Wan Namkung

TMEM16A Inhibitors Reveal TMEM16A as a Minor Component of Calcium-activated Chloride Channel Conductance in Airway and Intestinal Epithelial Cells

TMEM16A (ANO1) functions as a calcium-activated chloride channel (CaCC). We developed pharmacological tools to investigate the contribution of TMEM16A to CaCC conductance in human airway and intestinal epithelial cells. A screen of ∼110,000 compounds revealed four novel chemical classes of small molecule TMEM16A inhibitors that fully blocked TMEM16A chloride current with an IC50 < 10 μm, without interfering with calcium signaling. Following structure-activity analysis, the most potent inhibitor, an aminophenylthiazole (T16Ainh-A01), had an IC50 of ∼1 μm. Two distinct types of inhibitors were identified. Some compounds, such as tannic acid and the arylaminothiophene CaCCinh-A01, fully inhibited CaCC current in human bronchial and intestinal cells. Other compounds, including T16Ainh-A01 and digallic acid, inhibited total CaCC current in these cells poorly, but blocked mainly an initial, agonist-stimulated transient chloride current. TMEM16A RNAi knockdown also inhibited mainly the transient chloride current. In contrast to the airway and intestinal cells, all TMEM16A inhibitors fully blocked CaCC current in salivary gland cells. We conclude that TMEM16A carries nearly all CaCC current in salivary gland epithelium, but is a minor contributor to total CaCC current in airway and intestinal epithelia. The small molecule inhibitors identified here permit pharmacological dissection of TMEM16A/CaCC function and are potential development candidates for drug therapy of hypertension, pain, diarrhea, and excessive mucus production.

Synthetic ion transporters can induce apoptosis by facilitating chloride anion transport into cells

Anion transporters based on small molecules have received attention as therapeutic agents because of their potential to disrupt cellular ion homeostasis. However, a direct correlation between a change in cellular chloride anion concentration and cytotoxicity has not been established for synthetic ion carriers. Here we show that two pyridine diamide-strapped calix[4]pyrroles induce coupled chloride anion and sodium cation transport in both liposomal models and cells, and promote cell death by increasing intracellular chloride and sodium ion concentrations. Removing either ion from the extracellular media or blocking natural sodium channels with amiloride prevents this effect. Cell experiments show that the ion transporters induce the sodium chloride influx, which leads to an increased concentration of reactive oxygen species, release of cytochrome c from the mitochondria and apoptosis via caspase activation. However, they do not activate the caspase-independent apoptotic pathway associated with the apoptosis-inducing factor. Ion transporters, therefore, represent an attractive approach for regulating cellular processes that are normally controlled tightly by homeostasis.

Dynamic Regulation of CFTR Bicarbonate Permeability by [Cl−]i and Its Role in Pancreatic Bicarbonate Secretion

BACKGROUND & AIMS Pancreatic bicarbonate (HCO3-) secretion is important for a healthy pancreas as well as digestive physiology. However, how human pancreatic duct cells secrete copious amounts of HCO3- has long been a puzzle. Here, we report that a dynamic increase in the cystic fibrosis transmembrane conductance regulator (CFTR) HCO3- permeability by intracellular Cl- concentration ([Cl-]i)-sensitive mechanisms plays a pivotal role in pancreatic HCO3- secretion. METHODS The role of [Cl-]i-sensitive kinases in CFTR-mediated HCO3- transport was examined in heterologous expression systems, PANC1 human pancreatic duct cells, and human and guinea pig pancreatic tissues using an integrated molecular and physiologic approach. RESULTS In human pancreatic tissues, CFTR-positive duct cells abundantly expressed with-no-lysine (WNK1) kinase, oxidative stress-responsive kinase 1 (OSR1), and sterile 20/SPS1-related proline/alanine-rich kinase (SPAK), which are known to be activated by low [Cl-]i. Interestingly, CFTR activation rapidly decreased [Cl-]i in response to luminal Cl- depletion in polarized PANC1 human pancreatic duct cells. Notably, the WNK1-mediated OSR1 and SPAK activation by low [Cl-]i strongly increased CFTR HCO3- permeability in CFTR-transfected HEK 293T, PANC1, and guinea pig pancreatic duct cells, making CFTR primarily an HCO3- channel, which is essential for the secretion of pancreatic juice containing HCO3- at a concentration greater than 140 mmol/L. In contrast, OSR1 and SPAK activation inhibited CFTR-dependent Cl-/HCO3- exchange activity that may reabsorb HCO3- from the high HCO3--containing pancreatic juice. CONCLUSIONS These results indicate that the [Cl-]i-sensitive activation of the WNK1-OSR1/SPAK pathway is the molecular switch to generate HCO3--rich fluid in the human pancreatic duct.

Small-Molecule Screen Identifies Inhibitors of a Human Intestinal Calcium-Activated Chloride Channel

Calcium-activated chloride channels (CaCCs) are widely expressed in mammalian tissues, including intestinal epithelia, where they facilitate fluid secretion. Potent, selective CaCC inhibitors have not been available. We established a high-throughput screen for identification of inhibitors of a human intestinal CaCC based on inhibition of ATP/carbachol-stimulated iodide influx in HT-29 cells after lentiviral infection with the yellow fluorescent halide-sensing protein YFP-H148Q/I152L. Screening of 50,000 diverse, drug-like compounds yielded six classes of putative CaCC inhibitors, two of which, 3-acyl-2-aminothiophenes and 5-aryl-2-aminothiazoles, inhibited by >95% iodide influx in HT-29 cells in response to multiple calcium-elevating agonists, including thapsigargin, without inhibition of calcium elevation, calcium-calmodulin kinase II activation, or cystic fibrosis transmembrane conductance regulator chloride channels. These compounds also inhibited calcium-dependent chloride secretion in T84 human intestinal epithelial cells. Patch-clamp analysis indicated inhibition of CaCC gating, which, together with the calcium-calmodulin data, suggests that the inhibitors target the CaCC directly. Structure-activity relationships were established from analysis of more than 1800 analogs, with IC50 values of the best analogs down to ∼1 μM. Small-molecule CaCC inhibitors may be useful in pharmacological dissection of CaCC functions and in reducing intestinal fluid losses in CaCC-mediated secretory diarrheas.

Inhibition of Ca2+-activated Cl− channels by gallotannins as a possible molecular basis for health benefits of red wine and green tea

TMEM16A was found recently to be a calcium‐activated CU channel (CaCC). CaCCs perform important functions in cell physiology, including regulation of epithelial secretion, cardiac and neuronal excitability, and smooth muscle contraction. CaCC modulators are of potential utility for treatment of hypertension, diarrhea, and cystic fibrosis. Screening of drug and natural product collections identified tannic acid as an inhibitor of TMEM16A, with IC50 ∼ 6 µM and ∼100% inhibition at higher concentrations. Tannic acid inhibited CaCCs in multiple cell types but did not affect CFTR Cl− channels. Structure‐activity analysis indicated the requirement of gallic or digallic acid substituents on a macromolecular scaffold (gallotannins), as are present in green tea and red wine. Other polyphenolic components of teas and wines, including epicatechin, catechin, and malvidin‐3‐glucoside, poorly inhibited CaCCs. Remarkably, a 1000‐fold dilution of red wine and 100‐fold dilution of green tea inhibited CaCCs by >50%. Tannic acid, red wine, and green tea inhibited arterial smooth muscle contraction and intestinal Cl− secretion. Gallotannins are thus potent CaCC inhibitors whose biological activity provides a potential molecular basis for the cardioprotective and antisecretory benefits of red wine and green tea.—Namkung, W., Thiagarajah, J. R., Phuan, P.‐W., Verkman, A. S. Inhibition of Ca2+‐activated Cl− channels by gallotannins as a possible molecular basis for health benefits of red wine and green tea. FASEB J. 24, 4178–4186 (2010). www.fasebj.org

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 Ca2+. By patch‐clamp, N‐aroylaminothiazole “activators” (Eact) strongly increased Cl‐ current at 0 Ca2+, whereas tetrazolylbenzamide “potentiators” (Fact) were not active at 0 Ca2+ but reduced the EC50 for Ca2+‐dependent TMEM16A activation. Of 682 analogs tested, the most potent activator (Eact) and potentiator (Fact) produced large and more sustained CaCC Cl‐currents than general agonists of Ca2+ signaling, with EC50 3–6 μM and Cl‐ conductance comparable to that induced transiently by Ca2+‐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.—Namkung, W., Yao, Z., Finkbeiner, W. E., Verkman, A. S. Small‐molecule activators of TMEM16A, a calcium‐activated chloride channel, stimulate epithelial chloride secretion and intestinal contraction. FASEB J. 25, 4048–4062 (2011). www.fasebj.org

Dynamic regulation of cystic fibrosis transmembrane conductance regulator by competitive interactions of molecular adaptors.

Disorganized ion transport caused by hypo- or hyperfunctioning of the cystic fibrosis transmembrane conductance regulator (CFTR) can be detrimental and may result in life-threatening diseases such as cystic fibrosis or secretory diarrhea. Thus, CFTR is controlled by elaborate positive and negative regulations for an efficient homeostasis. It has been shown that expression and activity of CFTR can be regulated either positively or negatively by PDZ (PSD-95/discs large/ZO-1) domain-based adaptors. Although a positive regulation by PDZ domain-based adaptors such as EBP50/NHERF1 is established, the mechanisms for negative regulation of the CFTR by Shank2, as well as the effects of multiple adaptor interactions, are not known. Here we demonstrate a physical and physiological competition between EBP50-CFTR and Shank2-CFTR associations and the dynamic regulation of CFTR activity by these positive and negative interactions using the surface plasmon resonance assays and consecutive patch clamp experiments. Furthermore whereas EBP50 recruits a cAMP-dependent protein kinase (PKA) complex to CFTR, Shank2 was found to be physically and functionally associated with the cyclic nucleotide phosphodiesterase PDE4D that precludes cAMP/PKA signals in epithelial cells and mouse brains. These findings strongly suggest that balanced interactions between the membrane transporter and multiple PDZ-based adaptors play a critical role in the homeostatic regulation of epithelial transport and possibly the membrane transport in other tissues.

Crofelemer, an Antisecretory Antidiarrheal Proanthocyanidin Oligomer Extracted from Croton lechleri, Targets Two Distinct Intestinal Chloride Channels

Crofelemer, a purified proanthocyanidin oligomer extracted from the bark latex of Croton lechleri, is in clinical trials for secretory diarrheas of various etiologies. We investigated the antisecretory mechanism of crofelemer by determining its effect on the major apical membrane transport and signaling processes involved in intestinal fluid transport. Using cell lines and measurement procedures to isolate the effects on individual membrane transport proteins, crofelemer at 50 μM had little or no effect on the activity of epithelial Na+ or K+ channels or on cAMP or calcium signaling. Crofelemer inhibited the cystic fibrosis transmembrane regulator (CFTR) Cl− channel with maximum inhibition of ∼60% and an IC50 ∼7 μM. Crofelemer action at an extracellular site on CFTR produced voltage-independent block with stabilization of the channel closed state. Crofelemer did not affect the potency of glycine hydrazide or thiazolidinone CFTR inhibitors. Crofelemer action resisted washout, with <50% reversal of CFTR inhibition after 4 h. Crofelemer was also found to strongly inhibit the intestinal calcium-activated Cl− channel TMEM16A by a voltage-independent inhibition mechanism with maximum inhibition >90% and IC50 ∼6.5 μM. The dual inhibitory action of crofelemer on two structurally unrelated prosecretory intestinal Cl− channels may account for its intestinal antisecretory activity.

Suppression of Cerulein-Induced Cytokine Expression by Antioxidants in Pancreatic Acinar Cells

Reactive oxygen species (ROS) has been considered to be an important regulator in the development and pathogenesis of pancreatitis and an activator of the transcription factor, nuclear factor-κB (NF-κB), regulating inflammatory cytokine gene expression. NF-κB activation was demonstrated in cerulein pancreatitis, which rapidly induces an acute, edematous form of pancreatitis. This study aimed to investigate whether cerulein induced ROS generation, lipid peroxide and hydrogen peroxide production, NF-κB activation, and expression of cytokines (IL-1β, IL-6) in pancreatic acinar cells. An additional aim was to establish whether these alterations were inhibited by antioxidants such as glutathione, superoxide dismutase, and catalase and an inhibitor of NF-κB activation, pyrrolidine dithiocarbamate (PDTC). To determine the possible interactions of the antioxidants and PDTC with cerulein-induced signaling, Ca2+ signal and amylase release were monitored in the pancreatic acinar cells treated with cerulein in the presence or absence of either the antioxidants or PDTC. The results showed that cerulein generated ROS and increased lipid peroxide and hydrogen peroxide production in the acinar cells, as determined by dichlorofluorescein diacetate dye. This resulted in NF-κB activation and the induction of cytokine gene expression in the cells. The cerulein-induced NF-κB activation was in parallel to IκBα degradation. Cerulein also induced Ca2+ signals and amylase release in acinar cells. Both antioxidants (glutathione, superoxide dismutase, catalase) and PDTC inhibited the cerulein-induced, oxidant-mediated alterations but did not affect the cerulein-evoked Ca2+ signals and amylase release in acinar cells. In conclusion, ROS, generated by cerulein, activates NF-κB, resulting in the up-regulation of inflammatory cytokine gene expression in acinar cells. NF-κB inhibition by scavenging ROS might alleviate the inflammatory response in pancreatic acinar cells by suppressing cytokine gene expression.

Heterogeneity in the processing defect of SLC26A4 mutants

Background: Mutations in the SLC26A4 gene are responsible for Pendred syndrome and non-syndromic hearing loss (DFNB4). This study analysed non-synonymous SLC26A4 mutations newly identified in East Asians, as well as three common mutations in Caucasians, to characterise their molecular pathogenic mechanisms and to explore the possibility of rescuing their processing defects. Methods: A total of 11 non-synonymous disease associated mutations were generated and their effects on protein processing and on ion transporting activities were examined. Results: Most of the mutations caused retention of the SLC26A4 gene product (pendrin) in the intracellular region, while wild-type pendrin reached the plasma membrane. Accordingly, these mutations abolished complex glycosylation and Cl−/HCO3− exchange activities of pendrin. However, significant heterogeneity in the processing of mutant pendrin molecules was observed. Each mutant protein exhibited a different cellular localisation, a different degree of N-glycosylation, and a different degree of sensitivity to the treatments that rescue processing defects. For example, H723R-pendrin, the most common mutation in East Asians, was mostly expressed in endoplasmic reticulum (ER), and its defects in protein processing and ion transporting activities were restored considerably by low temperature incubation. On the other hand, L236P-pendrin, the most common mutation in Caucasians, was mainly in the centrosomal region and was temperature insensitive. Conclusion: These results indicate that the processing of pendrin mutant protein is determined by mutant specific mechanisms, and that a mutant specific method would be required to rescue the conformational defects of each folding mutant.