Activation Of TMEM16A Research Articles

Secreted Human CLCA1 Activates Calcium-Dependent Chloride Currents via Interaction with TMEM16A (Anoctamin 1)

The calcium-activated chloride channel (CaCC) regulator (CLCA) proteins were so named because their expression leads to generation of calcium-dependent chloride currents (ICaCC) in mammalian cells; however, the molecular identity of the channel(s) mediating these currents, and the mechanisms through which CLCA1 regulates their activity, have remained unknown. In a previous study, we showed that human CLCA1 is a secreted self-cleaving zincin metalloprotease, and the resulting N-terminal fragment increases ICaCC density in HEK293T cells (J. Biol. Chem. 287, 42138-49, 2012). Here, we found that the same type of currents are activated in untransfected cells co-cultured with CLCA1-transfected cells or exposed to CLCA1-conditioned medium, indicating that secreted CLCA1 can activate ICaCC in a paracrine fashion. Because CLCA1 and the CaCC TMEM16A (Anoctamin 1) are expressed in the same tissues, such as the airway epithelia, and are upregulated in the same pathologic states, such as asthma and COPD, we tested the hypothesis that CLCA1-modulated currents are carried by TMEM16A. We observed that CLCA1-induced whole-cell ICaCC in HEK293T cells overexpressing the protein is blocked in the presence of TMEM16A-specific inhibitors T16Ainh-A01 and N-((4-methoxy)-2-naphthyl)-5-nitroanhtranilic acid, and silenced in the presence of TMEM16A siRNA. By means of live cell flow cytometry assays using a novel fluorescent CLCA probe, we demonstrate that CLCA1 and TMEM16A physically interact at the cell surface, and our real-time qPCR, immunohistochemistry and confocal microscopy data suggest that CLCA1 drives TMEM16A surface expression. These results identify CLCA1 as the first secreted direct mediator of TMEM16A activity, and suggest that CLCA1 and TMEM16A operate together to generate ICaCC in multiple tissues.

Time for TMEM?

Time for TMEM?

Mechanosensitive Cl− secretion in biliary epithelium mediated through TMEM16A

Bile formation by the liver is initiated by canalicular transport at the hepatocyte membrane, leading to an increase in ductular bile flow. Thus, bile duct epithelial cells (cholangiocytes), which contribute to the volume and dilution of bile through regulated Cl(-) transport, are exposed to changes in flow and shear force at the apical membrane. The aim of the present study was to determine if fluid flow, or shear stress, is a signal regulating cholangiocyte transport. The results demonstrate that, in human and mouse biliary cells, fluid flow, or shear, increases Cl(-) currents and identify TMEM16A, a Ca(2+)-activated Cl(-) channel, as the operative channel. Furthermore, activation of TMEM16A by flow is dependent on PKCα through a process involving extracellular ATP, binding purinergic P2 receptors, and increases in intracellular Ca(2+) concentration. These studies represent the initial characterization of mechanosensitive Cl(-) currents mediated by TMEM16A. Identification of this novel mechanosensitive secretory pathway provides new insight into bile formation and suggests new therapeutic targets to enhance bile formation in the treatment of cholestatic liver disorders.

Proinflammatory cytokine secretion is suppressed by TMEM16A or CFTR channel activity in human cystic fibrosis bronchial epithelia

Cystic fibrosis (CF) is caused by the functional expression defect of the CF transmembrane conductance regulator (CFTR) chloride channel at the apical plasma membrane. Impaired bacterial clearance and hyperactive innate immune response are hallmarks of the CF lung disease, yet the existence of and mechanism accounting for the innate immune defect that occurs before infection remain controversial. Inducible expression of either CFTR or the calcium-activated chloride channel TMEM16A attenuated the proinflammatory cytokines interleukin-6 (IL-6), IL-8, and CXCL1/2 in two human respiratory epithelial models under air-liquid but not liquid-liquid interface culture. Expression of wild-type but not the inactive G551D-CFTR indicates that secretion of the chemoattractant IL-8 is inversely proportional to CFTR channel activity in cftr(∆F508/∆F508) immortalized and primary human bronchial epithelia. Similarly, direct but not P2Y receptor-mediated activation of TMEM16A attenuates IL-8 secretion in respiratory epithelia. Thus augmented proinflammatory cytokine secretion caused by defective anion transport at the apical membrane may contribute to the excessive and persistent lung inflammation in CF and perhaps in other respiratory diseases associated with documented down-regulation of CFTR (e.g., chronic obstructive pulmonary disease). Direct pharmacological activation of TMEM16A offers a potential therapeutic strategy to reduce the inflammation of CF airway epithelia.

Small‐molecule activators of TMEM16A, a calcium‐activated chloride channel, stimulate epithelial chloride secretion and intestinal contraction

TMEM16A (ANO1) is a calcium-activated chloride channel (CaCC) expressed in secretory epithelia, smooth muscle, and other tissues. Cell-based functional screening of ∼110,000 compounds revealed compounds that activated TMEM16A CaCC conductance without increasing cytoplasmic Ca(2+). By patch-clamp, N-aroylaminothiazole "activators" (E(act)) strongly increased Cl(-) current at 0 Ca(2+), whereas tetrazolylbenzamide "potentiators" (F(act)) were not active at 0 Ca(2+) but reduced the EC(50) for Ca(2+)-dependent TMEM16A activation. Of 682 analogs tested, the most potent activator (E(act)) and potentiator (F(act)) produced large and more sustained CaCC Cl(-) currents than general agonists of Ca(2+) signaling, with EC(50) 3-6 μM and Cl(-) conductance comparable to that induced transiently by Ca(2+)-elevating purinergic agonists. Analogs of activators were identified that fully inhibited TMEM16A Cl(-) conductance, providing further evidence for direct TMEM16A binding. The TMEM16A activators increased CaCC conductance in human salivary and airway submucosal gland epithelial cells, and IL-4 treated bronchial cells, and stimulated submucosal gland secretion in human bronchi and smooth muscle contraction in mouse intestine. Small-molecule, TMEM16A-targeted activators may be useful for drug therapy of cystic fibrosis, dry mouth, and gastrointestinal hypomotility disorders, and for pharmacological dissection of TMEM16A function.

CFTR induces extracellular acid sensing in Xenopus oocytes which activates endogenous Ca2+-activated Cl- conductance

The cystic fibrosis transmembrane conductance regulator (CFTR) produces a cyclic adenosine monophosphate (cAMP)-dependent Cl⁻ conductance of distinct properties that is essential for electrolyte secretion in human epithelial tissues. However, the functional consequences of CFTR expression are multifaceted, encompassing much more than simply supplying a cellular cAMP-regulated Cl⁻ conductance. When we expressed CFTR in Xenopus oocytes, we found that extracellular acidic pH activates a Ca²⁺-dependent outwardly rectifying Cl⁻ conductance that does not reflect CFTR activity. The proton-activated Cl⁻ conductance showed biophysical and pharmacological features of a Ca²⁺-dependent Cl⁻ conductance, most likely mediated by Xenopus TMEM16A. In contrast to the effects of extracellular acidification, intracellular acidification did not activate an endogenous Cl⁻ conductance. Proton/CFTR-mediated activation of human TMEM16A was also detected in HEK293 cells. The gating mutant G551D-CFTR conferred proton sensitivity, while deltaF508-CFTR enabled proton activation of TMEM16A only in Xenopus oocytes, which, unlike HEK293 cells, allow deltaF508-CFTR to be trafficked to the cell membrane. Activation of TMEM16A by lysophosphatidic acid was enhanced in the presence of CFTR but was additive with activation by extracellular protons. Because expression of CFTR-E1474X did not confer proton sensitivity, we propose that CFTR translocates a proton receptor to the plasma membrane via its PDZ-binding domain.

Regulation of TMEM16A by a novel mechanism involving PKC in biliary epithelium

TMEM16A is a Ca2+‐activated Cl− channel in the apical membrane of biliary epithelial cells (BECs), and contributes to biliary secretion and bile formation [Dutta et al. JBC, 2010]. Extracellular nucleotides activate TMEM16A via binding P2 receptors and increasing [Ca2+]i. While PKC contributes to ATP‐mediated secretion in BECs, the role in TMEM16A regulation is unknown.AimTo determine if PKC contributes to Cl− transport in BECs through regulation of TMEM16A.MethodsStudies were performed in human biliary Mz‐cha‐1 cells. Cl− currents were measured by whole‐cell patch clamp.ResultsTMEM16A Cl− currents were activated by exposure to extracellular ATP or increasing Ca2+ in the patch pipette. Currents had a reversal potential of 0 mV, time‐dependent activation and outward rectification. Currents were significantly inhibited by anti‐TMEM16A siRNA. In the presence of the PKC inhibitors Chelerithrine or Gö6976 Cl− currents were significantly inhibited. Conversely, activation by PMA resulted in large Cl− currents. Intracellular dialysis with recombinant PKCα activated Cl− currents with identical biophysical properties to TMEM16A.ConclusionPKCα couples ATP‐mediated signaling to TMEM16A activation in BECs. Modulation of kinase signaling pathways may represent a target for increasing biliary secretion and bile formation.[Studies are supported by AASLD/ALF Liver Scholar Award 2010, Children's Medical Center Foundation grant (Dutta), and NIH NIDDK DK078587]

Rotavirus toxin NSP4 induces diarrhea by activation of TMEM16A and inhibition of Na+ absorption

Rotavirus infection is the most frequent cause for severe diarrhea in infants, killing more than 600,000 every year. The nonstructural protein NSP4 acts as a rotavirus enterotoxin, inducing secretory diarrhea without any structural organ damage. Electrolyte transport was assessed in the colonic epithelium from pups and adult mice using Ussing chamber recordings. Western blots and immunocytochemistry was performed in intestinal tissues from wild-type and TMEM16A knockout mice. Ion channel currents were recorded using patch clamp techniques. We show that the synthetic NSP4(114-135) peptide uses multiple pro-secretory pathways to induce diarrhea, by activating the recently identified Ca2+ -activated Cl- channel TMEM16A, and by inhibiting Na+ absorption by the epithelial Na+ channel ENaC and the Na+ /glucose cotransporter SGLT1. Activation of secretion and inhibition of Na+ absorption by NSP4(114-135), respectively, could be potently suppressed by wheat germ agglutinin which probably competes with NSP4(114-135) for binding to an unknown glycolipid receptor. The present paper gives a clue as to mechanisms of rotavirus-induced diarrhea and suggests wheat germ agglutinin as a simple and effective therapy.

Calmodulin‐dependent activation of the epithelial calcium‐dependent chloride channel TMEM16A

TMEM16A (anoctamin 1, Ano1), a member of a family of 10 homologous proteins, has been shown to form an essential component of Ca(2+)-activated Cl(-) channels. TMEM16A-null mice exhibit severe defects in epithelial transport along with tracheomalacia and death within 1 mo after birth. Despite its outstanding physiological significance, the mechanisms for activation of TMEM16A remain obscure. TMEM16A is activated on increase in intracellular Ca(2+), but it is unclear whether Ca(2+) binds directly to the channel or whether additional components are required. We demonstrate that TMEM16A is strictly membrane localized and requires cytoskeletal interactions to be fully activated. Despite the need for cytosolic ATP for full activation, phosphorylation by protein kinases is not required. In contrast, the Ca(2+) binding protein calmodulin appears indispensable and interacts physically with TMEM16A. Openers of small- and intermediate-conductance Ca(2+)-activated potassium channels known to interact with calmodulin, such as 1-EBIO, DCEBIO, or riluzole, also activated TMEM16A. These results reinforce the use of these compounds for activation of electrolyte secretion in diseases such as cystic fibrosis.