RESUMO
Dysfunction of ENaC, the epithelial sodium channel that regulates salt and water reabsorption in epithelia, causes several human diseases, including cystic fibrosis (CF). To develop a global understanding of molecular regulators of ENaC traffic/function and to identify of candidate CF drug targets, we performed a large-scale screen combining high-content live-cell microscopy and siRNAs in human airway epithelial cells. Screening over 6,000 genes identified over 1,500 candidates, evenly divided between channel inhibitors and activators. Genes in the phosphatidylinositol pathway were enriched on the primary candidate list, and these, along with other ENaC activators, were examined further with secondary siRNA validation. Subsequent detailed investigation revealed ciliary neurotrophic factor receptor (CNTFR) as an ENaC modulator and showed that inhibition of (diacylglycerol kinase, iota) DGKι, a protein involved in PiP2 metabolism, downgrades ENaC activity, leading to normalization of both Na+ and fluid absorption in CF airways to non-CF levels in primary human lung cells from CF patients.
Assuntos
Fibrose Cística/tratamento farmacológico , Terapia de Alvo Molecular , Linhagem Celular , Células Cultivadas , Canais Epiteliais de Sódio/metabolismo , Humanos , Pulmão/citologia , Pulmão/metabolismo , RNA Interferente PequenoRESUMO
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.
Assuntos
Anoctamina-1/metabolismo , Anoctaminas/metabolismo , Cálcio/farmacologia , Membrana Celular/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Fosfolipídeos/metabolismo , Anoctamina-1/genética , Anoctaminas/genética , Apoptose , Hormônios e Agentes Reguladores de Cálcio/farmacologia , Células HEK293 , Humanos , Transporte de Íons , Proteínas de Neoplasias/genética , Fosfolipases A2/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Transdução de SinaisRESUMO
The Ca2+-activated Cl- channel TMEM16A [anoctamin (ANO)1] is homologous to yeast Ist2 and has been shown to tether the cortical endoplasmic reticulum (ER) to the plasma membrane. We therefore examined whether ANO1 and other members of the ANO family affect intracellular Ca2+ ([Ca2+]i) signals. It is shown that expression of ANO1 augments Ca2+ store release upon stimulation of GPCRs, whereas knockdown of ANO1, or lack of Ano1 expression in Ano1-/- animals, as shown in an earlier report, inhibits Ca2+ release. ANO6, and -10 show similar effects, whereas expression of ANO4, -8, and -9 attenuate filling of the ER store. The impact of ANO1 and -4 were examined in more detail. ANO1 colocalized and interacted with IP3R, whereas ANO4 colocalized with SERCA Ca2+ pumps and interacted with ORAI-1 channels, respectively. ANO1 Cl currents were rapidly activated exclusively through Ca2+ store release, and remained untouched by influx of extracellular Ca2+ In contrast expression of ANO4 caused a delayed activation of membrane-localized ANO6 channels, solely through store-operated Ca2+ entry via ORAI. Ca2+ signals were inhibited by knocking down expression of endogenous ANO1, -5, -6, and -10 and were also reduced in epithelial cells from Ano10-/- mice. The data suggest that ANOs affect compartmentalized [Ca2+]i signals, which may explain some of the cellular defects related to ANO mutations.-Cabrita, I., Benedetto, R., Fonseca, A., Wanitchakool, P., Sirianant, L., Skryabin, B. V., Schenk, L. K., Pavenstädt, H., Schreiber, R., Kunzelmann, K. Differential effects of anoctamins on intracellular calcium signals.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Membrana Celular/metabolismo , Canais de Cloreto/metabolismo , Retículo Endoplasmático/metabolismo , Animais , Canais de Cloreto/deficiência , Humanos , Espaço Intracelular/metabolismo , Camundongos , Proteínas de Transferência de Fosfolipídeos/metabolismoRESUMO
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.
Assuntos
Sinalização do Cálcio , Membrana Celular/metabolismo , Necrose/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Animais , Anoctaminas , Cálcio/metabolismo , Linhagem Celular , Camundongos , Células NIH 3T3 , Necrose/patologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
In response to cell swelling, volume-regulated anion channels (VRACs) participate in a process known as regulatory volume decrease (RVD). Only recently, first insight into the molecular identity of mammalian VRACs was obtained by the discovery of the leucine-rich repeats containing 8A (LRRC8A) gene. Here, we show that bestrophin 1 (BEST1) but not LRRC8A is crucial for volume regulation in human retinal pigment epithelium (RPE) cells. Whole-cell patch-clamp recordings in RPE derived from human-induced pluripotent stem cells (hiPSC) exhibit an outwardly rectifying chloride current with characteristic functional properties of VRACs. This current is severely reduced in hiPSC-RPE cells derived from macular dystrophy patients with pathologic BEST1 mutations. Disruption of the orthologous mouse gene (Best1(-/-)) does not result in obvious retinal pathology but leads to a severe subfertility phenotype in agreement with minor endogenous expression of Best1 in murine RPE but highly abundant expression in mouse testis. Sperm from Best1(-/-) mice showed reduced motility and abnormal sperm morphology, indicating an inability in RVD. Together, our data suggest that the molecular identity of VRACs is more complex--that is, instead of a single ubiquitous channel, VRACs could be formed by cell type- or tissue-specific subunit composition. Our findings provide the basis to further examine VRAC diversity in normal and diseased cell physiology, which is key to exploring novel therapeutic approaches in VRAC-associated pathologies.
Assuntos
Tamanho Celular , Canais de Cloreto/metabolismo , Proteínas do Olho/metabolismo , Modelos Biológicos , Epitélio Pigmentado da Retina/citologia , Sequência de Aminoácidos , Animais , Bestrofinas , Proteínas do Olho/genética , Feminino , Imunofluorescência , Técnicas de Silenciamento de Genes , Humanos , Canais Iônicos/deficiência , Canais Iônicos/genética , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Oócitos/metabolismo , Técnicas de Patch-Clamp , Espermatozoides/citologia , Estatísticas não Paramétricas , Xenopus laevisRESUMO
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.
Assuntos
Sinalização do Cálcio , Tamanho Celular , Linfócitos/metabolismo , Osmorregulação , Fosfolipases A2/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Potenciais de Ação , Animais , Anoctaminas , Células Cultivadas , Cloretos/metabolismo , Células HEK293 , Humanos , Linfócitos/citologia , Lisofosfolipídeos/metabolismo , Camundongos , Oócitos , Proteínas de Transferência de Fosfolipídeos/antagonistas & inibidores , Proteínas de Transferência de Fosfolipídeos/genéticaRESUMO
Since the discovery of TMEM16A (anoctamin 1, ANO1) as Ca(2+)-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(2+)-activated Cl(-) channel, it remains unclear whether all anoctamins form plasma membrane-localized or intracellular chloride channels. We find Ca(2+)-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(2+) signaling, we will discuss the role of these proteins in generating compartmentalized Ca(2+) signals, which may give a hint as to the broad range of cellular functions of anoctamins.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Proteínas de Neoplasias/metabolismo , Potenciais de Ação , Animais , Anoctamina-1 , Canais de Cloreto/genética , Humanos , Proteínas de Neoplasias/genéticaRESUMO
Volume regulation is an essential property of any living cell and needs to be tightly controlled. While different types of K(+) channels have been found to participate in the regulation of cell volume, the newly identified volume-regulated anion channel (VRAC) LRRC8 has been claimed to be essential for volume regulation. In unbiased genome-wide small interfering RNA (siRNA) screens, two independent studies identified LRRC8A/Swell1 as an essential component of VRAC, thus being indispensable for cellular volume regulation. We reanalyzed the role of LRRC8A for VRAC and regulatory volume decrease (RVD) in several cell types and under various conditions. While the role of LRRC8A for VRAC and its contribution to RVD is confirmed, we find that it is not essential for swelling-activated anion currents or cellular volume regulation, or apoptotic cell shrinkage. The contribution of LRRC8A is variable and largely depending on the cell type.
Assuntos
Tamanho Celular , Proteínas de Membrana/metabolismo , Apoptose , Células HEK293 , Células HeLa , Humanos , Potenciais da Membrana , Proteínas de Membrana/genéticaRESUMO
TMEM16A/anoctamin 1/ANO1 and VRAC/LRRC8 are independent chloride channels activated either by increase in intracellular Ca(2+) 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 Ca(2+) 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 Ca(2+) activated Cl(-) currents. Moreover, expression of LRRC8A enhanced Ca(2+) 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 Ca(2+) 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 Ca(2+) increase and/or through swelling induced autocrine release of ATP.
Assuntos
Canais de Cloreto/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Potenciais de Ação , Animais , Anoctamina-1 , Cálcio/metabolismo , Tamanho Celular , Cloretos/metabolismo , Exocitose , Células HCT116 , Células HEK293 , Humanos , Pressão Osmótica , Ligação Proteica , Transporte Proteico , XenopusRESUMO
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.
Assuntos
Apoptose , Canais de Cloreto/metabolismo , Animais , Anoctaminas , Ciclo Celular , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fenômenos Eletrofisiológicos , Células HCT116 , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Osmose , Permeabilidade , Proteínas de Transferência de Fosfolipídeos/metabolismo , Água/metabolismoRESUMO
Mutations in the NPHS2 gene are a major cause of steroid-resistant nephrotic syndrome, a severe human kidney disorder. The NPHS2 gene product podocin is a key component of the slit diaphragm cell junction at the kidney filtration barrier and part of a multiprotein-lipid supercomplex. A similar complex with the podocin ortholog MEC-2 is required for touch sensation in Caenorhabditis elegans. Although podocin and MEC-2 are membrane-associated proteins with a predicted hairpin-like structure and amino and carboxyl termini facing the cytoplasm, this membrane topology has not been convincingly confirmed. One particular mutation that causes kidney disease in humans (podocin(P118L)) has also been identified in C. elegans in genetic screens for touch insensitivity (MEC-2(P134S)). Here we show that both mutant proteins, in contrast to the wild-type variants, are N-glycosylated because of the fact that the mutant C termini project extracellularly. Podocin(P118L) and MEC-2(P134S) did not fractionate in detergent-resistant membrane domains. Moreover, mutant podocin failed to activate the ion channel TRPC6, which is part of the multiprotein-lipid supercomplex, indicative of the fact that cholesterol recruitment to the ion channels, an intrinsic function of both proteins, requires C termini facing the cytoplasmic leaflet of the plasma membrane. Taken together, this study demonstrates that the carboxyl terminus of podocin/MEC-2 has to be placed at the inner leaflet of the plasma membrane to mediate cholesterol binding and contribute to ion channel activity, a prerequisite for mechanosensation and the integrity of the kidney filtration barrier.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Membrana Celular/metabolismo , Barreira de Filtração Glomerular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mecanotransdução Celular , Proteínas de Membrana/metabolismo , Mutação de Sentido Incorreto , Substituição de Aminoácidos , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Linhagem Celular , Membrana Celular/genética , Membrana Celular/patologia , Colesterol/genética , Colesterol/metabolismo , Barreira de Filtração Glomerular/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Camundongos , Síndrome Nefrótica/congênito , Síndrome Nefrótica/genética , Síndrome Nefrótica/metabolismo , Síndrome Nefrótica/patologia , Proibitinas , Estrutura Terciária de ProteínaRESUMO
In a first genome-wide association study (GWAS) approach to anti-Borrelia seropositivity, we identified two significant single nucleotide polymorphisms (SNPs) (rs17850869, P = 4.17E-09; rs41289586, P = 7.18E-08). Both markers, located on chromosomes 16 and 3, respectively, are within or close to genes previously connected to spinocerebellar ataxia. The risk SNP rs41289586 represents a missense variant (R263H) of anoctamin 10 (ANO10), a member of a protein family encoding Cl(-) channels and phospholipid scramblases. ANO10 augments volume-regulated Cl(-) currents (IHypo) in Xenopus oocytes, HEK293 cells, lymphocytes and macrophages and controls volume regulation by enhancing regulatory volume decrease (RVD). ANO10 supports migration of macrophages and phagocytosis of spirochetes. The R263H variant is inhibitory on IHypo, RVD and intracellular Ca(2+) signals, which may delay spirochete clearance, thereby sensitizing adaptive immunity. Our data demonstrate for the first time that ANO10 has a central role in innate immune defense against Borrelia infection.
Assuntos
Infecções por Borrelia/genética , Infecções por Borrelia/imunologia , Borrelia/imunologia , Variação Genética , Macrófagos/metabolismo , Proteínas de Membrana/genética , Fases de Leitura Aberta , Animais , Anoctaminas , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Infecções por Borrelia/epidemiologia , Infecções por Borrelia/microbiologia , Estudos de Casos e Controles , Linhagem Celular , Tamanho Celular , Expressão Gênica , Estudo de Associação Genômica Ampla , Células HEK293 , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Macrófagos/patologia , Transtornos Mentais/genética , Transtornos Mentais/microbiologia , Oócitos , Fenótipo , Polimorfismo de Nucleotídeo Único , Estudos SoroepidemiológicosRESUMO
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(2+)-dependent and a volume-regulated Cl(-) channel; (2) it was claimed to be a Ca(2+)-regulated nonselective cation channel permeable for Ca(2+); (3) it was shown to be essential for Ca(2+)-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.
Assuntos
Cálcio/metabolismo , Cloretos/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Animais , Anoctaminas , Membrana Celular/metabolismo , Humanos , Transporte de Íons , Proteínas de Transferência de Fosfolipídeos/genética , Fosfolipídeos/metabolismoRESUMO
SLC26A11 (human)/Slc26a11 (mouse), also known as kidney brain anion transporter (KBAT), is a member of the SLC26 anion transporter family and shows abundant mRNA expression in the brain. However, its exact cellular distribution and subcellular localization in the brain and its functional identity and possible physiological roles remain unknown. Expression and immunostaining studies demonstrated that Slc26a11 is abundantly expressed in the cerebellum, with a predominant expression in Purkinje cells. Lower expression levels were detected in hippocampus, olfactory bulb, cerebral cortex, and subcortical structures. Patch clamp studies in HEK293 cells transfected with mouse cDNA demonstrated that Slc26a11 can function as a chloride channel that is active under basal conditions and is not regulated by calcium, forskolin, or co-expression with cystic fibrosis transmembrane regulator. Single and double immunofluorescent labeling studies demonstrated the localization of vacuolar (V) Hâº-ATPase and Slc26a11 (KBAT) in the plasma membrane in Purkinje cells. Functional studies in HEK293 cells indicated that transfection with Slc26a11 stimulated acid transport via endogenous V Hâº-ATPase. We conclude that Slc26a11 (KBAT) is prominently distributed in output neurons of various subcortical and cortical structures in the central nervous system, with specific expression in Purkinje cells and that it may operate as a chloride channel regulating acid translocation by Hâº-ATPase across the plasma membrane and in intracellular compartments.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Células de Purkinje/metabolismo , Animais , Proteínas de Transporte de Ânions/genética , Cálcio/farmacologia , Membrana Celular/metabolismo , Córtex Cerebral/metabolismo , Colforsina/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células HEK293 , Hipocampo/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Bulbo Olfatório/metabolismo , Especificidade de Órgãos , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transportadores de SulfatoRESUMO
Our previous study has shown that dihydroisosteviol (DHIS), a derivative of stevioside isolated from Stevia rebaudiana (Bertoni), inhibits cystic fibrosis transmembrane conductance regulator (CFTR)-mediated transepithelial chloride secretion across monolayers of human intestinal epithelial (T84) cells and prevents cholera toxin-induced intestinal fluid secretion in mouse closed loop models. In this study, we aimed to investigate a mechanism by which DHIS inhibits CFTR activity. Apical chloride current measurements in Fisher rat thyroid cells stably transfected with wild-type human CFTR (FRT-CFTR cells) and T84 cells were used to investigate mechanism of CFTR inhibition by DHIS. In addition, effect of DHIS on AMP-activated protein kinase (AMPK) activation was investigated using Western blot analysis. Surprisingly, it was found that DHIS failed to inhibit CFTR-mediated apical chloride current in FRT-CFTR cells. In contrast, DHIS effectively inhibited CFTR-mediated apical chloride current induced by a cell permeable cAMP analog CPT-cAMP and a direct CFTR activator genistein in T84 cell monolayers. Interestingly, this inhibitory effect of DHIS on CFTR was significantly (p<0.05) reduced by pretreatment with compound C, an AMPK inhibitor. AICAR, a known AMPK activator, was able to inhibit CFTR activity in both FRT-CFTR and T84 cells. Western blot analysis showed that DHIS induced AMPK activation in T84 cells, but not in FRT-CFTR cells. Our results indicate that DHIS inhibits CFTR-mediated chloride secretion in T84 cells, in part, by activation of AMPK activity. DHIS therefore represents a novel candidate of AMPK activators.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Diterpenos do Tipo Caurano/farmacologia , Ativadores de Enzimas/farmacologia , Células Epiteliais/efeitos dos fármacos , Animais , Linhagem Celular , Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Humanos , Mucosa Intestinal/citologia , Ratos , SteviaRESUMO
Overstimulation of cAMP-activated Cl- secretion can cause secretory diarrhea. Isoliquiritigenin (ISLQ) is a plant-derived chalcone that has a wide range of biological activities. The present study thus aimed to investigate the effect of ISLQ on cAMP-activated Cl- secretion in human intestinal epithelium, especially the underlying mechanism and therapeutic application. Short-circuit current analysis of human intestinal epithelial (T84) cell monolayers revealed that ISLQ dose-dependently inhibited cAMP-activated Cl- secretion with an IC50 of approximately 20 µM. ISLQ had no effect on either basal short-circuit current or Ca2+-activated Cl- secretion. Apical Cl- current analysis of T84 cell monolayers indicated that ISLQ blocked mainly the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels, but not other unidentified cAMP-dependent Cl- channels. ISLQ did not affect intracellular cAMP levels or cell viability. ISLQ completely abolished the cholera toxin-induced transepithelial Cl- secretion in T84 cells and reduced the cholera toxin-induced intestinal fluid secretion in mouse closed loop models by 90%. Similarly, ISLQ completely inhibited the cAMP-activated apical Cl- current across monolayers of Madin-Darby Canine Kidney (MDCK) cells and retarded cyst growth in MDCK cyst models by 90%. This study reveals a novel action of ISLQ as a potent CFTR inhibitor with therapeutic potential for treatment of cholera and polycystic kidney disease.
RESUMO
Overstimulation of cAMP-activated Cl(-) secretion can cause secretory diarrhea. Isoliquiritigenin (ISLQ) is a plant-derived chalcone that has a wide range of biological activities. The present study thus aimed to investigate the effect of ISLQ on cAMP-activated Cl(-) secretion in human intestinal epithelium, especially the underlying mechanism and therapeutic application. Short-circuit current analysis of human intestinal epithelial (T84) cell monolayers revealed that ISLQ dose-dependently inhibited cAMP-activated Cl(-) secretion with an IC(50) of approximately 20 µM. ISLQ had no effect on either basal short-circuit current or Ca(2+)-activated Cl(-) secretion. Apical Cl(-) current analysis of T84 cell monolayers indicated that ISLQ blocked mainly the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels, but not other unidentified cAMP-dependent Cl(-) channels. ISLQ did not affect intracellular cAMP levels or cell viability. ISLQ completely abolished the cholera toxin-induced transepithelial Cl(-) secretion in T84 cells and reduced the cholera toxin-induced intestinal fluid secretion in mouse closed loop models by 90%. Similarly, ISLQ completely inhibited the cAMP-activated apical Cl(-) current across monolayers of Madin-Darby Canine Kidney (MDCK) cells and retarded cyst growth in MDCK cyst models by 90%. This study reveals a novel action of ISLQ as a potent CFTR inhibitor with therapeutic potential for treatment of cholera and polycystic kidney disease.
Assuntos
Antidiarreicos/farmacologia , Chalconas/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Animais , Linhagem Celular , Cloretos/metabolismo , Cólera , Toxina da Cólera/farmacologia , Cães , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Doenças Renais PolicísticasRESUMO
PURPOSE: The present study examined the effects and mechanisms of actions of penta-m-digalloyl-glucose (PDG), a hydrolysable tannin extracted from Chinese gallnut, on cystic fibrosis transmembrane conductance regulator protein (CFTR). MATERIALS AND METHODS: Fisher rat thyroid cells stably expressing human CFTR (FRT cells) and human intestinal T84 cells were used as cell models to investigate the effects of PDG on chloride secretion using short-circuit current analysis. The mechanisms by which PDG affected chloride secretion were also examined. Finally, in vivo antidiarrheal efficacy and effects of PDG on intestinal fluid absorption were evaluated in mouse closed-loop models. RESULTS: In FRT cells, apical chloride current induced by forskolin, CPT-cAMP and apigenin were reversibly inhibited by PDG (IC50 approximately 10microM) without effects on intracellular cAMP content and cell viability. Similarly, in T84 cells, PDG effectively inhibited chloride secretion induced by forskolin and cholera toxin. However, it had no effect on calcium-induced chloride secretion. In mice, a single intraluminal injection of PDG (0.6 mg/kg) reduced cholera toxin-induced intestinal fluid secretion by 75% with no effect on intestinal fluid absorption. CONCLUSIONS: PDG represents a new class of CFTR inhibitors. Further development of this class of compounds may provide a new therapeutic intervention for diarrhea.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Diarreia/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Taninos Hidrolisáveis/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cloretos/metabolismo , Colo/citologia , AMP Cíclico/metabolismo , Medicamentos de Ervas Chinesas/química , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Taninos Hidrolisáveis/química , Camundongos , Camundongos Endogâmicos ICR , Plantas/química , RatosRESUMO
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 TNFα-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.
Assuntos
Anoctaminas/metabolismo , Sinalização do Cálcio , Deleção de Genes , Animais , Anoctaminas/deficiência , Apoptose , Caspase 3/metabolismo , Proliferação de Células , Tamanho Celular , Enterócitos/citologia , Enterócitos/metabolismo , Células HEK293 , Humanos , Macrófagos/metabolismo , Camundongos Knockout , Transporte Proteico , RatosRESUMO
Plasma membrane proteins are essential molecules in the cell which mediate interactions with the exterior milieu, thus representing key drug targets for present pharma. Not surprisingly, protein traffic disorders include a large range of diseases sharing the common mechanism of failure in the respective protein to reach the plasma membrane. However, specific therapies for these diseases are remarkably lacking. Herein, we report a robust platform for drug discovery applied to a paradigmatic genetic disorder affecting intracellular trafficking - Cystic Fibrosis. This platform includes (i) two original respiratory epithelial cellular models incorporating an inducible double-tagged traffic reporter; (ii) a plasma membrane protein traffic assay for high-throughput microscopy screening; and (iii) open-source image analysis software to quantify plasma membrane protein traffic. By allowing direct scoring of compounds rescuing the basic traffic defect, this platform enables an effective drug development pipeline, which can be promptly adapted to any traffic disorder-associated protein and leverage therapy development efforts.