ClC-5: ontogeny of an alternative chloride channel in respiratory epithelia

2002 ◽  
Vol 282 (3) ◽  
pp. L501-L507 ◽  
Author(s):  
Rebecca D. Edmonds ◽  
Ian V. Silva ◽  
William B. Guggino ◽  
Robert B. Butler ◽  
Pamela L. Zeitlin ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Lung Disease ◽  
Chloride Channel ◽  
Chloride Channels ◽  
Chloride Transport ◽  
Premature Death ◽  
Fetal Lung ◽  
Chloride Secretion ◽  
Normal Lung ◽  
Ribonuclease Protection Assay

Chloride transport is critical to many functions of the lung. Molecular defects in the best-known chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR), lead to impaired function of airway defensins, hydration of airway surface fluid, and mucociliary clearance leading to chronic lung disease, and premature death, but do not cause defects in lung development. We examined the expression of one member of the ClC family of volume- and voltage-regulated channels using the ribonuclease protection assay and Western blot analysis in rats. ClC-5 mRNA and protein are most strongly expressed in the fetal lung, and expression is maintained although downregulated postnatally. In addition, using immunocytochemistry, we find that ClC-5 is predominantly expressed along the luminal surface of the airway epithelium, suggesting that ClC-5 may participate in lung chloride secretion. Identifying candidate genes for critical ion transport functions is essential for understanding normal lung morphogenesis and the pathophysiology of several lung diseases. In addition, the manipulation of non-CFTR chloride channels may provide a viable approach for treating cystic fibrosis lung disease.

Inhibition of CLC-2 chloride channel expression interrupts expansion of fetal lung cysts

2004 ◽  
Vol 286 (2) ◽  
pp. L420-L426 ◽  
Author(s):  
Carol J. Blaisdell ◽  
Marcelo M. Morales ◽  
Ana Carolina Oliveira Andrade ◽  
Penelope Bamford ◽  
Michael Wasicko ◽  
...  
Keyword(s):  
Chloride Channel ◽  
Fluid Transport ◽  
Fetal Lung ◽  
Chloride Secretion ◽  
Normal Lung ◽  
Apical Surface ◽  
Lung Cysts ◽  
Lung Fluid ◽  
Lung Morphogenesis ◽  
Channel Expression

Normal lung morphogenesis is dependent on chloride-driven fluid transport. The molecular identity of essential fetal lung chloride channel(s) has not been elucidated. CLC-2 is a chloride channel, which is expressed on the apical surface of the developing respiratory epithelium. CLC-2-like pH-dependent chloride secretion exists in fetal airway cells. We used a 14-day fetal rat lung submersion culture model to examine the role of CLC-2 in lung development. In this model, the excised fetal lung continues to grow, secrete fluid, and become progressively cystic in morphology ( 26 ). We inhibited CLC-2 expression in these explants, using antisense oligonucleotides, and found that lung cyst morphology was disrupted. In addition, transepithelial voltage ( Vt) of lung explants transfected with antisense CLC-2 was inhibited with Vt = -1.5 ± 0.2 mV (means + SE) compared with -3.7 ± 0.3 mV (means + SE) for mock-transfected controls and -3.3 ± 0.3 mV (means + SE) for nonsense oligodeoxynucleotide-transfected controls. This suggests that CLC-2 is important for fetal lung fluid production and that it may play a role in normal lung morphogenesis.

Gestational and tissue-specific regulation of C1C-2 chloride channel expression

1996 ◽  
Vol 271 (5) ◽  
pp. L829-L837 ◽  
Author(s):  
C. B. Murray ◽  
S. Chu ◽  
P. L. Zeitlin
Keyword(s):  
Protein Expression ◽  
Chloride Channel ◽  
Chloride Channels ◽  
Chloride Secretion ◽  
The Body ◽  
Late Gestation ◽  
Ribonuclease Protection Assay ◽  
Mrna Transcripts ◽  
Specific Regulation ◽  
Ribonuclease Protection

Chloride channels supply critical functions in epithelial cells throughout the body. Although function of the volume- and voltage-gated C1C-2 is uncertain, its wide tissue distribution of mRNA suggests C1C-2 has important housekeeping functions. This study's objective was to identify the extent of not only C1C-2 mRNA expression but also protein expression as a measure of the capacity for C1C-2 chloride secretion in epithelial tissues. Using quantitative ribonuclease protection assay, we found that C1C-2 mRNA transcripts were abundant in fetal and postnatal brain, fetal kidney, liver, intestine, and lung. In contrast to brain, C1C-2 mRNA transcripts were downregulated during late gestation in lung, kidney, and intestine. The lung expressed the least C1C-2 mRNA. Immunoblotting demonstrated similar tissue- and gestation-dependent variations in C1C-2 protein expression. To determine if there is a correlation between the sites of C1C-2 protein expression and cystic fibrosis transmembrane conductance regulator (CFTR), another epithelial chloride channel, a polyclonal COOH-terminal C1C-2 antibody and an anti-R domain CFTR anti-body were used. C1C-2 and CFTR were expressed in different sites in lung and kidney.

scholarly journals Chloride transport modulators as drug candidates

2021 ◽  
Author(s):  
Alan S. Verkman ◽  
Luis J. V. Galietta
Keyword(s):  
Cystic Fibrosis ◽  
Drug Discovery ◽  
Small Molecule ◽  
Kidney Stones ◽  
Chloride Channel ◽  
Chloride Channels ◽  
Chloride Transport ◽  
Secretory Diarrhea ◽  
Small Molecule Modulators ◽  
Made In

Chloride transport across cell membranes is broadly involved in epithelial fluid transport, cell volume and pH regulation, muscle contraction, membrane excitability, and organellar acidification. The human genome encodes at least 53 chloride transporting proteins with expression in cell plasma or intracellular membranes, which include chloride channels, exchangers and cotransporters, some having broad anion specificity. Loss of function mutations in chloride transporters cause a wide variety of human diseases, including cystic fibrosis, secretory diarrhea, kidney stones, salt wasting nephropathy, myotonia, osteopetrosis, hearing loss and goiter. While impactful advances have been made in the past decade in drug treatment of cystic fibrosis using small molecule modulators of the defective cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, other chloride channels and solute carrier proteins (SLCs) represent relatively underexplored target classes for drug discovery. New opportunities have emerged for development of chloride transport modulators as potential therapeutics for secretory diarrheas, constipation, dry eye disorders, kidney stones, polycystic kidney disease, hypertension and osteoporosis. Approaches to chloride transport-targeted drug discovery are reviewed herein, with focus on chloride channel and exchanger classes in which recent preclinical advances have been made in the identification of small molecule modulators and in proof of concept testing in experimental animal models.

Perinatal regulation of the ClC-2 chloride channel in lung is mediated by Sp1 and Sp3

1999 ◽  
Vol 276 (4) ◽  
pp. L614-L624 ◽  
Author(s):  
Shijian Chu ◽  
Carol J. Blaisdell ◽  
Min-Zhi M. Liu ◽  
Pamela L. Zeitlin
Keyword(s):  
Chloride Channel ◽  
Fetal Lung ◽  
Airway Disease ◽  
Perinatal Period ◽  
Lung Epithelial Cells ◽  
Normal Lung ◽  
Mobility Shift ◽  
Channel Expression ◽  
Gc Box ◽  
Electrophoretic Mobility Shift Assays

Mechanisms responsible for regulation of pulmonary epithelial chloride-channel expression in the perinatal period are under investigation to better understand normal lung development and airway disease pathogenesis. The ClC-2 epithelial chloride channel is regulated by changes in pH and volume and is most abundant in lung during fetal development. In this study, we identify and sequence the ClC-2 promoter, which is GC rich and lacks a TATA box. By construction of a series of promoter-luciferase constructs, a 67-bp GC box-containing sequence in the promoter is shown to be critical to ClC-2 expression in primary and immortalized fetal lung epithelial cells. Electrophoretic mobility shift assays and antibody supershifts demonstrate that the Sp1 and Sp3 transcription factors are expressed in fetal lung nuclei and interact with the GC box sequences in the promoter. Immunoblotting techniques demonstrate that Sp1 and Sp3 are perinatally downregulated in the lung with the same temporal sequence as ClC-2 downregulation. This work suggests that Sp1 and Sp3 activate ClC-2 gene transcription and that reduction in Sp1 and Sp3 at birth explains perinatal downregulation of ClC-2 in the lung.

Developing bronchopulmonary epithelium of the human fetus secretes fluid

1992 ◽  
Vol 262 (3) ◽  
pp. L270-L279 ◽  
Author(s):  
P. B. McCray ◽  
J. D. Bettencourt ◽  
J. Bastacky
Keyword(s):  
Human Fetus ◽  
Chloride Transport ◽  
Fetal Lung ◽  
Fluid Secretion ◽  
Chloride Secretion ◽  
Beta Agonist ◽  
Lung Fluid ◽  
Luminal Area ◽  
Fluid Production ◽  
Human Fetal Lung

We studied human fetal lung tissue in submersion organ culture to determine whether the bronchopulmonary epithelium secretes fluid during development. In this system the acinar tubules continued to grow, secrete fluid, and become progressively dilated. Baseline transepithelial potential differences (psi t) of -0.5 to -11 mV (mean, -3.8 mV, lumen negative, n = 27) were measured with microelectrodes after 3-8 days in culture, suggesting active electrolyte transport. Bumetanide (500 microM), an inhibitor of chloride secretion in other systems, decreased the basal psi t from -5 +/- 1.5 to -3.2 +/- 1.6 (SE) mV (P less than 0.05, n = 6), suggesting that chloride transport contributed to the voltage. Isoproterenol (5 microM) increased the baseline psi t from -5.6 +/- 2.1 to -9.2 +/- 2.5 (SE) mV (P less than 0.05, n = 4). Subsequent addition of bumetanide inhibited the isoproterenol-induced stimulation of the psi t by 20% (P less than 0.05). 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate. (CPT-cAMP, 50 microM) and 3-isobutyl 1-methylxanthine (IBMX, 100 microM) had similar effects, causing an increase in the psi t from -2.2 +/- 0.5 to -8 +/- 1.6 (SE) mV, an effect that was inhibited by the addition of bumetanide (P less than 0.005, n = 6). Both isoproterenol and CPT-cAMP/IBMX produced significant increases in the percentage luminal area of the explants at 12 and 24 h after exposure compared with control. We conclude that 1) the developing bronchopulmonary epithelium (acinar tubules) contributes to lung fluid production in the human fetus, 2) fetal lung fluid secretion is chloride dependent, and 3) chloride secretion and fluid secretion may be stimulated by a beta-agonist and cAMP.

scholarly journals Defective organellar acidification as a cause of cystic fibrosis lung disease: reexamination of a recurring hypothesis

2009 ◽  
Vol 296 (6) ◽  
pp. L859-L867 ◽  
Author(s):  
Peter M. Haggie ◽  
A. S. Verkman
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Lung Disease ◽  
Alveolar Macrophages ◽  
Chloride Channels ◽  
Loss Of Function ◽  
Cellular Mechanisms ◽  
Sodium Efflux ◽  
Cystic Fibrosis Lung Disease ◽  
Respiratory Epithelial

The cellular mechanisms by which loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel produce cystic fibrosis (CF) lung disease remain uncertain. Defective organellar function has been proposed as an important determinant in the pathogenesis of CF lung disease. According to one hypothesis, reduced CFTR chloride conductance in organelles in CF impairs their acidification by preventing chloride entry into the organelle lumen, which is needed to balance the positive charge produced by proton entry. According to a different hypothesis, CFTR mutation hyperacidifies organelles by an indirect mechanism involving unregulated sodium efflux through epithelial sodium channels. There are reports of defective Golgi, endosomal and lysosomal acidification in CF epithelial cells, defective phagolysosomal acidification in CF alveolar macrophages, and organellar hyperacidification in CF respiratory epithelial cells. The common theme relating too high or low organellar pH to cellular dysfunction and CF pathogenesis is impaired functioning of organellar enzymes, such as those involved in ceramide metabolism and protein processing in epithelial cells and antimicrobial activity in alveolar macrophages. We review here the evidence for defective organellar acidification in CF. Significant technical and conceptual concerns are discussed regarding the validity of data showing too high/low organellar pH in CF cells, and rigorous measurements of organellar pH in CF cells are reviewed that fail to support defective organellar acidification in CF. Indeed, there is an expanding body of evidence supporting the involvement of non-CFTR chloride channels in organellar acidification. We conclude that biologically significant involvement of CFTR in organellar acidification is unlikely.

In vivo role of CLC chloride channels in the kidney

2000 ◽  
Vol 279 (5) ◽  
pp. F802-F808 ◽  
Author(s):  
Shinichi Uchida
Keyword(s):  
Chloride Channel ◽  
Chloride Channels ◽  
Cellular Localization ◽  
Chloride Transport ◽  
Kidney Diseases ◽  
Cell Volume Regulation ◽  
Special Focus ◽  
Loss Of Function ◽  
Intracellular Vesicles

Chloride channels in the kidney are involved in important physiological functions such as cell volume regulation, acidification of intracellular vesicles, and transepithelial chloride transport. Among eight mammalian CLC chloride channels expressed in the kidney, three (CLC-K1, CLC-K2, and CLC-5) were identified to be related to kidney diseases in humans or mice. CLC-K1 mediates a transepithelial chloride transport in the thin ascending limb of Henle's loop and is essential for urinary concentrating mechanisms. CLC-K2 is a basolateral chloride channel in distal nephron segments and is necessary for chloride reabsorption. CLC-5 is a chloride channel in intracellular vesicles of proximal tubules and is involved in endocytosis. This review will cover the recent advances in research on the CLC chloride channels of the kidney with a special focus on the issues most necessary to understand their physiological roles in vivo, i.e., their intrarenal and cellular localization and their phenotypes of humans and mice that have their loss-of-function mutations.

Chloride channels and cystic fibrosis of the pancreas

1995 ◽  
Vol 15 (6) ◽  
pp. 531-541 ◽  
Author(s):  
M. A. Gray ◽  
J. P. Winpenny ◽  
B. Verdon ◽  
H. McAlroy ◽  
B. E. Argent
Keyword(s):  
Cystic Fibrosis ◽  
Chloride Channel ◽  
Chloride Channels ◽  
Secretory Activity ◽  
Caucasian Population ◽  
Pharmacological Intervention ◽  
Apical Plasma Membrane ◽  
Pancreatic Dysfunction ◽  
Strong Candidate ◽  
Cl Conductance

Cystic fibrosis (CF) affects approximately 1 in 2000 people making it one of the commonest fatal, inherited diseases in the Caucasian population. CF is caused by mutations in a cyclic AMP-regulated chloride channel known as CFTR, which is found on the apical plasma membrane of many exocrine epithelial cells. In the CF pancreas, dysfunction of the CFTR reduces the secretory activity of the tubular duct cells, which leads to blockage of the ductal system and eventual fibrosis of the whole gland. One possible approach to treating the disease would be to activate an alternative chloride channel capable of bypassing defective CFTR. A strong candidate for this is a chloride channel regulated by intracellular calcium, which has recently been shown to protect the pancreas in transgenic CF mice. Pharmacological intervention directed at activating this calcium-activated Cl− conductance might provide a possible therapy to treat the problems of pancreatic dysfunction in CF.

Chloride channels of intracellular organelles and their potential role in cystic fibrosis.

1992 ◽  
Vol 172 (1) ◽  
pp. 245-266 ◽  
Author(s):  
Q al-Awqati ◽  
J Barasch ◽  
D Landry
Keyword(s):  
Cystic Fibrosis ◽  
Fusion Protein ◽  
Cdna Library ◽  
Chloride Channel ◽  
Chloride Channels ◽  
Kidney Cortex ◽  
Affinity Column ◽  
Channel Activity ◽  
Planar Bilayers ◽  
Intracellular Organelles

Chloride channels were previously purified from bovine kidney cortex membranes using a drug affinity column. Reconstitution of the purified proteins into artificial liposomes and planar bilayers yielded chloride channels. A 64 x 10(3) M(r) protein, p64, identified as a component of this chloride channel, was used to generate antibodies which depleted solubilized kidney membranes of all chloride channel activity. This antibody has now been used to identify a clone, H2B, from a kidney cDNA library. Antibodies, affinity-purified against the fusion protein of H2B, from a kidney cDNA library. Antibodies, affinity-purified against the fusion protein of H2B, also depleted solubilized kidney cortex from all chloride channel activity. The predicted amino acid sequence of p64 shows that it contains two and possibly four putative transmembrane domains and potential phosphorylation sites by protein kinases A and C. There was no significant homology to other protein (or DNA) sequences in the data base including other anion channels or the cystic fibrosis transmembrane conductance regulator. The protein is expressed in all cells tested and probably represents the chloride channel of intracellular organelles. Cystic fibrosis (CF) is associated with a defect in a cyclic-AMP-activated chloride channel in secretory epithelia which leads to decreased fluid secretion. In addition, many mucus glycoproteins show decreased sialylation but increased sulfation. We have recently shown that the pH of intracellular organelles is more alkaline in CF cells, an abnormality that is due to defective chloride conductance in the vesicle membranes. We postulate that the defect in the intracellular chloride channel, and hence the alkalization, could explain the glycosylation abnormalities since the pH optimum of Golgi sialyltransferase is acid while that of focusyl- and sulfotransferases is alkaline. Defects in sialyation of glycolipids might also generate receptors for Pseudomonas, which is known to colonize the respiratory tract of CF patients.

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