Mucoviscidosis (Cystic Fibrosis), Molecular Cell Biology of
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Cystic fibrosis (CF) is the most common autosomal recessive disorder in Caucasians, affecting ∼1:2500 children, with a carrier frequency of 1:25. The causative gene, named CF transmembrane conductance regulator (CFTR), encodes a chloride channel in epithelial cells. Abnormal transport of chloride and sodium ions affects water movement across epithelia, leading to pathophysiological consequences in various organs including the respiratory, gastrointestinal and reproductive tract, the pancreas, and liver. The CF phenotype is rather heterogeneous due to many different mutations in CFTR and the influence of modifier genes. Chronic bacterial lung infections stimulate inflammatory defense mechanisms, leading to extensive tissue remodeling. The resulting emphysema and fibrosis mainly determine the reduced life expectancy in individuals with CF. Owing to improved symptomatic treatment strategies, including better nutrition and antibiotic therapy, the prognosis of CF individuals has considerably improved and many children now reach adult life. Research is focused on the development of pharmacological drugs correcting ion channel dysfunction, anti-inflammatory drugs and vaccines to prevent airway infections. The causative gene replacement therapy has not yet been successfully applied in CF patients.Keywords:
Chloride channel
Respiratory tract
Chloride channel
PDZ domain
Apical membrane
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Cystic fibrosis (CF) causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) lead to mislocalization of CFTR protein from the brush border membrane of epithelial tissues and/or its dysfunction as a chloride channel. In initial reports, it was proposed that certain channels from the ClC family of chloride channels may provide compensatory or alternative pathways for epithelial chloride secretion in tissues from cystic fibrosis patients. In the present work, we provide the first evidence that ClC-4 protein is functionally expressed on the surface of the intestinal epithelium and hence, is appropriately localized to act as a therapeutic target in this CF-affected tissue. We show using confocal and electron microscopy that ClC-4 co-localizes with CFTR in the brush border membrane of the epithelium lining intestinal crypts in mouse and human tissues. In Caco-2 cells, a cell line thought to model human enterocytes, ClC-4 protein is expressed on the cell surface and also partially co-localizes with EEA1 and transferrin, marker molecules of early and recycling endosomes, respectively. Hence, like CFTR, ClC-4 may cycle between the plasma membrane and endosomal compartment. Furthermore, we show that ClC-4 functions as a chloride channel on the surface of these epithelial cells as antisense ClC-4 cDNA expression reduced the amplitude of endogenous chloride currents by 50%. These studies provide the first evidence that ClC-4 is endogenously expressed and may be functional in the brush border membrane of enterocytes and hence should be considered as a candidate channel to provide an alternative pathway for chloride secretion in the gastrointestinal tract of CF patients. Cystic fibrosis (CF) causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) lead to mislocalization of CFTR protein from the brush border membrane of epithelial tissues and/or its dysfunction as a chloride channel. In initial reports, it was proposed that certain channels from the ClC family of chloride channels may provide compensatory or alternative pathways for epithelial chloride secretion in tissues from cystic fibrosis patients. In the present work, we provide the first evidence that ClC-4 protein is functionally expressed on the surface of the intestinal epithelium and hence, is appropriately localized to act as a therapeutic target in this CF-affected tissue. We show using confocal and electron microscopy that ClC-4 co-localizes with CFTR in the brush border membrane of the epithelium lining intestinal crypts in mouse and human tissues. In Caco-2 cells, a cell line thought to model human enterocytes, ClC-4 protein is expressed on the cell surface and also partially co-localizes with EEA1 and transferrin, marker molecules of early and recycling endosomes, respectively. Hence, like CFTR, ClC-4 may cycle between the plasma membrane and endosomal compartment. Furthermore, we show that ClC-4 functions as a chloride channel on the surface of these epithelial cells as antisense ClC-4 cDNA expression reduced the amplitude of endogenous chloride currents by 50%. These studies provide the first evidence that ClC-4 is endogenously expressed and may be functional in the brush border membrane of enterocytes and hence should be considered as a candidate channel to provide an alternative pathway for chloride secretion in the gastrointestinal tract of CF patients. cystric fibrosis cystic fibrosis transmembrane conductance regulator glutathione S-transferase reverse transcriptase phosphate-buffered saline green fluorescent protein The disease cystic fibrosis (CF)1 affects the epithelium lining multiple organs including the respiratory tract, the gastrointestinal tract, sweat ducts, and the reproductive organs (1Sheppard D.N. Welsh M.J. Physiol. Rev. 1999; 79: S23-45Crossref PubMed Scopus (813) Google Scholar). Normally, the protein product of the CF gene, the cystic fibrosis transmembrane conductance regulator (CFTR) resides on the apical surface of these epithelia. However, the most common disease causing mutation in CFTR (i.e. CFTRΔF508) promotes misfolding, leading to its mislocalization and degradation in intracellular compartments (2Cheng S.H. Gregory R.J. Marshall J. Paul S. Souza D.W. White G.A. O'Riordan C.R. Smith A.E. Cell. 1990; 63: 827-834Abstract Full Text PDF PubMed Scopus (1427) Google Scholar). Alternatively, other mutations lead to alterations in CFTR function as a phosphorylation and nucleotide-regulated anion channel (3Welsh M.J. Smith A.E. Cell. 1993; 73: 1251-1254Abstract Full Text PDF PubMed Scopus (1235) Google Scholar). The anion channel function of CFTR is thought to provide the primary driving force for fluid transport and clearance of mucus and bacteria and lack of this function is the major cause for mucus obstruction in CF-affected organs. As proposed initially by Clarkeet al. (4Clarke L.L. Grubb B.R. Yankaskas J.R. Cotton C.U. McKenzie A. Boucher R.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 479-483Crossref PubMed Scopus (312) Google Scholar), non-CFTR chloride channels could compensate for lack of CFTR in certain tissues if they were appropriately localized on the apical, brush border membrane of the epithelium and could be opened under physiological conditions. In fact, we recently reported that increased basal chloride secretion correlates with amelioration of disease severity in intestinal tissues of a subpopulation ofCftr-deficient mice (5Gyomorey K. Rozmahel R. Bear C.E. Pediatr. Res. 2000; 48: 731-734Crossref PubMed Scopus (17) Google Scholar). Therefore, there is a compelling rationale for studying the expression of other chloride channels that may mediate chloride secretion across the gastrointestinal epithelium as these proteins may provide a strategic therapeutic target for treatment of cystic fibrosis. There are nine mammalian members of the superfamily of voltage-gated ClC channels (6Jentsch T.J. Friedrich T. Schriever A. Yamada H. Pflugers Arch. 1999; 437: 783-795Crossref PubMed Scopus (297) Google Scholar). ClC-1, ClC-2, ClCKa, and ClCKb are closely related, ClC-3, ClC-4, and ClC-5 form another arm of the family and finally, ClC-6 and ClC-7 comprise a distinct branch. Some of these family members exhibit quite a restricted tissue expression and hence are unlikely to contribute to chloride transport across the epithelium of the gastrointestinal tract, i.e. ClC-1 is only expressed in muscle tissue and ClCKa and ClCKb are expressed exclusively in the kidney (7Waldegger S. Jentsch T.J. J. Am. Soc. Nephrol. 2000; 11: 1331-1339PubMed Google Scholar, 8Uchida S. Am. J. Physiol. Renal Physiol. 2000; 279: F802-808Crossref PubMed Google Scholar, 9Kieferle S. Fong P. Bens M. Vandewalle A. Jentsch T.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6943-6947Crossref PubMed Scopus (246) Google Scholar). On the other hand, ClC-2, ClC-3, ClC-4, ClC-5, ClC-6, and ClC-7 are relatively widely distributed. However, most of these family members, with the exception of ClC-2 and possibly ClC-3, are thought to be primarily expressed on intracellular membranes (6Jentsch T.J. Friedrich T. Schriever A. Yamada H. Pflugers Arch. 1999; 437: 783-795Crossref PubMed Scopus (297) Google Scholar, 10Vandewalle A. Cluzeaud F. Peng K.C. Bens M. Luchow A. Gunther W. Jentsch T.J. Am. J. Physiol. Cell Physiol. 2001; 280: C373-381Crossref PubMed Google Scholar,11Wang S.S. Devuyst O. Courtoy P.J. Wang X.T. Wang H. Wang Y. Thakker R.V. Guggino S. Guggino W.B. Hum. Mol. Genet. 2000; 9: 2937-2945Crossref PubMed Scopus (271) Google Scholar). We have recently reported that ClC-2 is endogenously expressed at a unique location in intestinal epithelia, in proximity to the tight junctions at the apical boundary between interacting differentiated enterocytes (12Gyomorey K. Yeger H. Ackerley C. Garami E. Bear C.E. Am. J. Physiol. Cell Physiol. 2000; 279: C1787-1794Crossref PubMed Google Scholar, 13Mohammad-Panah R. Gyomorey K. Rommens J. Choudhury M. Li C. Wang Y. Bear C.E. J. Biol. Chem. 2001; 276: 8306-8313Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Furthermore, we showed using an antisense strategy that endogenous ClC-2 channels in intestinal epithelial cells can mediate chloride flux. Hence, we proposed that ClC-2 contributes to chloride secretion across certain epithelia. These findings were substantiated in a recent report describing Clc-2 knockout mice, in which the chloride current across the retinal pigment epithelium was decreased in the Clc-2 knockout animals (14Bosl M.R. Stein V. Hubner C. Zdebik A.A. Jordt S.E. Mukhopadhyay A.K. Davidoff M.S. Holstein A.F. Jentsch T.J. EMBO J. 2001; 20: 1289-1299Crossref PubMed Scopus (261) Google Scholar). However, as the authors suggest, the impact of disrupting ClC-2 in intestinal tissue may only be apparent in Cftr-null animals. At present, the localization and physiological role of ClC-3 remains controversial as some studies suggest that ClC-3 functions in intracellular vesicles (6Jentsch T.J. Friedrich T. Schriever A. Yamada H. Pflugers Arch. 1999; 437: 783-795Crossref PubMed Scopus (297) Google Scholar, 15Friedrich T. Breiderhoff T. Jentsch T.J. J. Biol. Chem. 1999; 274: 896-902Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar) and others support a role for ClC-3 channels on the plasma membrane (16Duan D. Winter C. Cowley S. Hume J.R. Horowitz B. Nature. 1997; 390: 417-421Crossref PubMed Scopus (412) Google Scholar, 17Shimada K. Li X. Xu G. Nowak D.E. Showalter L.A. Weinman S.A. Am. J. Physiol. Gastrointest. Liver Physiol. 2000; 279: G268-276Crossref PubMed Google Scholar, 18Huang P. Liu J. Di A. Robinson N.C. Musch M.W. Kaetzel M.A. Nelson D.J. J. Biol. Chem. 2001; 276: 20093-20100Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). However, in our own immunolocalization studies of ClC-3 expression in the human intestinal cell line, Caco-2, we found that ClC-3 protein was predominantly expressed in intracellular vesicles (13Mohammad-Panah R. Gyomorey K. Rommens J. Choudhury M. Li C. Wang Y. Bear C.E. J. Biol. Chem. 2001; 276: 8306-8313Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Mutations in ClCN5 are associated with Dent's disease in humans, a kidney disease characterized by proteinuria and hypercalcuria (6Jentsch T.J. Friedrich T. Schriever A. Yamada H. Pflugers Arch. 1999; 437: 783-795Crossref PubMed Scopus (297) Google Scholar, 7Waldegger S. Jentsch T.J. J. Am. Soc. Nephrol. 2000; 11: 1331-1339PubMed Google Scholar, 19Devuyst O. Christie P.T. Courtoy P.J. Beauwens R. Thakker R.V. Hum. Mol. Genet. 1999; 8: 247-257Crossref PubMed Scopus (252) Google Scholar, 20Lloyd S.E. Gunther W. Pearce S.H. Thomson A. Bianchi M.L. Bosio M. Craig I.W. Fisher S.E. Scheinman S.J. Wrong O. Jentsch T.J. Thakker R.V. Hum. Mol. Genet. 1997; 6: 1233-1239Crossref PubMed Scopus (143) Google Scholar). Recently, examination of the native expression of ClC-5 in differentiated epithelial tissues revealed that this channel resides predominantly in intracellular membranes. In fact Clc-5 knockout mice predominantly show a defect in endocytosis of protein from lumen of the renal proximal tubule (11Wang S.S. Devuyst O. Courtoy P.J. Wang X.T. Wang H. Wang Y. Thakker R.V. Guggino S. Guggino W.B. Hum. Mol. Genet. 2000; 9: 2937-2945Crossref PubMed Scopus (271) Google Scholar). Similarily, Vandewalleet al. (10Vandewalle A. Cluzeaud F. Peng K.C. Bens M. Luchow A. Gunther W. Jentsch T.J. Am. J. Physiol. Cell Physiol. 2001; 280: C373-381Crossref PubMed Google Scholar) showed that ClC-5 protein localizes in intracellular compartments, i.e. endosomes and Golgi in the rat intestinal mucosa. Hence, in light of these immunohistochemical studies of native tissue, it seems unlikely that ClC-5 will contribute significantly to chloride currents across the apical membrane of the intestinal epithelium. ClC-4 message is known to be expressed in brain, skeletal muscle, heart, kidney (21Jentsch T.J. Gunther W. Pusch M. Schwappach B. J. Physiol. 1995; 482: 19S-25SCrossref PubMed Scopus (203) Google Scholar, 22Adler D.A. Rugarli E.I. Lingenfelter P.A. Tsuchiya K. Poslinski D. Liggitt H.D. Chapman V.M. Elliott R.W. Ballabio A. Disteche C.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9244-9248Crossref PubMed Scopus (63) Google Scholar, 23Van Slegtenhorst M.A. Bassi M.T. Borsani G. Wappenaar M.C. Ferrero G.B. Conciliis L. Rugali E.I. Grillo A. Franco B. Zoghbi H.Y. Ballabio A. Hum. Mol. Genet. 1994; 3: 547-552Crossref PubMed Scopus (102) Google Scholar), and intestine (24Gyomorey K. Garami E. Galley K. Rommens J. Bear C. Pflugers Arch. 2001; 443: S103-S106Crossref PubMed Scopus (25) Google Scholar), however, the subcellular distribution and function of ClC-4 protein in these tissues has yet to be determined. In our present work, we provide the first evidence that ClC-4 is endogenously expressed in the apical plasma membrane of murine and human enterocytes. Furthermore, we show using an antisense strategy, that endogenous ClC-4 protein mediates chloride currents across the plasma membrane of Caco-2 cells, cells that model human enterocytes. From the prospective of our current knowledge about the ClC family of chloride channels, these findings were unexpected. It was predicted that the subcellular distribution of ClC-4 would be similar to that of the related channel proteins, ClC-5 and ClC-3, and reside primarily in the membranes of intracellular organelles. To our knowledge, ClC-4 is the first chloride channel protein shown to co-localize with CFTR on the brush border membrane of intestinal crypt epithelial cells endogenously where it could mediate chloride currents into the gut lumen. Hence, these data strongly support a role for ClC-4 in intestinal chloride secretion and suggest that it may be capable of functionally complementing CFTR in vivo. Caco-2 cells were obtained from the American Type Culture Collection (Manassas, VA). They were grown in Earl's α-minimum essential medium (Wisent Inc., Montreal, Canada) containing 10% fetal calf serum, with 2 mm glutamine, 100 units of penicillin G, and 100 μg/ml streptomycin sulfate at 37 °C in an atmosphere of 5% CO2. For patch clamp studies, cells were used 1–2 days after plating onto 35-mm coverslips (Fisher). For analysis of ClC-4 localization in fully differentiated epithelia, Caco-2 cells were seeded on semipermeable filters (Corning Costar) and grown for 4 days past confluency, conditions previously reported to induced a differentiated monolayer (25Sood R. Bear C. Auerbach W. Reyes E. Jensen T. Kartner N. Riordan J.R. Buchwald M. EMBO J. 1992; 11: 2487-2494Crossref PubMed Scopus (61) Google Scholar). Caco-2 cell cultures at 40% confluency (undifferentiated cells) were transfected with antisense ClC-4 cDNA or vector control using Lipofectin (Invitrogen) and the recommended transfection protocol was followed as previously described (13Mohammad-Panah R. Gyomorey K. Rommens J. Choudhury M. Li C. Wang Y. Bear C.E. J. Biol. Chem. 2001; 276: 8306-8313Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Tissues were obtained from adult male rats (Wistar) fed a standard diet. Fragments (0.5 cm long) of ileum were removed, rapidly frozen in liquid nitrogen, and kept at −80 °C until used. Portions of a small human bowel biopsy deemed normal by a pathologist were used for electron microscopic studies. Expression of ClC-4 protein in mouse tissues and Caco-2 cells was determined by immunoblotting as described in our previous papers (12Gyomorey K. Yeger H. Ackerley C. Garami E. Bear C.E. Am. J. Physiol. Cell Physiol. 2000; 279: C1787-1794Crossref PubMed Google Scholar, 13Mohammad-Panah R. Gyomorey K. Rommens J. Choudhury M. Li C. Wang Y. Bear C.E. J. Biol. Chem. 2001; 276: 8306-8313Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). In brief, tissues from mouse were homogenized and then centrifuged at 2,000 × g to pellet the nuclei. The supernatant was further centrifuged at 100,000 ×g to yield a membrane pellet. Caco-2 cells were homogenized and centrifuged at 80,000 × g for 10 min at 4 °C to isolate a crude membrane preparation. 50 μg of each preparation was analyzed by SDS-polyacrylamide gel electrophoresis (8% gel) using anti-ClC-4 antibody at a concentration of 11.8 μg/ml. This polyclonal antibody was generated against a GST fusion peptide containing amino acids (1–52) of mouse ClC-4 (ClC-4 cDNA obtained from E. Rugarli, Milano, Italy). The antiserum was pre-adsorbed to a GST-coupled matrix to remove anti-GST antibodies. For the competition studies, the anti-ClC-4 antibody was preincubated with a 4.8-fold excess of the antigenic fusion peptide containing peptide from ClC-4, or a GST fusion protein containing residues 1–52 of hClC-5 overnight at 4 °C before incubation (hClC-5 cDNA kindly provided by T. Jentsch, Hamburg, Germany). Immunoreactive protein was detected using the ECL system (Amersham Bioscience, Inc.). Total RNA was isolated from Caco-2 cell monolayers by dissolution in guanidinium isothiocyanate and centrifugation through a CsCl cushion (26MacDonald R.J. Swift G.H. Przybyla A.E. Chirgwin J.M. Methods Enzymol. 1987; 152: 219-227Crossref PubMed Scopus (532) Google Scholar). Total RNA (5 μg) was analyzed on agarose gels (1%) containing 0.6m formaldehyde and transferred to Hybond-N membranes (Amersham Bioscience Inc.). Blots were cross-linked with UV radiation and hybridized with mouse-specific ClC-4 cDNA fragments radiolabeled by random priming (27Feinberg A.P. Vogelstein B. Anal. Biochem. 1983; 132: 6-13Crossref PubMed Scopus (16651) Google Scholar). Final conditions of washing included 0.2 × SSC (sodium chloride/sodium citrate) with 0.1% SDS at 60 °C. The blots were exposed to X-Omat film (Kodak) for 24–72 h at 0.1% SDS at 70 °C with on intensifying screen. Expression of ClC-4 was analyzed by reverse transcription-PCR, using the primers with following sequences: human ClC-4, sense (5′-TCCTCGATGAGCCGTTCCCTGATGT-3′) and antisense (5′-AGGATGTACATTAAGTAATTCAGA-3′), producing a PCR product of 402 bp. The sequence identity of the PCR products was confirmed by BLAST sequence data base search. The antisense murine ClC-4 was generated by cloning the ClC-4 open reading frame withBamHI (5′) and EcoRI (3′) into the eukaryotic vector pCDNA 3.1(−) (Promega, Madison, WI) such that the reversed restriction sites on this vector would reverse the orientation of the open reading frame to create the antisense plasmid. The murine ClC-4 sequence shares 87% sequence identity with the human sequence. Caco-2 cells were washed with PBS (phosphate-buffered saline), fixed with paraformaldehyde AM (4% in PBS), and permeabilized with 0.05% Triton X-100 in PBS. Blocking was done by using 5% normal goat serum in PBS for 1 h prior to primary antibody incubation. Primary antibodies were dissolved in blocking solution as follows. Mouse anti-endosomal early antigen 1 (1:200, Transduction Laboratories), mouse anti-giantin (1/1000, kind gift of H. P. Hauri, Basel, Switzerland), rabbit anti-ClC-3 (1/10, Alomone Labs Ltd., Jerusalem, Israel), rabbit anti-ClC-5 (1:150, kind gift of T. Jentsch, Hamburg, Germany), and rabbit anti-ClC-4 antibody (1/200). The cells were then incubated for 2.5 h at room temperature in primary antibody and then rinsed in PBS and incubated with Cy3-conjugated or fluorescein isothiocyanate-conjugated anti-rabbit or anti-mouse secondary antibodies (1:1000, Molecular Probes) and washed again before mounting. For double labeling experiments, two primary antibodies developed in different species were applied together, followed by the simultaneous detection using Cy3 and fluorescein isothiocyanate-coupled secondary antibodies. In some experiments we loaded Caco-2 cells for 1 h at 37 °C with 50 μg/ml transferrin conjugated to iron and tetramethylrhodamine (transferrin-Fe2+-Rhd, Molecular Probes) diluted in Earl's α-minimum essential medium without serum. Following fixation, cells we labeled with the ClC-4 antibody. For immunofluorescence labeling of rat tissues, cryosections (5 μm thick) of intestine were fixed in ice-cold methanol for 10 min prior to blocking. Samples were then processed for fluorescence as above. Slides were viewed with a ×63 objective on a Carl Zeiss LSM 510 equipped with an Axiovert 100 confocal microscopy. Portions of small bowel from a normal human biopsy, ileum from mice, and Caco-2 cells grown on semi-permeable filters were collected and prepared for freeze substitution and Lowicryl HM20 embedding (28Haagsman H.P. Elfring R.H. van Buel B.L. Voorhout W.F. Biochem. J. 1991; 275: 273-276Crossref PubMed Scopus (40) Google Scholar). The sections were incubated with the ClC-4 antibody diluted 1:100 in PBS, 0.5% bovine serum albumin. Sections were then labeled as previously described (12Gyomorey K. Yeger H. Ackerley C. Garami E. Bear C.E. Am. J. Physiol. Cell Physiol. 2000; 279: C1787-1794Crossref PubMed Google Scholar). Controls included the omission of either the primary or secondary antibody or a peptide competition. In double labeling experiments, sections of human small bowel were first labeled with the ClC-4 antibody and then with a monoclonal antibody against the R-domain of CFTR (1/100, R&D, Minneapolis, MN). Following ClC-4 labeling the sections were incubated for an hour in the CFTR antibody diluted 1:10 with PBS/bovine serum albumin. The sections were then labeled with goat anti-mouse IgG 5 nm complex, washed thoroughly with distilled water, and stained with uranyl acetate and lead citrate prior to examination in a JEOL JEM 1230 transmission electron microscope (JEOL USA Inc., Peabody, MA). Images were acquired with an AMT CCD digital camera (AMT Corp., Danvers, MA). Caco-2 cells were microinjected with plasmids at day 1 after plating on glass coverslips for patch clamp experiments as described (13Mohammad-Panah R. Gyomorey K. Rommens J. Choudhury M. Li C. Wang Y. Bear C.E. J. Biol. Chem. 2001; 276: 8306-8313Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Plasmids were diluted to a final concentration of 300 μg/ml for antisense ClC-4 and antisense ClC-2. Fluorescein isothiocyanate-labeled dextran (0.5%, Sigma) was also added to the injection medium to identify successfully microinjected cells. Caco-2 cell membrane currents were measured using conventional whole cell patch clamp technique as described (13Mohammad-Panah R. Gyomorey K. Rommens J. Choudhury M. Li C. Wang Y. Bear C.E. J. Biol. Chem. 2001; 276: 8306-8313Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Data were collected and analyzed with an Axopatch-200A amplifier and pCLAMP software (Axon Instruments, Foster City, CA). The bath solution contained (in mm): 140N-methyl-d-glutamine chloride, 2 MgCl2, 2 CaCl2, 5 HEPES, while the pipette solution contained (in mm) 140N-methyl-d-glutamine chloride, 2 MgCl2, 2 EGTA, and 5 HEPES. Both pipette and bath solutions were adjusted to pH 7.4. The tip resistance was 3–5 MΩ when filled with the pipette solution. Patch clamp measurements are presented as the mean ± S.E. Statistical analyses were performed using the Student's t test. Probabilities (p) of 0.05 or less were considered statistically significant. To assess ClC-4 protein expression, we raised a polyclonal antibody against a GST fusion protein containing residues 1–52 of the amino terminus of mouse ClC-4. As expected, given the abundant ClC-4 message expression in the rodent brain (22Adler D.A. Rugarli E.I. Lingenfelter P.A. Tsuchiya K. Poslinski D. Liggitt H.D. Chapman V.M. Elliott R.W. Ballabio A. Disteche C.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9244-9248Crossref PubMed Scopus (63) Google Scholar), our antibody recognized a broad, prominent signal in immunoblots of mouse brain lysate (Fig.1i, A). The predominant band corresponds to a protein of ∼90 kDa in molecular mass, close to the ClC-4 mass predicted from the primary sequence of 83 kDa. This signal is specific for ClC-4 as it is competed by the fusion peptide of the amino terminus of ClC-4 (residues 1–52) but not a fusion protein containing the amino terminus (residues 1–52) of the closely related channel protein, ClC-5 (Fig. 1i, B). Using this polyclonal antibody, we detected a prominent 97-kDa band in immunoblots of mouse ileum suggesting that ClC-4 protein is also endogenously expressed in intestinal tissue (Fig. 1ii). ClC-4 mRNA and protein could also be detected in the human intestinal epithelial cell line, Caco-2. ClC-4 mRNA in Caco-2 cells was detected as a 4.7-kb transcript by Northern analysis, using a mouse ClC-4 specific probe (Fig. 1iii, A). A smaller, as yet unidentified, transcript of less than 1.9 kb was also detected in Caco-2 cells. ClC-4 mRNA expression in Caco-2 cells was confirmed using RT-PCR analysis with sequence specific primers to human ClC-4 (Fig. 1iii, B). ClC-4 protein expression in Caco-2 cells was detected by immunoblotting using the polyclonal antibody described above. As in the immunoblots of mouse intestine, ClC-4 protein was detected as a predominant 97-kDa protein in Caco-2 cells, confirming that ClC-4 is also expressed in human intestinal epithelial cells (Fig. 1iii, C). The distribution of ClC-5 protein in rat intestinal tissue has been previously described by Vandewalle et al. (10Vandewalle A. Cluzeaud F. Peng K.C. Bens M. Luchow A. Gunther W. Jentsch T.J. Am. J. Physiol. Cell Physiol. 2001; 280: C373-381Crossref PubMed Google Scholar) using a ClC-5-specific polyclonal antibody originally characterized by Gunther et al. (29Gunther W. Luchow A. Cluzeaud F. Vandewalle A. Jentsch T.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8075-8080Crossref PubMed Scopus (385) Google Scholar). We also observed that ClC-5 specific staining was primarily perinuclear in cross-sections of intestinal crypts, showing a Golgi-like pattern of distribution (Fig. 2i,A). ClC-5 specific signal also appeared to be localized close to the luminal membrane of the intestinal crypt. It was previously reported that this juxtaluminal membrane distribution of ClC-5 may be localized in early endosomes (10Vandewalle A. Cluzeaud F. Peng K.C. Bens M. Luchow A. Gunther W. Jentsch T.J. Am. J. Physiol. Cell Physiol. 2001; 280: C373-381Crossref PubMed Google Scholar). Therefore, we processed cryosections of rat ileum for double immunofluorescence labeling using the polyclonal ClC-5 antibody and a monoclonal antibody against EEA1, one of the most specific early endosomal markers (30Wilson J.M. de Hoop M. Zorzi N. Toh B.H. Dotti C.G. Parton R.G. Mol. Biol. Cell. 2000; 11: 2657-2671Crossref PubMed Scopus (153) Google Scholar) (Fig. 2i, A, middle, green). The immunolabeled ClC-5 protein appeared to co-localize with immunolabeled EEA1 protein in a distinct ring around the apical membrane (Fig. 2i, A, right, yellow). These results provide a benchmark for our comparative studies of ClC-4 distribution in this tissue. Overall, the pattern observed for ClC-4-specific staining of the intestinal mucosa is different from that observed for ClC-5. Cross-sections of intestinal crypts were most intensely labeled around the luminal membrane of the gland suggesting the presence of ClC-4 in the apical membrane and/or subapical membrane compartment (Fig.2i, B, left, red). The apical or luminal membrane of the crypt can be readily discerned by differential interference contrast microscopy (Fig. 2i, B, middle and right). To determine if ClC-4 protein is located in early endosomes, we performed a double labeling experiment, wherein, intestinal sections were labeled with both our ClC-4 antibody and the EEA1 monoclonal antibody described above (Fig. 2i, C, middle, green). These images show that the two patterns partially overlap (Fig. 2i, C, right, yellow), suggesting that ClC-4 protein, like ClC-5 is (at least transiently) localized in early endosomes. Furthermore, double labeling of intestinal crypts with ClC-4 antibody (red) and a Golgi marker (giantin, green) clearly shows the lack of overlap and the definite apical polarization of ClC-4 (Fig. 2i, D). Our immunofluorescence studies on rat intestinal tissue suggest that ClC-4 is localized in proximity to the apical membrane of intestinal epithelial cells. The confocal images do not have the resolution necessary to determine whether ClC-4 protein is inserted in the apical plasma membrane. Therefore, we examined ultra-thin sections of freeze-substituted mouse and human intestinal tissues labeled with ClC-4 antibody (described above) by electron microscopy. Fig.2ii, A, shows that in mouse tissue, immunogold-labeled ClC-4 is localized primarily along the brush border membrane and the apical cytoplasm of an absorptive cell from the crypt of the ileum. In adjacent goblet cells, ClC-4 labeling was found predominantly in clusters in the apical cytoplasm (Fig. 2ii, B). Double labeling of a section of human small bowel with gold-labeled ClC-4 antibody (large grains, arrows) and CFTR antibody (small gold grains) shows that both of these proteins co-localize primarily on the brush border membrane of human enterocytes (Fig. 2ii, C). ClC-4 labeling intensity was also detected in the apical cytoplasm and membrane of goblet cells in human tissue (Fig. 2ii, D). The ClC-4 labeling pattern in mouse enterocytes (Fig.2iii, A) and goblet cells (Fig. 2iii, B) as well as in human enterocytes (Figs. 2iii, C) and goblet cells (Fig. 2iii, D) was effectively competed with the ClC-4 fusion protein against which the ClC-4 antibody was raised, supporting its specificity. Clearly, as ClC-4 was found in the apical membrane of the absorptive cells in close proximity to CFTR, it was next necessary to determine whether ClC-4 is functional as a chloride channel in the plasma membrane of enterocytes. We suggest that the Caco-2 cell line is an effective model for analysis of ClC-4 function in intestinal epithelia, as ClC-4 localization in confluent Caco-2 monolayers is identical to that observed in the native epithelia.
Chloride channel
Brush border
Apical membrane
Transport protein
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Cystic fibrosis transmembrane conductance regulator (CFTR) is an important chloride ion channel. Some researches indicate CFTR contains five domains:two membrane-spanning domains (MSDs) forming chloride ion channel and two nucleotide-binding domains (NBDs) regulating channal open or close and one regulatory domain regulating channel activity.All these domains cooperated to regulate chloride ion transport. Mutation of CFTR which leads to cystic fibrosis will influence cell function.This review gives an outline of CFTR chloride channel and the channel-related disease,cystic fibrosis.
Chloride channel
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The cystic fibrosis transmembrane conductance regulator (CFTR), which forms adenosine 3',5'-monophosphate (cAMP)-regulated chloride channels, is defective in patients with cystic fibrosis. This protein contains two putative nucleotide binding domains (NBD1 and NBD2) and an R domain. CFTR in which the R domain was deleted (CFTR delta R) conducted chloride independently of the presence of cAMP. However, sites within CFTR other than those deleted also respond to cAMP, because the chloride current of CFTR delta R increased further in response to cAMP stimulation. In addition, deletion of the R domain suppressed the inactivating effect of a mutation in NBD2 (but not NBD1), a result which suggests that NBD2 interacts with the channel through the R domain.
Chloride channel
Cyclic nucleotide-binding domain
Cyclic adenosine monophosphate
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This study is to investigate the activation effect of butyl-p-hydroxybenzoate (Bpb) on cAMP-dependent cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel gating. A stably transfected Fischer rat thyroid (FRT) epithelial cell lines co-expressing human CFTR and a green fluorescent protein mutant with ultra-high halide sensitivity (EYFP) were used to measure CFTR-mediated iodide influx rates. Bpb was identified as an effective activator of wild-type CFTR chloride channel, it can correct delta F508-CFTR gating defects but not processing defect. Bpb can't potentiate G551D-CFTR channel gating. The activity was reversible and dose-dependent. The study also provided clues that Bpb activates CFTR chloride channel through a direct binding mechanism. Our study identified Bpb as a novel structure CFTR activator. Bpb may be useful for probing CFTR channel gating mechanisms and as a lead compound to develop pharmacological therapy for CFTR-related disease.
Chloride channel
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Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a plasma membrane-localized chloride channel. Some mutations in CFTR, including one which affects most patients (ΔF508-CFTR), prevent CFTR from exiting the endoplasmic reticulum (ER) where it is synthesized. To examine whether normal and mutant CFTRs function as chloride channels when they reside in the ER, the patch clamp technique was used to measure currents in the outer membrane of nuclei isolated from mammalian cells expressing CFTR. Both ΔF508-CFTR as well as CFTR were revealed to function as cAMP-regulated chloride channels in native ER membrane. These results represent the first demonstrations of functional activity of CFTR in the biosynthetic pathway and suggest that conformational changes in the mutant protein, although recognized by ER-retention mechanisms, do not necessarily affect CFTR chloride channel properties, which may have implications for pathophysiology and therapeutic interventions in cystic fibrosis. Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a plasma membrane-localized chloride channel. Some mutations in CFTR, including one which affects most patients (ΔF508-CFTR), prevent CFTR from exiting the endoplasmic reticulum (ER) where it is synthesized. To examine whether normal and mutant CFTRs function as chloride channels when they reside in the ER, the patch clamp technique was used to measure currents in the outer membrane of nuclei isolated from mammalian cells expressing CFTR. Both ΔF508-CFTR as well as CFTR were revealed to function as cAMP-regulated chloride channels in native ER membrane. These results represent the first demonstrations of functional activity of CFTR in the biosynthetic pathway and suggest that conformational changes in the mutant protein, although recognized by ER-retention mechanisms, do not necessarily affect CFTR chloride channel properties, which may have implications for pathophysiology and therapeutic interventions in cystic fibrosis.
Chloride channel
ΔF508
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SUMMARY 1. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) result in the primary defect observed in patients with cystic fibrosis. 2. The CFTR is a member of the ATPase‐binding cassette (ABC) transporter family but, unlike other members of this group, CFTR conducts a chloride current that is activated by cAMP. 3. In epithelial cells, the cAMP‐stimulated chloride current is conducted by both CFTR and the outwardly rectifying chloride channel (ORCC). 4. The present review summarizes the current knowledge of the properties of the two channels, as well as their relationship. Because the gene encoding the ORCC has not been identified, a discussion as to possible candidates for this chloride channel is included.
Chloride channel
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Chloride channel
Vas deferens
Loss function
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Chloride channel
Epithelial sodium channel
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Chloride channels are widely expressed and play important roles in cell volume regulation, transepithelial transport, intracellular pH regulation, and membrane excitability. Most chloride channels have yet to be identified at a molecular level. The ClC gene family and the cystic fibrosis transmembrane conductance regulator (CFTR) are distinct chloride channels expressed in many cell types, and mutations in their genes are the cause of several diseases including myotonias, cystic fibrosis, and kidney stones. Because of their molecular definition and roles in disease, these channels have been studied intensively over the past several years. The focus of this review is on recent studies that have provided new insights into the mechanisms governing the opening and closing, i.e. gating, of the ClC and CFTR chloride channels.
Chloride channel
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