2+ sensitivity between ANO1 and ANO6.
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Anoctaminas/química , Anoctaminas/metabolismo , Calcio/metabolismo , Proteínas de Transferencia de Fosfolípidos/química , Proteínas de Transferencia de Fosfolípidos/metabolismo , Secuencia de Aminoácidos , Anoctaminas/genética , Motivos EF Hand , Células HEK293 , Humanos , Modelos Biológicos , Simulación de Dinámica Molecular , Mutación/genética , Proteínas de Transferencia de Fosfolípidos/genética , Dominios Proteicos , Relación Estructura-ActividadRESUMEN
Neuronal Ca2+ entry elicited by electrical activity contributes to information coding via activation of K+ and Cl- channels. While Ca2+-dependent K+ channels have been extensively studied, the molecular identity and role of Ca2+-activated Cl- channels (CaCCs) remain unclear. Here, we demonstrate that TMEM16F governs a Ca2+-activated Cl- conductance in spinal motoneurons. We show that TMEM16F is expressed in synaptic clusters facing pre-synaptic cholinergic C-boutons in α-motoneurons of the spinal cord. Mice with targeted exon deletion in Tmem16f display decreased motor performance under high-demanding tasks attributable to an increase in the recruitment threshold of fast α-motoneurons. Remarkably, loss of TMEM16F function in a mouse model of amyotrophic lateral sclerosis (ALS) significantly reduces expression of an activity-dependent early stress marker and muscle denervation, delays disease onset, and preserves muscular strength only in male ALS mice. Thus, TMEM16F controls motoneuron excitability and impacts motor resistance as well as motor deterioration in ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Esclerosis Amiotrófica Lateral/fisiopatología , Anoctaminas/metabolismo , Actividad Motora , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Proteínas de Transferencia de Fosfolípidos/metabolismo , Terminales Presinápticos/patología , Médula Espinal/patología , Animales , Biomarcadores/metabolismo , Canales de Cloruro/metabolismo , Colina/metabolismo , Progresión de la Enfermedad , Exones/genética , Células HEK293 , Humanos , Masculino , Ratones Endogámicos C57BL , Condicionamiento Físico Animal , Receptores Muscarínicos/metabolismo , Eliminación de Secuencia/genéticaRESUMEN
Ca2+ activated Cl- channels (TMEM16A; ANO1) support cell proliferation and cancer growth. Expression of TMEM16A is strongly enhanced in different types of malignomas. In contrast, TMEM16F (ANO6) operates as a Ca2+ activated chloride/nonselective ion channel and scrambles membrane phospholipids to expose phosphatidylserine at the cell surface. Both phospholipid scrambling and cell swelling induced through activation of nonselective ion currents appear to destabilize the plasma membrane thereby causing cell death. There is growing evidence that activation of TMEM16F contributes to various forms of regulated cell death. In the present study, we demonstrate that ferroptotic cell death, occurring during peroxidation of plasma membrane phospholipids activates TMEM16F. Ferroptosis was induced by erastin, an inhibitor of the cystine-glutamate antiporter and RSL3, an inhibitor of glutathione peroxidase 4 (GPX4). Cell death was largely reduced in the intestinal epithelium, and in peritoneal macrophages isolated from mice with tissue-specific knockout of TMEM16F. We show that TMEM16F is activated during erastin and RSL3-induced ferroptosis. In contrast, inhibition of ferroptosis by ferrostatin-1 and by inhibitors of TMEM16F block TMEM16F currents and inhibit cell death. We conclude that activation of TMEM16F is a crucial component during ferroptotic cell death, a finding that may be useful to induce cell death in cancer cells.
RESUMEN
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.
Asunto(s)
Anoctamina-1/metabolismo , Anoctaminas/metabolismo , Membrana Celular/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Animales , Anoctamina-1/análisis , Anoctamina-1/genética , Anoctaminas/análisis , Anoctaminas/genética , Línea Celular , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/análisis , Exocitosis , Células HEK293 , Humanos , Ratones Noqueados , Proteínas de Transferencia de Fosfolípidos/análisis , Proteínas de Transferencia de Fosfolípidos/genéticaRESUMEN
Dysregulation of the disintegrin-metalloproteinase ADAM10 may contribute to the development of diseases including tumorigenesis and Alzheimer's disease. The mechanisms underlying ADAM10 sheddase activation are incompletely understood. Here, we show that transient exposure of the negatively charged phospholipid phosphatidylserine (PS) is necessarily required. The soluble PS headgroup was found to act as competitive inhibitor of substrate cleavage. Overexpression of the Ca2+-dependent phospholipid scramblase Anoctamin-6 (ANO6) led to increased PS externalization and substrate release. Transfection with a constitutively active form of ANO6 resulted in maximum sheddase activity in the absence of any stimulus. Calcium-dependent ADAM10 activation could not be induced in lymphocytes of patients with Scott syndrome harbouring a missense mutation in ANO6. A putative PS-binding motif was identified in the conserved stalk region. Replacement of this motif resulted in strong reduction of sheddase activity. In conjunction with the recently described 3D structure of the ADAM10 extracellular domain, a model is advanced to explain how surface-exposed PS triggers ADAM10 sheddase function.
Asunto(s)
Proteína ADAM10/metabolismo , Membrana Celular/metabolismo , Activación Enzimática , Proteína ADAM10/química , Secuencia de Aminoácidos , Animales , Anoctaminas/metabolismo , Biomarcadores , Células COS , Línea Celular , Chlorocebus aethiops , Eritrocitos/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Fosfoserina/metabolismo , Conejos , Relación Estructura-ActividadRESUMEN
ADAM17, a prominent member of the "Disintegrin and Metalloproteinase" (ADAM) family, controls vital cellular functions through cleavage of transmembrane substrates including TGF-alpha, Amphiregulin (AREG) and TNF-Receptor 1 (TNFR1). We recently presented evidence that surface exposure of phosphatidylserine (PS) is pivotal for ADAM17 to exert sheddase activity. Anoctamin-6 (ANO6) has Ca2+-dependent phospholipid scramblase activity and it followed that the functions of ANO6 and ADAM17 might be linked. We report that overexpression of ANO6 in HEK293T cells led to increased Ca2+-mediated PS-exposure that was indeed accompanied by enhanced release of AREG and TGF-alpha. The effect was not observed when cells were treated with the PKC-dependent ADAM17 activator PMA. Transformation of cells with a constitutively active ANO6 mutant led to spontaneous PS-exposure and to the release of ADAM17-substrates in the absence of any stimuli. Inhibitor experiments indicated that ANO6-mediated enhancement of substrate cleavage simultaneously broadened the spectrum of participating metalloproteinases. In complementary experiments, siRNA-mediated downregulation of ANO6 was shown to decrease ionophore-mediated release of TNFR1 in human umbilical vein endothelial cells (HUVECs). We conclude that ANO6, by virtue of its scramblase activity, may play a role as an important regulator of the ADAM-network in the plasma membrane.
Asunto(s)
Proteínas ADAM/metabolismo , Anoctaminas/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Fosfolípidos/metabolismo , Proteína ADAM17/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Ionomicina/farmacología , Modelos Biológicos , Mutación , Factor de Crecimiento Transformador alfa/metabolismoRESUMEN
The increase in migratory ability of pancreatic ductal adenocarcinoma cells is a key event in the development of metastasis to the lymph nodes and distant organs. Although the C-C motif chemokine receptor 7 (CCR7) and its ligand, C-C motif chemokine ligand 21 (CCL21), have been revealed to serve an important role in tumor migration, their precise roles and potential underlying mechanisms remain largely unknown. The present study revealed that overexpression of CCR7 significantly promoted BxPC-3 cell migration, accompanied by the induction of anoctamin 6 (ANO6) expression, indicating that ANO6 is a downstream target of CCR7 signaling. Furthermore, the level of phosphorylated extracellular signal-regulated kinase (ERK) was significantly increased in CCR7-overexpressing BxPC-3 cells, indicating that ERK may be a potential mediator of CCR7-regulated ANO6 expression in BxPC-3 cells. To characterize the receptor-mediated pathway, a specific ERK inhibitor, U0126, was used, which reduced BxPC-3 cell migration and the expression of ANO6. In summary, the results of the present study demonstrate that CCR7 promoted BxPC-3 cell migration by regulating ANO6 expression perhaps via activation of the ERK signaling pathway.
RESUMEN
Anoctamin 6 (ANO6/TMEM16F) is a recently identified membrane protein that has both phospholipid scramblase activity and anion channel function activated by relatively high [Ca2+]i. In addition to the low sensitivity to Ca2+, the activation of ANO6 Cl- conductance is very slow (>3-5â¯min to reach peak level at 10⯵M [Ca2+]i), with subsequent inactivation. In a whole-cell patch clamp recording of ANO6 current (IANO6,w-c), disruption of the actin cytoskeleton with cytochalasin-D (cytoD) significantly accelerated the activation kinetics, while actin filament-stabilizing agents (phalloidin and jasplakinolide) commonly inhibited IANO6,w-c. Inside-out patch clamp recording of ANO6 (IANO6,i-o) showed immediate activation by raising [Ca2+]i. We also found that intracellular ATP (3â¯mM MgATP in pipette solution) decelerated the activation of IANO6,w-c, and also prevented the inactivation of IANO6,w-c. However, the addition of cytoD still accelerated both activation and inactivation of IANO6,w-c. We conclude that the actin cytoskeleton and intracellular ATP play major roles in the Ca2+-dependent activation and inactivation of IANO6,w-c, respectively.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Adenosina Trifosfato/metabolismo , Anoctaminas/metabolismo , Calcio/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Células HEK293 , Humanos , Técnicas de Placa-ClampRESUMEN
The Ca2+-activated phospholipid scramblase and ion channel TMEM16F is expressed in podocytes of renal glomeruli. Podocytes are specialized cells that form interdigitating foot processes as an essential component of the glomerular filter. These cells, which participate in generation of the primary urine, are often affected during primary glomerular diseases, such as glomerulonephritis and secondary hypertensive or diabetic nephropathy, which always leads to proteinuria. Because the function of podocytes is known to be controlled by intracellular Ca2+ signaling, it is important to know about the role of Ca2+-activated TMEM16F in these cells. To that end, we generated an inducible TMEM16F knockdown in the podocyte cell line AB8, and produced a conditional mouse model with knockout of TMEM16F in podocytes and renal epithelial cells of the nephron. We found that knockdown of TMEM16F did not produce proteinuria or any obvious phenotypic changes. Knockdown of TMEM16F affected cell death of tubular epithelial cells but not of glomerular podocytes when analyzed in TUNEL assays. Surprisingly, and in contrast to other cell types, TMEM16F did not control intracellular Ca2+ signaling and was not responsible for Ca2+-activated whole cell currents in podocytes. TMEM16F levels in podocytes were enhanced after inhibition of the endolysosomal pathway and after treatment with angiotensin II. Renal knockout of TMEM16F did not compromise renal morphology and serum electrolytes. Taken together, in contrast to other cell types, such as platelets, bone cells, and immune cells, TMEM16F shows little effect on basal properties of podocytes and does not appear to be essential for renal function.
Asunto(s)
Anoctaminas/genética , Proteínas de Transferencia de Fosfolípidos/genética , Podocitos/metabolismo , Potenciales de Acción , Animales , Anoctaminas/metabolismo , Apoptosis , Señalización del Calcio , Células HEK293 , Humanos , Ratones , Fenotipo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Podocitos/fisiologíaRESUMEN
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.
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Anoctamina-1/metabolismo , Anoctaminas/metabolismo , Calcio/farmacología , Membrana Celular/metabolismo , Lípidos de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Fosfolípidos/metabolismo , Anoctamina-1/genética , Anoctaminas/genética , Apoptosis , Hormonas y Agentes Reguladores de Calcio/farmacología , Células HEK293 , Humanos , Transporte Iónico , Proteínas de Neoplasias/genética , Fosfolipasas A2/metabolismo , Proteínas de Transferencia de Fosfolípidos/genética , Transducción de SeñalRESUMEN
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.
Asunto(s)
Anoctaminas/metabolismo , Apoptosis , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Animales , Anoctaminas/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células HEK293 , Células HeLa , Humanos , Ratones , Proteínas de Transferencia de Fosfolípidos/genética , Especies Reactivas de Oxígeno/metabolismo , XenopusRESUMEN
Nanosecond pulsed electric fields (nsPEF) are emerging as a novel modality for cell stimulation and tissue ablation. However, the downstream protein effectors responsible for nsPEF bioeffects remain to be established. Here we demonstrate that nsPEF activate TMEM16F (or Anoctamin 6), a protein functioning as a Ca2+-dependent phospholipid scramblase and Ca2+-activated chloride channel. Using confocal microscopy and patch clamp recordings, we investigated the relevance of TMEM16F activation for several bioeffects triggered by nsPEF, including phosphatidylserine (PS) externalization, nanopore-conducted currents, membrane blebbing, and cell death. In HEK 293 cells treated with a single 300-ns pulse of 25.5 kV/cm, Tmem16f expression knockdown and TMEM16F-specific inhibition decreased nsPEF-induced PS exposure by 49 and 42%, respectively. Moreover, the Tmem16f silencing significantly decreased Ca2+-dependent chloride channel currents activated in response to the nanoporation. Tmem16f expression also affected nsPEF-induced cell blebbing, with only 20% of the silenced cells developing blebs compared with 53% of the control cells. This inhibition of cellular blebbing correlated with a 25% decrease in cytosolic free Ca2+ transient at 30 s after nanoporation. Finally, in TMEM16F-overexpressing cells, a train of 120 pulses (300 ns, 20 Hz, 6 kV/cm) decreased cell survival to 34% compared with 51% in control cells (*, p < 0.01). Taken together, these results indicate that TMEM16F activation by nanoporation mediates and enhances the diverse cellular effects of nsPEF.
Asunto(s)
Anoctaminas/metabolismo , Apoptosis/efectos de la radiación , Calcio/metabolismo , Membrana Celular/fisiología , Electricidad , Nanotecnología , Fosfatidilserinas/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Relación Dosis-Respuesta en la Radiación , Células HEK293 , Humanos , Canales Iónicos/metabolismoRESUMEN
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 TNFα, 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.
Asunto(s)
Señalización del Calcio , Membrana Celular/metabolismo , Necrosis/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Animales , Anoctaminas , Calcio/metabolismo , Línea Celular , Ratones , Células 3T3 NIH , Necrosis/patología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The anoctamin (ANO, TMEM16) family of Ca2+-activated Cl- channels consists of ten members with different cellular functions (ANO1-10). ANO1 is a Ca2+-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 Ca2+-dependent scrambling of membrane phospholipids in platelets, red blood cells, and lymphocytes. ANO10 modulates Ca2+ signals in macrophages and has a role in cerebellar ataxia and other neurological disorders. All three anoctamins have been proposed to control intracellular Ca2+ signals. Anoctamins may also form the basolateral Ca2+-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 Ca2+-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 Ca2+-dependent Cl- secretion in RPE, whereas ANO6 and ANO10 may have different functions, such as modulating Ca2+ signals.
Asunto(s)
Proteínas de la Membrana/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Adenosina Trifosfato/metabolismo , Animales , Calcio/metabolismo , Bovinos , Línea Celular , Membrana Celular/metabolismo , Células Cultivadas , Cloruros/metabolismo , Humanos , Proteínas de la Membrana/genética , Ratones , Ácido Niflúmico/farmacología , Epitelio Pigmentado de la Retina/efectos de los fármacos , PorcinosRESUMEN
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 P2Y7 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.
Asunto(s)
Apoptosis/fisiología , Canales de Cloruro/metabolismo , Necrosis/patología , Canales de Potasio/metabolismo , Animales , Anoctaminas , Membrana Celular/patología , Proliferación Celular/fisiología , Tamaño de la Célula , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Neoplasias/patología , Proteínas de Transferencia de Fosfolípidos/metabolismo , Piroptosis/fisiologíaRESUMEN
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(2+) influx is activated and thus ANO6 is stimulated during cell swelling by local Ca(2+) increase probably in functional nanodomains near the plasma membrane. This leads to stimulation of phospholipase A2 (PLA2) 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(2+) supports activation of ANO6, but is not required when PLA2 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 PLA2. 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.