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Secretory diarrhea is one of the most common causes of death world-wide especially in children under 5 years old. Isoliquiritigenin (ISLQ), a plant-derived chalcone, has previously been shown to exert anti-secretory action in vitro and in vivo by inhibiting CFTR Cl - channels. However, its CFTR inhibition potency is considerably low (IC 50  > 10 μM) with unknown mechanism of action. This study aimed to identify novel chalcone derivatives with improved potency and explore their mechanism of action. Screening of 27 chalcone derivatives identified CHAL-025 as the most potent chalcone analog that reversibly inhibited CFTR-mediated Cl - secretion in T84 cells with an IC 50 of ∼1.5 μM. As analyzed by electrophysiological and biochemical analyses, the mechanism of CFTR inhibition by CHAL-025 is through AMP-activated protein kinase (AMPK), a negative regulator of CFTR activity. Furthermore, Western blot analyses and molecular dynamics (MD) results suggest that CHAL-025 activates AMPK by binding at the allosteric site of an upstream kinase calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ). Interestingly, CHAL-025 inhibited both cholera toxin (CT) and bile acid-induced Cl - secretion in T84 cells and prevented CT-induced intestinal fluid secretion in mice. Therefore, CHAL-025 represents a promising anti-diarrheal agent that inhibits CFTR Cl - channel activity via CaMKKβ-AMPK pathways., (Copyright © 2019 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Background: Although pharmacological treatment has increased the average life expectancy of patients with cystic fibrosis, the median survival of females is shorter than that of males. In vitro and in vivo studies have shown that estrogens play a relevant role in the disease progression. The aim of this study was to investigate the effects of 17β-estradiol and tamoxifen citrate (TMX) on calcium-activated chloride channel (CaCC) currents in human bronchial epithelial cells carrying the ΔPhe508-CFTR mutation both in homozygosis and in heterozygosis., Methods: Perforated patch clamp experiments were performed on single cells of the immortalized cell lines CFBE and IB3-1. Gramicidin (10 or 20 μM) was added to the electrode solution to reach the whole cell configuration. The electrical stimulation protocol consisted of square voltages ranging from - 80 to + 80 mV, in steps of 20 mV and with a duration of 800 msec., Results: The presence of 17β-estradiol significantly reduced the CaCC currents, both in basal conditions and in the presence of ATP (100 μM). The addition of TMX (10 μM) completely restored the currents abolished by 17β-estradiol, in basal conditions and after stimulation with ATP in both CFBE and IB3-1 cells. TMX had a strong, direct action on membrane current density, which significantly increased more than 4-fold in both cases. The membrane current stimulation produced by TMX was further enhanced by the addition of ATP. CFBE cells incubated for 24 h with 3 μM VX-809 (a CFTR corrector) and then acutely stimulated with VX-770 (a CFTR potentiator) in the presence of forskolin, showed an increase of chloride currents which were abolished by Inh-172. The chloride current density induced by TMX + ATP was, on average, greater than that obtained with VX-809 + VX-770 + forskolin. The currents elicited by TMX + ATP were abolished by the addition of NPPB, a CaCC inhibitor. The combined administration of TMX/ATP and VXs/FSK had an additional effect on chloride currents., Conclusions: Our results show that TMX restores CaCC currents inhibited by 17ß-estradiol and directly activates the transmembrane chloride currents potentiated by ATP, an effect which is mutation independent. The combined effect of TMX with current used treatments for cystic fibrosis could be of benefit to patients.

Background/aims: Sputum symptoms are commonly seen in the elderly. This study aimed to identify an efficacious expectorant treatment stratagem through evaluating the secretion-promoting activation and cystic fibrosis transmembrane conductance regulator (CFTR) expression of the bioactive herbal monomer naringenin., Methods: Vectorial Cl- transport was determined by measuring short-circuit current (ISC) in rat airway epithelium. cAMP content was measured by ELISA in primary cultured epithelial cells and Calu-3 cells. CFTR expression in Calu-3 cells was determined by qPCR., Results: Addition of naringenin to the basolateral side of the rat airway led to a concentration-dependent sustained increase in ISC. The current was suppressed when exposed to Cl--free solution or by bumetanide, BaCl2, and DPC but not by DIDS and IBMX. Forskolin-induced ISC increase and CFTRinh-172/MDL-12330A-induced ISC inhibition were not altered by naringenin. Intracellular cAMP content was significantly increased by naringenin. With lipopolysaccharide stimulation, CFTR expression was significantly reduced, and naringenin dose-dependently enhanced CFTR mRNA expression., Conclusion: These results demonstrate that naringenin has the ability to stimulate Cl- secretion, which is mediated by CFTR through a signaling pathway by increasing cAMP content. Moreover, naringenin can increase CFTR expression when organism CFTR expression is seriously hampered. Our data suggest a potentially effective treatment strategy for sputum., (© 2017 The Author(s). Published by S. Karger AG, Basel.)

Background & Aims: Excessive consumption of ethanol is one of the most common causes of acute and chronic pancreatitis. Alterations to the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) also cause pancreatitis. However, little is known about the role of CFTR in the pathogenesis of alcohol-induced pancreatitis., Methods: We measured CFTR activity based on chloride concentrations in sweat from patients with cystic fibrosis, patients admitted to the emergency department because of excessive alcohol consumption, and healthy volunteers. We measured CFTR levels and localization in pancreatic tissues and in patients with acute or chronic pancreatitis induced by alcohol. We studied the effects of ethanol, fatty acids, and fatty acid ethyl esters on secretion of pancreatic fluid and HCO3(-), levels and function of CFTR, and exchange of Cl(-) for HCO3(-) in pancreatic cell lines as well as in tissues from guinea pigs and CFTR knockout mice after administration of alcohol., Results: Chloride concentrations increased in sweat samples from patients who acutely abused alcohol but not in samples from healthy volunteers, indicating that alcohol affects CFTR function. Pancreatic tissues from patients with acute or chronic pancreatitis had lower levels of CFTR than tissues from healthy volunteers. Alcohol and fatty acids inhibited secretion of fluid and HCO3(-), as well as CFTR activity, in pancreatic ductal epithelial cells. These effects were mediated by sustained increases in concentrations of intracellular calcium and adenosine 3',5'-cyclic monophosphate, depletion of adenosine triphosphate, and depolarization of mitochondrial membranes. In pancreatic cell lines and pancreatic tissues of mice and guinea pigs, administration of ethanol reduced expression of CFTR messenger RNA, reduced the stability of CFTR at the cell surface, and disrupted folding of CFTR at the endoplasmic reticulum. CFTR knockout mice given ethanol or fatty acids developed more severe pancreatitis than mice not given ethanol or fatty acids., Conclusions: Based on studies of human, mouse, and guinea pig pancreata, alcohol disrupts expression and localization of the CFTR. This appears to contribute to development of pancreatitis. Strategies to increase CFTR levels or function might be used to treat alcohol-associated pancreatitis., (Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette transporter superfamily that functions as an epithelial chloride channel. Gating of the CFTR ion conduction pore involves a conserved irreversible cyclic mechanism driven by ATP binding and hydrolysis at two cytosolic nucleotide-binding domains (NBDs): formation of an intramolecular NBD dimer that occludes two ATP molecules opens the pore, whereas dimer disruption after ATP hydrolysis closes it. CFTR dysfunction resulting from inherited mutations causes CF. The most common CF mutation, deletion of phenylalanine 508 (ΔF508), impairs both protein folding and processing and channel gating. Development of ΔF508 CFTR correctors (to increase cell surface expression) and potentiators (to enhance open probability, Po) is therefore a key focus of CF research. The practical utility of 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), one of the most efficacious potentiators of ΔF508 CFTR identified to date, is limited by its pore-blocking side effect. NPPB-mediated stimulation of Po is unique in that it involves modulation of gating transition state stability. Although stabilization by NPPB of the transition state for pore opening enhances both the rate of channel opening and the very slow rate of nonhydrolytic closure, because of CFTR's cyclic gating mechanism, the net effect is Po stimulation. In addition, slowing of ATP hydrolysis by NPPB delays pore closure, further enhancing Po. Here we show that NPPB stimulates gating at a site outside the pore and that these individual actions of NPPB on CFTR are fully attributable to one or the other of its two complementary molecular parts, 3-nitrobenzoate (3NB) and 3-phenylpropylamine (3PP), both of which stimulate Po: the pore-blocking 3NB selectively stabilizes the transition state for opening, whereas the nonblocking 3PP selectively slows the ATP hydrolysis step. Understanding structure-activity relationships of NPPB might prove useful for designing potent, clinically relevant CFTR potentiators., (© 2014 Csanády and Töröcsik.)

It has been difficult to separate/identify the roles of ClC-2 and CFTR in Cl(-) transport studies. Using pharmacological agents, we aimed to differentiate functionally between ClC-2 and CFTR Cl(-) channel currents. Effects of CFTR inhibitor 172 (CFTRinh172), N-(4-methylphenylsulfonyl)-N'-(4-trifluoromethylphenyl)urea (DASU-02), and methadone were examined by whole cell patch clamp on Cl(-) currents in recombinant human ClC-2/human embryonic kidney 293 (ClC-2/HEK293) cells stably transformed with Epstein-Barr nuclear antigen 1 (hClC-2/293EBNA) and human CFTR/HEK293 (hCFTR/HEK293) cells and by short-circuit current (Isc) measurements in T84 cells. Lubiprostone and forskolin-IBMX were used as activators. CFTRinh172 inhibited forskolin-IBMX-stimulated recombinant human CFTR (hCFTR) and lubiprostone-stimulated recombinant human ClC-2 (hClC-2) Cl(-) currents in a concentration-dependent manner equipotently. DASU-02 inhibited forskolin-IBMX-stimulated Cl(-) currents in hCFTR/HEK293 cells, but not lubiprostone-stimulated Cl(-) currents in hClC-2/293EBNA cells. In T84 cells with basolateral nystatin or 1-ethyl-2-benzimidazolinone (1-EBIO), lubiprostone-stimulated and forskolin-IBMX-cyclosporin A (FICA)-stimulated Isc components were observed. CFTRinh172 inhibited major portions of both components. DASU-02 had no effect on lubiprostone-stimulated Isc but partially inhibited FICA-stimulated Isc. T84 cells in which ClC-2 or CFTR was knocked down using siRNAs were constructed. T84 ClC-2 knockdown cells did not respond to lubiprostone but did respond to forskolin-IBMX in a methadone-insensitive, DASU-02-sensitive manner, indicating CFTR function. T84 CFTR knockdown cells responded separately to lubiprostone and forskolin-IBMX in a methadone-sensitive and DASU-02-insensitive manner, indicating ClC-2 function. Low lubiprostone concentrations activated ClC-2, but not CFTR, and both channels were activated by forskolin-IBMX but have different inhibitor sensitivities. Methadone, but not DASU-02, inhibited ClC-2. DASU-02, but not methadone, inhibited CFTR. In T84 cells, both ClC-2 and CFTR are present and likely play roles in Cl(-) secretion., (Copyright © 2014 the American Physiological Society.)

Diarrheal diseases constitute a significant global health burden and are a major cause of childhood mortality and morbidity. Treatment of diarrheal disease has centered on the replacement of fluid and electrolyte losses using oral rehydration solutions. Although oral rehydration solutions have been highly successful, significant mortality and morbidity due to diarrheal disease remains. Secretory diarrheas, such as those caused by bacterial and viral enterotoxins, result from activation of cyclic nucleotide and/or Ca(2+) signaling pathways in intestinal epithelial cells, enterocytes, which increase the permeability of Cl(-) channels at the lumen-facing membrane. Additionally, there is often a parallel reduction in intestinal Na(+) absorption. Inhibition of enterocyte Cl(-) channels, including the cystic fibrosis transmembrane conductance regulator and Ca(2+)-activated Cl(-) channels, represents an attractive strategy for antisecretory drug therapy. High-throughput screening of synthetic small-molecule collections has identified several classes of Cl(-) channel inhibitors that show efficacy in animal models of diarrhea but remain to be tested clinically. In addition, several natural product extracts with Cl(-) channel inhibition activity have shown efficacy in diarrhea models. However, a number of challenges remain to translate the promising bench science into clinically useful therapeutics, including efficiently targeting orally administered drugs to enterocytes during diarrhea, funding development costs, and carrying out informative clinical trials. Nonetheless, Cl(-) channel inhibitors may prove to be effective adjunctive therapy in a broad spectrum of clinical diarrheas, including acute infectious and drug-related diarrheas, short bowel syndrome, and congenital enteropathies., (Copyright © 2014. Published by Elsevier Inc.)

Purpose: Diarrhea and dehydration caused by enteric infections is a major factor of morbidity and mortality worldwide. Secretory diarrhea can be devastating especially among infants, children, and HIV infected people and can result in death of more than 50% of its victims for without adequate rehydration, patients are at maximum risk during the first 6-18 hours. Hence, it is a leading cause of morbidity and mortality worldwide. Diarrhea is experienced by over 50% of AIDS patients at some time or other during the course of their illness, which is an important cause of increased morbidity and mortality in them. Currently, the standard-of-care therapy focuses only on rehydration therapy to combat dehydration and antibiotic therapy that targets the infectious agent only. Though, antimicrobial drugs have been the key treatment for diarrhea but, with the emergence of resistant strains the search for novel targets/drugs is on, for diarrhea still continues to kill millions., Methods: A literature search was done using secretory diarrhea and Crofelemer, as key words using PubMed (Medline), ProQuest, Cochrane Library, Medscape and Google Scholar search engines from January 2012 to December 2014. The types of articles included in this review were original research, review papers, recent patents and editorials from various medical schools across the globe. Though, it was practically not possible to include all studies, one can marvel at all the proclaimed mechanism of action of Crofelemer in this study., Results: Crofelemer, a channel blocker of intestinal chloride channels such as the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and the Calcium Activated Chloride Channels (CaCCs) plays significant roles in providing symptomatic relief in secretory diarrhea., Conclusion: Crofelemer is a first-in-class agent that possesses a unique mechanism of action through dual inhibition of both the intestinal chloride channels in secretory diarrhea.

We previously developed cell-based kinetics assays of chloride channel modulators utilizing genetically encoded yellow fluorescent proteins. Fluorescence platereader-based high-throughput screens yielded small-molecule activators and inhibitors of the cAMP-activated chloride channel CFTR and calcium-activated chloride channels, including TMEM16A. Here, we report a microfluidics platform for single-shot determination of concentration-activity relations in which a 1.5 × 1.5 mm square area of adherent cultured cells is exposed for 5-10 min to a pseudo-logarithmic gradient of test compound generated by iterative, two-component channel mixing. Cell fluorescence is imaged following perfusion with an iodide-containing solution to give iodide influx rate at each location in the image field, thus quantifying modulator effects over a wide range of concentrations in a single measurement. IC50 determined for CFTR and TMEM16A activators and inhibitors by single-shot microfluidics were in agreement with conventional plate reader measurements. The microfluidics approach developed here may accelerate the discovery and characterization of chloride channel-targeted drugs.

In clinical trials, methadone, but not morphine, appeared to prevent beneficial effects of lubiprostone, a ClC-2 Cl(-) channel activator, on opioid-induced constipation. Effects of methadone and morphine on lubiprostone-stimulated Cl(-) currents were measured by short circuit current (Isc) across T84 cells. Whole cell patch clamp of human ClC-2 (hClC-2) stably expressed in HEK293 cells and in a high expression cell line (HEK293EBNA) as well as human CFTR (hCFTR) stably expressed in HEK293 cells was used to study methadone and morphine effects on recombinant hClC-2 and hCFTR Cl(-) currents. Methadone but not morphine inhibited lubiprostone-stimulated Isc in T84 cells with half-maximal inhibition at 100 nM. Naloxone did not affect lubiprostone stimulation or methadone inhibition of Isc. Lubiprostone-stimulated Cl(-) currents in hClC-2/HEK293 cells, but not forskolin/IBMX-stimulated Cl(-) currents in hCFTR/HEK293 cells, were inhibited by methadone, but not morphine. HEK293EBNA cells expressing hClC-2 showed time-dependent, voltage-activated, CdCl2-inhibited Cl(-) currents in the absence (control) and the presence of lubiprostone. Methadone, but not morphine, inhibited control and lubiprostone-stimulated hClC-2 Cl(-) currents with half-maximal inhibition at 100 and 200-230 nM, respectively. Forskolin/IBMX-stimulated hClC-2 Cl(-) currents were also inhibited by methadone. Myristoylated protein kinase inhibitor (a specific PKA inhibitor) inhibited forskolin/IBMX- but not lubiprostone-stimulated hClC-2 Cl(-) currents. Methadone caused greater inhibition of lubiprostone-stimulated currents added before patching (66.1 %) compared with after patching (28.7 %). Methadone caused inhibition of lubiprostone-stimulated Cl(-) currents in T84 cells and control; lubiprostone- and forskolin/IBMX-stimulated recombinant hClC-2 Cl(-) currents may be the basis for reduced efficacy of lubiprostone in methadone-treated patients.

Purpose: We investigated the role that the cystic fibrosis transmembrane conductance regulator (CFTR) may play in Cl(-) transport in the acinar and ductal epithelial cells of rabbit lacrimal gland (LG)., Methods: Primary cultured LG acinar cells were processed for whole-cell patch-clamp electrophysiological recording of Cl(-) currents by using perfusion media with high and low [Cl(-)], 10 µM forskolin and 100 µM 3-isobutyl-1-methylxanthine (IBMX), the non-specific Cl(-) channel blocker 4,4'-disothiocyanostilbene-2, 2' sulphonic acid (DIDS; 100 µM) and CFTRinh-172 (10 µM), a specific blocker for CFTR. Ex vivo live cell imaging of [Cl(-)] changes in duct cells was performed on freshly dissected LG duct with a multiphoton confocal laser scanning microscope using a Cl(-) sensitive fluorescence dye, N-[ethoxycarbonylmethyl]-6-methoxy-quinolinium bromide., Results: Whole-cell patch-clamp studies demonstrated the presence of Cl(-) current in isolated acinar cells and revealed that this Cl(-) current was mediated by CFTR channel. Live cell imaging also showed the presence of CFTR-mediated Cl(-) transport across the plasma membrane of duct cells., Conclusions: Our previous data showed the presence of CFTR in all acinar and duct cells within the rabbit LG, with expression most prominent in the apical membranes of duct cells. The present study demonstrates that CFTR is actively involved in Cl(-) transport in both acinar cells and epithelial cells from duct segments, suggesting that CFTR may play a significant role in LG secretion.

Cystic fibrosis (CF) is a frequent autosomal recessive disease caused by mutations that impair the CF transmembrane conductance regulator (CFTR) protein function. CFTR is a chloride channel activated by cyclic AMP (cAMP) via protein kinase A (PKA) and ATP hydrolysis. We describe here a method to measure CFTR activity in a monolayer of cultured cells using a fluorescence spectrophotometer and the chloride-sensitive probe 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Modifying a slice holder, the spectrophotometer quartz cuvette was converted in a perfusion chamber, allowing measurement of CFTR activity in real time, in a monolayer of T84 colon carcinoma cells. The SPQ Stern-Volmer constant (K(Cl(-))) for chloride in water solution was 115.0 ± 2.8M(-1), whereas the intracellular (K(Cl(-))) was 17.8 ± 0.8 M(-1), for T84 cells. A functional analysis was performed by measuring CFTR activity in T84 cells. The CFTR transport inhibitors CFTR(inh)-172 (5 μM) and glibenclamide (100 μM) showed a significant reduction (P<0.05) in CFTR activity. This simple method allows measuring CFTR activity in a very simple, reproducible, and sensitive way., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Pruritus, also known as itch, is a sensation that causes a desire to scratch. Prolonged scratching exacerbates skin lesions in several skin diseases such as atopic dermatitis. Here, we identify the cystic fibrosis transmembrane conductance regulator (CFTR/Cftr), an integral membrane protein that mediates transepithelial chloride transport, as a determinant factor in mice for the susceptibility to several cutaneous symptoms during mite infestation. Mice that endogenously express dysfunctional Cftr (Cftr(ΔF508/ΔF508)) show significant increase of scratching behavior and skin fibrosis after mite exposure. These phenotypes were due to the increased expression of nerve growth factor (NGF) that augments the sensitization of peripheral nerve fibers. Moreover, protein gene product 9.5 (PGP9.5)-positive neurites were abundant in the epidermis of mite-infested Cftr(ΔF508/ΔF508) mice. Furthermore, mite-infested Cftr(+/+) mice orally administered with a chloride channel inhibitor glibenclamide had higher scratching count and increased level of NGF than vehicle-treated mice. Consistently, mite extract-exposed primary and transformed human keratinocytes, treated with CFTR inhibitor, had significantly higher level of NGF mRNA compared with vehicle-treated, mite extract-exposed cells. These results reveal that CFTR in keratinocytes plays a critical role for the regulation of peripheral nerve function and pruritus sensation, and suggest that Cftr(ΔF508/ΔF508) mice may serve as a novel mouse model that represents NGF-dependent generation of pruritus.

Previous reports point out to a functional relationship of the cystic fibrosis transmembrane conductance regulator (CFTR) and Ca(2+) activated Cl(-) channels (CaCC). Recent findings showing that TMEM16A forms the essential part of CaCC, prompted us to examine whether CFTR controls TMEM16A. Inhibition of endogenous CaCC by activation of endogenous CFTR was found in 16HBE human airway epithelial cells, which also express TMEM16A. In contrast, CFBE airway epithelial cells lack of CFTR expression, but express TMEM16A along with other TMEM16-proteins. These cells produce CaCC that is inhibited by overexpression and activation of CFTR. In HEK293 cells coexpressing TMEM16A and CFTR, whole cell currents activated by IMBX and forskolin were significantly reduced when compared with cells expressing CFTR only, while the halide permeability sequence of CFTR was not changed. Expression of TMEM16A, but not of TMEM16F, H or J, produced robust CaCC, which that were inhibited by CaCCinh-A01 and niflumic acid, but not by CFTRinh-172. TMEM16A-currents were attenuated by additional expression of CFTR, and were completely abrogated when additionally expressed CFTR was activated by IBMX and forskolin. On the other hand, CFTR-currents were attenuated by additional expression of TMEM16A. CFTR and TMEM16A were both membrane localized and could be coimmunoprecipitated. Intracellular Ca(2+) signals elicited by receptor-stimulation was not changed during activation of CFTR, while ionophore-induced rise in [Ca(2+)](i) was attenuated after stimulation of CFTR. The data indicate that both CFTR and TMEM16 proteins are separate molecular entities that show functional and molecular interaction., (Copyright © 2011 S. Karger AG, Basel.)

Two highly potent and selective cystic fibrosis (CF) transmembrane regulator (CFTR) inhibitors have been identified by high-throughput screening: the thiazolidinone CFTR(inh)-172 [3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]- 2-thioxo-4-thiazolidinone] and the glycine hydrazide GlyH-101 [N-(2-naphthalenyl)-((3,5-dibromo-2,4-dihydroxyphenyl)methylene)glycine hydrazide]. Inhibition of the CFTR chloride channel by these compounds has been suggested to be of pharmacological interest in the treatment of secretory diarrheas and polycystic kidney disease. In addition, functional inhibition of CFTR by CFTR(inh)-172 has been proposed to be sufficient to mimic the CF inflammatory profile. In the present study, we investigated the effects of the two compounds on reactive oxygen species (ROS) production and mitochondrial membrane potential in several cell lines: the CFTR-deficient human lung epithelial IB3-1 (expressing the heterozygous F508del/W1282X mutation), the isogenic CFTR-corrected C38, and HeLa and A549 as non-CFTR-expressing controls. Both inhibitors were able to induce a rapid increase in ROS levels and depolarize mitochondria in the four cell types, suggesting that these effects are independent of CFTR inhibition. In HeLa cells, these events were associated with a decrease in the rate of oxygen consumption, with GlyH-101 demonstrating a higher potency than CFTR(inh)-172. The impact of CFTR inhibitors on inflammatory parameters was also tested in HeLa cells. CFTR(inh)-172, but not GlyH-101, induced nuclear translocation of nuclear factor-kappaB (NF-kappaB). CFTR(inh)-172 slightly decreased interleukin-8 secretion, whereas GlyH-101 induced a slight increase. These results support the conclusion that CFTR inhibitors may exert nonspecific effects regarding ROS production, mitochondrial failure, and activation of the NF-kappaB signaling pathway, independently of CFTR inhibition.

The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in many segments of the mammalian nephron, where it may interact with and modulate the activity of a variety of apical membrane proteins, including the renal outer medullary potassium (ROMK) K(+) channel. However, the expression of CFTR in apical cell membranes or its function as a Cl(-) channel in native renal epithelia has not been demonstrated. Here, we establish that CFTR forms protein kinase A (PKA)-activated Cl(-) channels in the apical membrane of principal cells from the cortical collecting duct obtained from mice. These Cl(-) channels were observed in cell-attached apical patches of principal cells after stimulation by forskolin/3-isobutyl-1-methylxanthine. Quiescent Cl(-) channels were present in patches excised from untreated tubules because they could be activated after exposure to Mg-ATP and the catalytic subunit of PKA. The single-channel conductance, kinetics, and anion selectivity of these Cl(-) channels were the same as those of recombinant mouse CFTR channels expressed in Xenopus laevis oocytes. The CFTR-specific closed-channel blocker CFTR(inh)-172 abolished apical Cl(-) channel activity in excised patches. Moreover, apical Cl(-) channel activity was completely absent in principal cells from transgenic mice expressing the DeltaF508 CFTR mutation but was present and unaltered in ROMK-null mice. We discuss the physiologic implications of open CFTR Cl(-) channels on salt handling by the collecting duct and on the functional CFTR-ROMK interactions in modulating the metabolic ATP-sensing of ROMK.

Chloride channels represent a relatively under-explored target class for drug discovery as elucidation of their identity and physiological roles has lagged behind that of many other drug targets. Chloride channels are involved in a wide range of biological functions, including epithelial fluid secretion, cell-volume regulation, neuroexcitation, smooth-muscle contraction and acidification of intracellular organelles. Mutations in several chloride channels cause human diseases, including cystic fibrosis, macular degeneration, myotonia, kidney stones, renal salt wasting and hyperekplexia. Chloride-channel modulators have potential applications in the treatment of some of these disorders, as well as in secretory diarrhoeas, polycystic kidney disease, osteoporosis and hypertension. Modulators of GABA(A) (gamma-aminobutyric acid A) receptor chloride channels are in clinical use and several small-molecule chloride-channel modulators are in preclinical development and clinical trials. Here, we discuss the broad opportunities that remain in chloride-channel-based drug discovery.

The cystic fibrosis transmembrane conductance regulator (CFTR) represents the main Cl(-) channel in the apical membrane of epithelial cells for cAMP-dependent Cl(-) secretion. Here we report on the synthesis and screening of a small library of nontoxic alpha-aminoazaheterocycle-methylglyoxal adducts, inhibitors of wild-type (WT) CFTR and G551D-, G1349D-, and F508del-CFTR Cl(-) channels. In whole-cell patch-clamp experiments of Chinese hamster ovary (CHO) cells expressing WT-CFTR, we recorded rapid and reversible inhibition of forskolin-activated CFTR currents in the presence of the adducts 5a and 8a,b at 10 pM concentrations. Using iodide efflux experiments, we compared concentration-dependent inhibition of CFTR with glibenclamide (IC(50) = 14.7 microM), 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl-)methylene]-2-thioxo-4-thiazolidinone (CFTR(inh)-172) (IC(50) = 1.2 microM), and alpha-aminoazaheterocycle-methylglyoxal adducts and identified compounds 5a (IC(50) = 71 pM), 8a,b (IC(50) = 2.5 nM), and 7a,b (IC(50) = 3.4 nM) as the most potent inhibitors of WT-CFTR channels. Similar ranges of inhibition were also found when these compounds were evaluated on CFTR channels with the cystic fibrosis mutations F508del (in temperature-corrected human airway epithelial F508del/F508del CF15 cells)-, G551D-, and G1349D-CFTR (expressed in CHO and COS-7 cells). No effect of compound 5a was detected on the volume-regulated or calcium-regulated iodide efflux. Picomolar inhibition of WT-CFTR with adduct 5a was also found using a 6-methoxy-N-(3-sulfopropyl)-quinolinium fluorescent probe applied to the human tracheobronchial epithelial cell line 16HBE14o-. Finally, we found comparable inhibition by 5a or by CFTR(inh)-172 of forskolin-dependent short-circuit currents in mouse colon. To the best of our knowledge, these new nontoxic alpha-aminoazaheterocycle-methylglyoxal adducts represent the most potent compounds reported to inhibit CFTR chloride channels.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane protein that belongs to the same family as multidrug resistance-associated proteins whose main function is to expel xenobiotics and physiological organic anions from the cell interior. Despite the overall structural similarity with these membrane proteins, CFTR is not an active transporter but is instead a Cl- channel. We have tested the ability of known inhibitors of multidrug resistance-associated proteins to affect CFTR Cl- currents. We have found that sulfinpyrazone, probenecid, and benzbromarone are also inhibitors of CFTR activity, with a mechanism involving blockage of the channel pore.

Transepithelial fluctuation analysis (noise analysis) provides valuable information about the density and single-channel properties of ion channels in intact epithelia. Here we investigate cystic fibrosis transmembrane conductance regulator (CFTR)-dependent chloride (Cl-) secretion in T84 human colonic epithelia by inducing noise using the diarylsulfonylurea DASU-01, a low-affinity open-channel blocker of CFTR. Our data indicate that the apical membrane of maximally stimulated T84 epithelia has a very high Cl- conductance generated by approximately 7000 active CFTR channels per cell with open probability (Po) of approximately 0.4 and single-channel amplitude (i) of approximately 0.1 pA. Similar experiments might provide important information about how drugs regulate CFTR in intact epithelia.

The thiazolidinone CFTR(inh)-172 was identified recently as a potent and selective blocker of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. Here, we characterized the CFTR(inh)-172 inhibition mechanism by patch-clamp and short-circuit analysis using cells stably expressing wild-type and mutant CFTRs. CFTR(inh)-172 did not alter CFTR unitary conductance (8 pS), but reduced open probability by >90% with K(i) approximately 0.6 microM. This effect was due to increased mean channel closed time without changing mean channel open time. Short-circuit current experiments indicated similar CFTR(inh)-172 inhibitory potency (K(i) approximately 0.5 microM) for inhibition of Cl(-) current in wild-type, G551D, and G1349D CFTR; however, K(i) was significantly reduced to 0.2 microM for DeltaF508 CFTR. Our studies provide evidence for CFTR inhibition by CFTR(inh)-172 by a mechanism involving altered CFTR gating.

The active absorption of fluid from the airspaces of the lung is important for the resolution of clinical pulmonary edema. Although ENaC channels provide a major route for Na(+) absorption, the route of Cl(-) transport has been unclear. We applied a series of complementary approaches to define the role of Cl(-) transport in fluid clearance in the distal airspaces of the intact mouse lung, using wild-type and cystic fibrosis Delta F508 mice. Initial studies in wild-type mice showed marked inhibition of fluid clearance by Cl(-) channel inhibitors and Cl(-) ion substitution, providing evidence for a transcellular route for Cl(-) transport. In response to cAMP stimulation by isoproterenol, clearance was inhibited by the CFTR inhibitor glibenclamide in both wild-type mice and the normal human lung. Although isoproterenol markedly increased fluid absorption in wild-type mice, there was no effect in Delta F508 mice. Radioisotopic clearance studies done at 23 degrees C (to block active fluid absorption) showed approximately 20% clearance of (22)Na in 30 min both without and with isoproterenol. However, the clearance of (36)Cl was increased by 47% by isoproterenol in wild-type mice but was not changed in Delta F508 mice, providing independent evidence for involvement of CFTR in cAMP-stimulated Cl(-) transport. Further, CFTR played a major role in fluid clearance in a mouse model of acute volume-overload pulmonary edema. After infusion of saline (40% body weight), the lung wet-to-dry weight ratio increased by 28% in wild-type versus 64% in Delta F508 mice. These results provide direct evidence for a functionally important role for CFTR in the distal airspaces of the lung.

The role of the cystic fibrosis transmembrane conductance regulator (CFTR) in the renal cortical collecting duct (CCD) has not yet been fully elucidated. Here, we investigated the effects of deamino-8-D-arginine vasopressin (dDAVP) and isoproterenol (ISO) on NaCl transport in primary cultured CCDs microdissected from normal [CFTR(+/+)] and CFTR-knockout [CFTR(-/-)] mice. dDAVP stimulated the benzamyl amiloride (BAm)-sensitive transport of Na(+) assessed by the short-circuit current (I(sc)) method in both CFTR(+/+) and CFTR(-/-) CCDs to a very similar degree. Apical addition of 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) or glibenclamide partially inhibited the rise in I(sc) induced by dDAVP and ISO in BAm-treated CFTR(+/+) CCDs, whereas dDAVP, ISO, and NPPB did not alter I(sc) in BAm-treated CFTR(-/-) CCDs. dDAVP stimulated the apical-to-basal flux and, to a lesser extent, the basal-to-apical flux of (36)Cl(-) in CFTR(+/+) CCDs. dDAVP also increased the apical-to-basal (36)Cl(-) flux in CFTR(-/-) CCDs but not the basal-to-apical (36)Cl(-) flux. These results demonstrate that CFTR mediates the cAMP-stimulated component of secreted Cl(-) in mouse CCD.

The primary secretion formed in various exocrine glands has a [K+] 2-5 times that of plasma. In this study we measured the transepithelial flux of 36Cl-, 22Na+ and 42K+ across the frog skin and applied the single-channel patch-clamp technique to the apical membrane of frog skin gland acini to investigate the pathway taken by K+ secreted by the glands. Transepithelial K+ secretion was active and was driven by a larger force than the secretion of Na+. When driving Na+ through the epithelium by clamping the transepithelial potential to 100 mV (apical solution reference), blockers of cellular secretion (apical 5-nitro-2-(3-phenylpropylamino)benzoate or basolateral quinine or furosemide) decreased K+ secretion but left Na+ secretion unaffected. We conclude that K+ follows a transcellular pathway across the epithelium. Patch-clamp analysis of the apical membrane of microdissected gland acini revealed a population of voltage- and calcium-activated K+ channels of the maxi K+ type. In cell-attached patches these channels were activated by membrane potential depolarisation or exposure to prostaglandin E2 and had a permeability of 3.6 +/- 0.3 x 10(-13) cm3 s-1, giving a calculated conductance of 170 pS with 125 mM K+ on both sides of the membrane. In inside-out patches the channels were activated by increasing intracellular [Ca2+] from 10(-7) to 10(-6) M and were blocked by Ba2+ added to the cytoplasmic side. Exposure of inside-out patches containing the maxi K+ channel to ATP on the inside activated cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels, confirming that both channels are co-localised to the apical membrane. We interpret these findings in terms of a model where transepithelial NaCl secretion can be supported in part by an apical K+ conductance.

1. Anion binding within the pores of wild-type and mutant cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels, expressed in two different mammalian cell lines, was assayed using patch clamp recording. Specifically, experiments measured both the conductance of different anions and the ability of other permeant anions to block Cl- permeation through the pore. 2. Under symmetrical ionic conditions, wild-type CFTR channels showed the conductance sequence Cl- > NO3- > Br- > or = formate > F- > SCN- congruent to ClO4-. 3. High SCN- conductance was not observed, nor was there an anomalous mole fraction effect of SCN- on conductance under the conditions used. Iodide currents could not be measured under symmetrical ionic conditions, but under bi-ionic conditions I- conductance appeared low. 4. Chloride currents through CFTR channels were blocked by low concentrations (10 mM) of SCN-, I- and ClO4-, implying relatively tight binding of these anions within the pore. 5. Two mutations in CFTR which alter the anion permeability sequence, F337S and T338A, also altered the anion conductance sequence. Furthermore, block by SCN-, I- and ClO4- were weakened in both mutants. Both these effects are consistent with altered anion binding within the pore. 6. The effects of mutations on anion permeability and relative anion conductance suggested that, for most anions, increased permeability was associated with increased conductance. This indicates that the CFTR channel pore does not achieve its anion selectivity by selective anion binding within the mutated region. Instead, it is suggested that entry of anions into the region around F337 and T338 facilitates their passage through the pore. In wild-type CFTR channels, anion entry into this crucial pore region is probably dominated by anion hydration energies.

Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is inhibited by a broad range of intracellular organic anions. Here it is shown, using patch clamp recording from CFTR-transfected mammalian cell lines, that the fatty acids butyrate and 4-phenylbutyrate cause a voltage-dependent block of CFTR Cl(-) currents when applied to the cytoplasmic face of membrane patches, with apparent K(d)s (at 0 mV) of 29.6 mM for butyrate and 6.6 mM for 4-phenylbutyrate. At the single channel level, both these fatty acids caused an apparent reduction in CFTR current amplitude, suggesting a kinetically fast blocking mechanism. The concentration-dependence of block suggests that CFTR-mediated Cl(-) currents in vivo may be affected by both 4-phenylbutyrate used in the treatment of various diseases, including cystic fibrosis, and by butyrate produced endogenously within the colonic lumen.

Cystic fibrosis (CF) is an autosomal genetic disease associated with impaired epithelial ion transport. Mutations in the CF gene alter the primary sequence of the CF transmembrane conductance regulator (CFTR). Several therapeutic modalities have been proposed for CF patients, including the phytoestrogen genistein. Experiments were completed in cellular and subcellular systems to evaluate the impact of naturally occurring and synthetic estrogens on epithelial ion transport, and specifically on the CF protein CFTR. 17beta-Estradiol, a naturally occurring estrogen, caused a rapid and reversible inhibition of forskolin-stimulated chloride secretion across T84 epithelial cell monolayers with a K(i) of 8 microM. In addition, 17alpha-estradiol, a stereoisomer that fails to bind and activate nuclear estrogen receptors was equipotent with 17beta-estradiol, arguing against a genomic-mediated mechanism of action. Synthetic estrogens, including diethylstilbesterol and the antiestrogen tamoxifen likewise inhibited forskolin-stimulated ion transport. Aldosterone, dexamethasone, and cholesterol were without effect at the highest concentrations tested (>/=1 mM). Studies indicated that diethylstilbesterol and other synthetic estrogens that inhibited anion secretion in intact monolayers likewise inhibited CFTR chloride channel activity with similar concentration dependencies in excised membrane patches. Experiments with radioactive photoactivatable estrogen derivatives demonstrated that these compounds bind directly to CFTR expressed in insect cells. Taken together, the data suggest that estrogens can interact directly with CFTR to alter anion transport.

Blockers of CFTR with well-characterized kinetics and mechanism of action will be useful as probes of pore structure. We have studied the mechanism of block of CFTR by the arylaminobenzoates NPPB and DPC. Block of macroscopic currents by NPPB and DPC exhibited similar voltage-dependence, suggestive of an overlapping binding region. Kinetic analysis of single-channel currents in the presence of NPPB indicate drug-induced closed time constants averaging 2.2 msec at -100 mV. The affinity for NPPB calculated from single-channel block, K(D) = 35 microm, exceeds that for other arylaminobenzoates studied thus far. These drugs do not affect the rate of activation of wild-type (WT) channels expressed in oocytes, consistent with a simple mechanism of block by pore occlusion, and appear to have a single binding site in the pore. Block by NPPB and DPC were affected by pore-domain mutations in different ways. In contrast to its effects on block by DPC, mutation T1134F-CFTR decreased the affinity and reduced the voltage-dependence for block by NPPB. We also show that the alteration of macroscopic block by NPPB and DPC upon changes in bath pH is due to both direct effects (i.e., alteration of voltage-dependence) and indirect effects (alteration of cytoplasmic drug loading). These results indicate that both NPPB and DPC block CFTR by entering the pore from the cytoplasmic side and that the structural requirements for binding are not the same, although the binding regions within the pore are similar. The two drugs may be useful as probes for overlapping regions in the pore.

The protein defective in cystic fibrosis (CF), the CF transmembrane-conductance regulator (CFTR), functions as an epithelial chloride channel and as a regulator of separate ion channels. Although the consequences that disease-causing mutations have on the chloride-channel function have been studied extensively, little is known about the effects that mutations have on the regulatory function. To address this issue, we transiently expressed CFTR-bearing mutations associated with CF or its milder phenotype, congenital bilateral absence of the vas deferens, and determined whether mutant CFTR could regulate outwardly rectifying chloride channels (ORCCs). CFTR bearing a CF-associated mutation in the first nucleotide-binding domain (NBD1), DeltaF508, functioned as a chloride channel but did not regulate ORCCs. However, CFTR bearing disease-associated mutations in other domains retained both functions, regardless of the associated phenotype. Thus, a relationship between loss of CFTR regulatory function and disease severity is evident for NBD1, a region of CFTR that appears important for regulation of separate channels.

1. The isoflavone genistein may either stimulate or inhibit cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. To investigate how genistein inhibits CFTR, we studied CFTR Cl- channels in excised inside-out membrane patches from cells expressing wild-type human CFTR. 2. Addition of genistein (100 microM) to the intracellular solution caused a small decrease in single-channel current amplitude (i), but a large reduction in open probability (Po). 3. Single-channel analysis of channel block suggested that genistein (100 microM) may inhibit CFTR by two mechanisms: first, it may slow the rate of channel opening and second, it may block open channels. 4. Acidification of the intracellular solution relieved channel block, suggesting that the anionic form of genistein may inhibit CFTR. 5. Genistein inhibition of CFTR Cl- currents was weakly voltage dependent and unaffected by changes in the extracellular Cl- concentration. 6. Channel block was relieved by pyrophosphate (5 mM) and ATP (5 mM), two agents that interact with the nucleotide-binding domains (NBDs) of CFTR to greatly stimulate channel activity. 7. ATP (5 mM) prevented the genistein-induced decrease in Po, but was without effect on the genistein-induced decrease in i. 8. The genistein-induced decrease in i was voltage dependent, whereas the genistein-induced decrease in Po was voltage independent. 9. The data suggest that genistein may inhibit CFTR by two mechanisms. First, it may interact with NBD1 to potently inhibit channel opening. Second, it may bind within the CFTR pore to weakly block Cl- permeation.

The effect of genistein on anion secretion via cystic fibrosis transmembrane conductance regulator (CFTR) in cultured rat cauda epididymal epithelia was studied by short-circuit current (Isc) technique. Genistein added apically stimulated a concentration-dependent rise in Isc due to Cl(-) and HCO(3)(-) secretion. The genistein-induced Isc was observed in basolaterally permeabilized monolayers, suggesting that the Isc response was mediated by the apical anion channel. The response could be blocked by the nonspecific Cl(-) channel blocker, diphenylamine-2-carboxylate (DPC), but not by the Ca(2+)-activated Cl(-) channel blocker, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Genistein did not increase intracellular cAMP, but H-89, a protein kinase A inhibitor, completely abolished the Isc response to genistein. Moreover, pretreatment of the tissues with MDL-12330A, an adenylate cyclase inhibitor, markedly attenuated the Isc response to genistein, but the response was restored upon the addition of exogenous cAMP. Ca(2+), protein kinase C, tyrosine kinase, and protein phosphatase signalling pathways were not involved in the action of genistein. It is speculated that genistein stimulates anion secretion by direct interaction with CFTR. This requires a low level of phosphorylation of CFTR by basal protein kinase A activity. It is suggested that genistein may provide therapeutic benefit to male infertility associated with cystic fibrosis.

Previous studies demonstrated that ACh-induced liquid secretion by porcine bronchi is driven by active Cl(-) and HCO(-)(3) secretion. The present study was undertaken to determine whether this process was localized to submucosal glands and mediated by the cystic fibrosis transmembrane conductance regulator (CFTR). When excised, cannulated, and treated with ACh, porcine bronchi secreted 15.6 +/- 0.6 microliter. cm(-2). h(-1). Removal of the surface epithelium did not significantly affect the rate of secretion, indicating that the source of the liquid was the submucosal glands. Pretreatment with diphenylamine-2-carboxylate, a relatively nonselective Cl(-)-channel blocker, significantly reduced liquid secretion by 86%, whereas pretreatment with DIDS, which inhibits a variety of Cl(-) channels but not CFTR, had no effect. When bronchi were pretreated with glibenclamide or 5-nitro-2-(3-phenylpropylamino)benzoic acid (both inhibitors of CFTR), the rate of ACh-induced liquid secretion was significantly reduced by 39 and 91%, respectively, compared with controls. Agents that blocked liquid secretion also caused disproportionate reductions in HCO(-)(3) secretion. Polyclonal antibodies to the CFTR bound preferentially to submucosal gland ducts and the surface epithelium, suggesting that this channel was localized to these sites. These data suggest that ACh-induced gland liquid secretion by porcine bronchi is driven by active secretion of both Cl(-) and HCO(-)(3) and is mediated by the CFTR.

To examine a possible relation between the swelling-induced ATP release pathway and the volume-sensitive Cl(-) channel, we measured the extracellular concentration of ATP released upon osmotic swelling and whole-cell volume-sensitive Cl(-) currents in a human epithelial cell line, Intestine 407, which lacks expression of cystic fibrosis transmembrane conductance regulator (CFTR). Significant release of ATP was observed within several minutes after a hypotonic challenge (56-80% osmolality) by the luciferin/luciferase assay. A carboxylate analogue Cl(-) channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoate, suppressed ATP release in a concentration-dependent manner with a half-maximal inhibition concentration of 6.3 microM. However, swelling-induced ATP release was not affected by a stilbene-derivative Cl(-) channel blocker, 4-acetamido-4'-isothiocyanostilbene at 100 microM. Glibenclamide (500 microM) and arachidonic acid (100 microM), which are known to block volume-sensitive outwardly rectifying (VSOR) Cl(-) channels, were also ineffective in inhibiting the swelling-induced ATP release. Gd(3+), a putative blocker of stretch-activated channels, inhibited swelling-induced ATP release in a concentration-dependent manner, whereas the trivalent lanthanide failed to inhibit VSOR Cl(-) currents. Upon osmotic swelling, the local ATP concentration in the immediate vicinity of the cell surface was found to reach approximately 13 microM by a biosensor technique using P2X(2) receptors expressed in PC12 cells. We have raised antibodies that inhibit swelling-induced ATP release from Intestine 407 cells. Earlier treatment with the antibodies almost completely suppressed swelling-induced ATP release, whereas the activity of VSOR Cl(-) channel was not affected by pretreatment with the antibodies. Taking the above results together, the following conclusions were reached: first, in a CFTR-lacking human epithelial cell line, osmotic swelling induces ATP release and increases the cell surface ATP concentration over 10 microM, which is high enough to stimulate purinergic receptors; second, the pathway of ATP release is distinct from the pore of the volume-sensitive outwardly rectifying Cl(-) channel; and third, the ATP release is not a prerequisite to activation of the Cl(-) channel.

1. Hypoglycaemia-inducing sulphonylureas, such as glibenclamide, inhibit cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. In search of modulators of CFTR, we investigated the effects of the non-sulphonylurea hypoglycaemic agents meglitinide, repaglinide, and mitiglinide (KAD-1229) on CFTR Cl- channels in excised inside-out membrane patches from C127 cells expressing wild-type human CFTR. 2. When added to the intracellular solution, meglitinide and mitiglinide inhibited CFTR Cl- currents with half-maximal concentrations of 164+/-19 microM and 148+/-36 microM, respectively. However, repaglinide only weakly inhibited CFTR Cl- currents. 3. To understand better how non-sulphonylurea hypoglycaemic agents inhibit CFTR, we studied single channels. Channel blockade by both meglitinide and mitiglinide was characterized by flickery closures and a significant decrease in open probability (Po). In contrast, repaglinide was without effect on either channel gating or Po, but caused a small decrease in single-channel current amplitude. 4. Analysis of the dwell time distributions of single channels indicated that both meglitinide and mitiglinide greatly decreased the open time of CFTR. Mitiglinide-induced channel closures were about 3-fold longer than those of meglitinide. 5. Inhibition of CFTR by meglitinide and mitiglinide was voltage-dependent: at positive voltages channel blockade was relieved. 6. The data demonstrate that non-sulphonylurea hypoglycaemic agents inhibit CFTR. This indicates that these agents have a wider specificity of action than previously recognized. Like glibenclamide, non-sulphonylurea hypoglycaemic agents may inhibit CFTR by occluding the channel pore and preventing Cl- permeation.

1. The goals of this study were to identify the structural components required for arylaminobenzoate block of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and to determine the involvement of two positively charged amino acid residues, found within the channel, in drug binding. 2. Wild-type and mutant CFTR chloride channels were expressed in Xenopus oocytes and CFTR currents measured using the two microelectrode voltage clamp. Block of the wild-type CFTR current by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) occurred in a voltage-dependent manner with preferential inhibition of the inward currents (Kd = 166 microM at -90 mV). 3. Removal of the phenyl ring from the aliphatic chain of NPPB, with the compound 2-butylamino-5-nitrobenzoic acid, caused only a small change in CFTR inhibition (Kd = 243 microM), while addition of an extra phenyl ring at this position (5-nitro-2-(3,3-diphenylpropylamino)-benzoic acid) increased drug potency (Kd = 58 microM). In contrast, removal of the benzoate ring (2-amino-4-phenylbutyric acid) or the 5-nitro group (2-(3-phenylpropylamino)-benzoic acid) of NPPB severely limited drug block of the wild-type channel. 4. NPPB inhibition of CFTR currents in oocytes expressing the mutants K335E and R347E also occurred in a voltage-dependent manner. However, the Kds for NPPB block were increased to 371 and 1573 microM, for the K335E and R347E mutants, respectively. 5. NPPB block of the inward wild-type CFTR current was reduced in the presence of 10 mM of the permeant anion SCN-. 6. These studies present the first step in the development of high affinity probes to the CFTR channel.

Previous studies have demonstrated that Cl- secretion by the mouse endometrial epithelium is under neurohormonal influence. The present study characterized the Cl- conductance activated by a number of agonists in the mouse endometrial epithelial cells using the whole-cell voltage-clamp technique. Adrenaline (1 microM), prostaglandin (PG) E2 (5-10 microM), and PGF2alpha (100 microM) activated a whole-cell current that exhibited a linear I-V relationship as well as time- and voltage-independent characteristics. However, the current magnitude varied with different agonists. The agonist-activated current could be mimicked by an adenylate cyclase activator, forskolin (10 microM), and suppressed by an adenylate cyclase inhibitor, MDL12330A, suggesting the involvement of cAMP. Current characteristics remained the same after cation replacement, leaving Cl- as the major permeant ion species in the solutions. The reversal potential of the agonist-induced current was close to the equilibrium potential of Cl- in the presence of a Cl- gradient, indicating the activation of Cl- conductance. The agonist-induced current was inhibited by the Cl- channel blocker diphenylamine 2,2'-dicarboxylic acid (DPC), but not by the Cl- channel blocker 4,4'-diisothiocyanatostibene-2, 2'-disulfonic acid (DIDS). The anion selectivity sequence of the current was NO3->Br->Cl->I-. The observed electrophysiological properties of the agonist-induced Cl- conductance were consistent with those reported for the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated Cl- channel expressed in many epithelia. The expression of CFTR in the mouse endometrial cells was also demonstrated by Western blot analysis. It appears that neurohormonal regulation of the uterine fluid in the mouse endometrium converges on the cAMP-activated Cl- channel, presumably CFTR.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, which mediates transepithelial Cl- transport in a variety of epithelia, including airway, intestine, pancreas, and sweat duct. In some but not all epithelial cells, cAMP stimulates Cl- secretion in part by increasing the number of CFTR Cl- channels in the apical plasma membrane. Because the mechanism whereby cAMP stimulates CFTR Cl- secretion is cell-type specific, our goal was to determine whether cAMP elevates CFTR-mediated Cl- secretion across serous airway epithelial cells by stimulating the insertion of CFTR Cl- channels from an intracellular pool into the apical plasma membrane. To this end we studied Calu-3 cells, a human airway cell line with a serous cell phenotype. Serous cells in human airways, such as Calu-3 cells, express high levels of CFTR, secrete antibiotic-rich fluid, and play a critical role in airway function. Moreover, dysregulation of CFTR-mediated Cl- secretion in serous cells is thought to contribute to the pathophysiology of cystic fibrosis lung disease. We report that cAMP activation of CFTR-mediated Cl- secretion across human serous cells involves stimulation of CFTR channels present in the apical plasma membrane and does not involve the recruitment of CFTR from an intracellular pool to the apical plasma membrane.

The cystic fibrosis transmembrane conductance regulator (CFTR) forms a chloride-selective channel. Residues from the 12 putative membrane-spanning segments form at least part of the channel lining. We need to identify the channel-lining residues in order to understand the structural basis for the channel's functional properties. Using the substituted-cysteine-accessibility method we mutated to cysteine, one at a time, 24 consecutive residues (Asp192-Ile215) in the M3 membrane-spanning segment. Cysteines substituted for His199, Phe200, Trp202, Ile203, Pro205, Gln207, Leu211, and Leu214 reacted with charged, sulfhydryl-specific reagents that are derivatives of methanethiosulfonate (MTS). We infer that these residues are on the water-accessible surface of the protein and probably form a portion of the channel lining. When plotted on an alpha-helical wheel the exposed residues from Gln207 to Leu214 lie within an arc of 60 degrees; the exposed residues in the cytoplasmic half (His199-Ile203) lie within an arc of 160 degrees. We infer that the secondary structures of the extracellular and cytoplasmic halves of M3 are alpha-helical and that Pro205, in the middle of the M3 segment, may bend the M3 segment, moving the cytoplasmic end of the segment in toward the central axis of the channel. The bend in the M3 segment may help to narrow the channel lumen near the cytoplasmic end. In addition, unlike full-length CFTR, the current induced by the deletion construct, Delta259, is inhibited by the MTS reagents, implying that the channel structure of Delta259 is different than the channel structure of wild-type CFTR.

The cystic fibrosis transmembrane conductance regulator (CFTR) forms an anion-selective channel involved in epithelial chloride transport. Recent studies have provided new insights into the structural determinants of the channel's functional properties, such as anion selectivity, single-channel conductance, and gating. Using the scanning-cysteine-accessibility method we identified 7 residues in the M1 membrane-spanning segment and 11 residues in and flanking the M6 segment that are exposed on the water-accessible surface of the protein; many of these residues may line the ion-conducting pathway. The pattern of the accessible residues suggests that these segments have a largely alpha-helical secondary structure with one face exposed in the channel lumen. Our results suggest that the residues at the cytoplasmic end of the M6 segment loop back into the channel, narrowing the lumen, and thereby forming both the major resistance to ion movement and the charge-selectivity filter.

cAMP-dependent phosphorylation activates the cystic fibrosis transmembrane conductance regulator (CFTR) in epithelia. However, the protein phosphatase (PP) that dephosphorylates and inactivates CFTR in airway and intestinal epithelia, two major sites of disease, is not certain. We found that in airway and colonic epithelia, neither okadaic acid nor FK506 prevented inactivation of CFTR when cAMP was removed. These results suggested that a phosphatase distinct from PP1, PP2A, and PP2B was responsible. Because PP2C is insensitive to these inhibitors, we tested the hypothesis that it regulates CFTR. We found that PP2Calpha is expressed in airway and T84 intestinal epithelia. To test its activity on CFTR, we generated recombinant human PP2Calpha and found that it dephosphorylated CFTR and an R domain peptide in vitro. Moreover, in cell-free patches of membrane, addition of PP2Calpha inactivated CFTR Cl- channels; reactivation required readdition of kinase. Finally, coexpression of PP2Calpha with CFTR in epithelia reduced the Cl- current and increased the rate of channel inactivation. These results suggest that PP2C may be the okadaic acid-insensitive phosphatase that regulates CFTR in human airway and T84 colonic epithelia. It has been suggested that phosphatase inhibitors could be of therapeutic value in cystic fibrosis; our data suggest that PP2C may be an important phosphatase to target.

The activity of the Cl(-)-conductive pathways, their regulation by protein kinase A (PKA) and their relationship to the cystic fibrosis transmembrane regulator (CFTR) protein were assessed in rat kidney cortical brush-border-membrane vesicles (cBBMV) and outer medullary vesicles (OMV) by measuring the rate of valinomycin-induced microsomal swelling by light scattering in the presence of an inward Cl- gradient. Valinomycin increased the rate of swelling of cBBMV and OMV, which is consistent with the presence of a Cl(-)-conductive pathway. PKA further increased these rates. This effect was blocked by the inhibitor of protein kinase A, suggesting that phosphorylation by PKA activates these pathways. Four anion-transport inhibitors were tested ¿N-phenylanthranilic acid (PhNHPhCOOH), 5-nitro-2-(3-phenylpropylamino)benzoic acid [N(PhPrNH2)BzOH], glybenclamide and 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid¿. Ph2COOH and 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid inhibited the basal Cl(-)-conductive pathways, while PKA-treated microsomes were sensitive also to N(PhPrNH2)BzOH and glybenclamide, suggesting that additional Cl- pathways were activated by phosphorylation. The pharmacological properties of these pathways were similar to those of the CFTR Cl- channel. Two anti-CFTR mAbs inhibited PKA-activated valinomycin-induced swelling in cBBMV and OMV, while immunoblot analysis of the corresponding proteins with the same antibodies indicated the presence of a 170-kDa protein. The results thus indicate the presence of a PKA-activated Cl(-)-conductive pathway in cBBMV and OMV, and suggest that CFTR protein is involved in PKA-activated Cl- fluxes in these vesicles.

We have described a preparation of Necturus maculosus gallbladder (NGB) epithelium yielding isolated cells that retain structural and functional polarity ("figure-eight" cells). These cells have a normal membrane voltage and remain polarized for several hours after isolation. Apical and basolateral membrane domains are differentially labeled with hydrophobic fluorescent dyes; freeze-fracture electron microscopy reveals two distinct membrane domains separated by tight junctions; ZO-1, Na+/H+ exchanger (NHE3), and Na(+)-K(+)-ATPase are present in the junctional, apical, and basolateral region, respectively; and cell-attached patch-clamp experiments reveal different K+ currents in the two membrane domains [R. J. Torres, G. A. Altenberg, J. A. Copello, G. Zampighi, and L. Reuss, Am. J. Physiol. 270 (Cell Physiol. 39): C1864-C1874, 1996]. Here, we show that NGB epithelial cells express a protein cross-reactive with an antibody against human cystic fibrosis transmembrane conductance regulator (CFTR). In figure-eight cells, immunoreactivity was restricted to the apical membrane domain. Using intracellular microelectrodes and a novel method of regional superfusion, we found that control cells have high K+ conductances in both membranes and a small basolateral Cl- conductance, similar to findings in the epithelium. Activation of adenylate cyclase with forskolin elicited a large apical membrane Cl- conductance and membrane depolarization. Whole cell patch-clamp studies yielded a forskolin-activated linear Cl- current, with high Cl-/aspartate selectivity. In conclusion, 1) figure-eight cells maintain the conductive membrane properties present in the epithelium, including polarized expression of adenosine 3',5'-cyclic monophosphate (cAMP)-activated Cl- channels, and 2) the cAMP-activated Cl- conductance is underlied by a CFTR homologue.

Recent studies from our laboratory indicate that members of the ATP-binding cassette (ABC) family of transporters, including P-glycoprotein and cystic fibrosis transmembrane conductance regulator (CFTR), are ATP-permeable channels. The physiological relevance of this novel transport mechanism is largely unknown. In the present study, intra- and extracellular ATP content, cellular ATP release, and O2 consumption before and after adenosine 3',5'-cyclic monophosphate (cAMP) stimulation were determined to assess the role of CFTR in the transport of ATP under physiological conditions. The functional expression of CFTR by the stable transfection of mouse mammary carcinoma cells, C1271, with human epithelial CFTR cDNA resulted in a stimulated metabolism, since both basal and cAMP-inducible O2 consumption were increased compared with mock-transfected cells. The stimulated (but not basal) O2 consumption was inhibited by diphenyl-2-carboxylic acid (DPC), a known inhibitor of CFTR. CFTR expression was also associated with the cAMP-activated and DPC-inhibitable release of intracellular ATP. The recovery of intracellular ATP from complete depletion after metabolic poisoning was also assessed under basal and cAMP-stimulated conditions. The various maneuvers indicate that CFTR may be an important contributor to the release of cellular ATP, which may help modify signal transduction pathways associated with secretory Cl- movement or other related processes. Changes in the CFTR-mediated delivery of nucleotides to the extracellular compartment may play an important role in the onset and reversal of the cystic fibrosis phenotype.