The Chloride Channel ClC-4 Contributes to Endosomal Acidification and Trafficking
2003; Elsevier BV; Volume: 278; Issue: 31 Linguagem: Inglês
10.1074/jbc.m304357200
ISSN1083-351X
AutoresRaha Mohammad-Panah, Rene E. Harrison, Sonja U. Dhani, Cameron Ackerley, Ling‐Jun Huan, Yanchun Wang, Christine E. Bear,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoMutations in the gene coding for the chloride channel ClC-5 cause Dent's disease, a disease associated with proteinuria and renal stones. Studies in ClC-5 knockout mice suggest that this phenotype is related to defective endocytosis of low molecular weight proteins and membrane proteins by the renal proximal tubule. In this study, confocal micrographs of proximal tubules and cultured epithelial cells revealed that the related protein ClC-4 is expressed in endosomal membranes suggesting that this channel may also contribute to the function of this organelle. In support of this hypothesis, specific disruption of endogenous ClC-4 expression by transfection of ClC-4 antisense cDNA acidified endosomal pH and altered transferrin trafficking in cultured epithelial cells to the same extent as the specific disruption of ClC-5. Both channels can be co-immunoprecipitated, arguing that they may partially contribute to endosomal function as a channel complex. These studies prompt future investigation of the role of ClC-4 in renal function in health and in Dent's disease. Future studies will assess whether the severity of Dent's disease relates not only to the impact of particular mutations on ClC-5 but also on the consequences of those mutations on the functional expression of ClC-4. Mutations in the gene coding for the chloride channel ClC-5 cause Dent's disease, a disease associated with proteinuria and renal stones. Studies in ClC-5 knockout mice suggest that this phenotype is related to defective endocytosis of low molecular weight proteins and membrane proteins by the renal proximal tubule. In this study, confocal micrographs of proximal tubules and cultured epithelial cells revealed that the related protein ClC-4 is expressed in endosomal membranes suggesting that this channel may also contribute to the function of this organelle. In support of this hypothesis, specific disruption of endogenous ClC-4 expression by transfection of ClC-4 antisense cDNA acidified endosomal pH and altered transferrin trafficking in cultured epithelial cells to the same extent as the specific disruption of ClC-5. Both channels can be co-immunoprecipitated, arguing that they may partially contribute to endosomal function as a channel complex. These studies prompt future investigation of the role of ClC-4 in renal function in health and in Dent's disease. Future studies will assess whether the severity of Dent's disease relates not only to the impact of particular mutations on ClC-5 but also on the consequences of those mutations on the functional expression of ClC-4. There are nine members of the ClC family of chloride channels in mammals, and several members have been implicated in congenital diseases. For example, mutations in ClCN1 cause congenital myotonia (1Steinmeyer K. Klocke R. Ortland C. Gronemeier M. Jockusch H. Grunder S. 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Genet. 2003; 4: 527-532Crossref Scopus (300) Google Scholar); ClCN5 is mutated in Dent's disease, a renal disease characterized by low molecular weight proteinuria, hypercalciuria, and in some severe cases renal failure (6Lloyd 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 (142) Google Scholar, 7Jentsch T.J. Friedrich T. Schriever A. Yamada H. Pfluegers Arch. 1999; 437: 783-795Crossref PubMed Scopus (294) Google Scholar, 8Devuyst O. Christie P.T. Courtoy P.J. Beauwens R. Thakker R.V. Hum. Mol. Genet. 1999; 8: 247-257Crossref PubMed Scopus (251) Google Scholar, 9Waldegger S. Jentsch T.J. J. Am. Soc. Nephrol. 2000; 11: 1331-1339PubMed Google Scholar, 10Yu A.S. Curr. Opin. Nephrol. Hypertens. 2001; 10: 415-420Crossref PubMed Scopus (17) Google Scholar, 11Sasaki Y. Nagai J. Kitahara Y. Takai N. Murakami T. Takano M. Biochem. Biophys. Res. Commun. 2001; 282: 212-218Crossref PubMed Scopus (23) Google Scholar); and mutations in ClCN7 are associated with osteopetrosis (12Kornak U. Kasper D. Bosl M.R. Kaiser E. Schweizer M. Schulz A. Friedrich W. Delling G. Jentsch T.J. Cell. 2001; 104: 205-215Abstract Full Text Full Text PDF PubMed Scopus (813) Google Scholar). Each of these particular chloride channels belongs to distinct subgroups of the ClC family, defined on the basis of their degree of sequence similarity. ClC-1 is grouped with ClC-2, ClC-Ka, and ClC-Kb (7Jentsch T.J. Friedrich T. Schriever A. Yamada H. Pfluegers Arch. 1999; 437: 783-795Crossref PubMed Scopus (294) Google Scholar). ClC-7 is most closely related to ClC-6 (7Jentsch T.J. Friedrich T. Schriever A. Yamada H. Pfluegers Arch. 1999; 437: 783-795Crossref PubMed Scopus (294) Google Scholar). ClC-5, ClC-4, and ClC-3 form a distinct subgroup, sharing close to 80% sequence identity. The subgroup of ClC channels, including ClC-3, ClC-4, and ClC-5, is thought to function in intracellular compartments. For example, ClC-3 is localized in synaptic vesicles in neurons where it has been shown to contribute to vesicular acidification probably by providing an electrical shunt permissive to V-type ATPase activity (13Stobrawa S.M. Breiderhoff T. Takamori S. Engel D. Schweizer M. Zdebik A.A. Bosl M.R. Ruether K. Jahn H. Draguhn A. Jahn R. Jentsch T.J. Neuron. 2001; 29: 185-196Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar). In non-neuronal tissue, the shorter isoform (ClC-3A) has been localized to late endosomes and lysosomes where it is presumed to function in regulating the pH of these compartments (14Gentzsch M. Cui L. Mengos A. Chang X.B. Chen J.H. Riordan J.R. J. Biol. Chem. 2003; 278: 6440-6449Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 15Li X. Wang T. Zhao Z. Weinman S.A. Am. J. Physiol. 2002; 282: C1483-C1491Crossref PubMed Scopus (117) Google Scholar). The function of the longer Golgi-localized isoform (ClC-3B) has yet to be determined (14Gentzsch M. Cui L. Mengos A. Chang X.B. Chen J.H. Riordan J.R. J. Biol. Chem. 2003; 278: 6440-6449Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). ClC-5 is localized primarily in early endosomes in native tissues of rodent renal proximal tubules and in various heterologous expression systems (16Gunther 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, 17Schwake M. Friedrich T. Jentsch T.J. J. Biol. Chem. 2001; 276: 12049-12054Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Disruption of Clcn5 in mice leads to defective fluid phase and receptor-mediated endocytosis by the renal proximal tubule, arguing that ClC-5 contributes to endocytosis in vivo (16Gunther 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, 18Gunther W. Piwon N. Jentsch T.J. Pfluegers Arch. 2003; 445: 456-462Crossref PubMed Scopus (164) Google Scholar, 19Piwon N. Gunther W. Schwake M. Bosl M.R. Jentsch T.J. Nature. 2000; 408: 369-373Crossref PubMed Scopus (483) Google Scholar, 20Wang 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 (272) Google Scholar). On the basis of these studies, it has been hypothesized that ClCN5 mutations may lead to low molecular weight proteinuria and hypercalciuria in patients with Dent's disease because of defective internalization of protein and membrane receptors from the apical surface of the renal proximal tubule (19Piwon N. Gunther W. Schwake M. Bosl M.R. Jentsch T.J. Nature. 2000; 408: 369-373Crossref PubMed Scopus (483) Google Scholar). Furthermore, it was proposed that ClC-5 normally contributes to endocytosis by facilitating endosomal acidification, a phenomenon essential for appropriate vesicular trafficking (21van Weert A.W. Dunn K.W. Gueze H.J. Maxfield F.R. Stoorvogel W. J. Cell Biol. 1995; 130: 821-834Crossref PubMed Scopus (299) Google Scholar, 22Presley J.F. Mayor S. McGraw T.E. Dunn K.W. Maxfield F.R. J. Biol. Chem. 1997; 272: 13929-13936Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 23Marshansky V. Ausiello D.A. Brown D. Curr. Opin. Nephrol. Hypertens. 2002; 11: 527-537Crossref PubMed Scopus (91) Google Scholar). As for ClC-3A, it has been suggested that ClC-5 may provide an electrical shunt for charge dissipation, thereby permitting endosomal acidification through the action of V-type ATPases (15Li X. Wang T. Zhao Z. Weinman S.A. Am. J. Physiol. 2002; 282: C1483-C1491Crossref PubMed Scopus (117) Google Scholar, 24Jentsch T.J. Stein V. Weinreich F. Zdebik A.A. Physiol. Rev. 2002; 82: 503-568Crossref PubMed Scopus (1061) Google Scholar). In fact, endosomal vesicles purified from Clcn5 knockout mice exhibited slower rates of acidification than vesicles purified from their wild type siblings (18Gunther W. Piwon N. Jentsch T.J. Pfluegers Arch. 2003; 445: 456-462Crossref PubMed Scopus (164) Google Scholar). However, a direct contribution of ClC-5 to this function in vivo has not yet been demonstrated. ClC-4 shares 78% sequence identity with ClC-5, and these two channels exhibit almost identical channel properties when studied in heterologous expression systems (25Friedrich T. Breiderhoff T. Jentsch T.J. J. Biol. Chem. 1999; 274: 896-902Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). However, unlike ClC-5, very little is known about the native expression and function of ClC-4 in epithelial cells. Recently, we generated a specific antibody against ClC-4, and we showed that in rodent and human intestinal epithelia, ClC-4 channels co-localize with the cystic fibrosis transmembrane conductance regulator in the apical membrane and in subapical vesicles (26Mohammad-Panah R. Ackerley C. Rommens J. Choudhury M. Wang Y. Bear C.E. J. Biol. Chem. 2002; 277: 566-574Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Significantly, a proportion of the intracellular vesicles bearing ClC-4 appeared to co-localize with the endosomal marker EEA1 (26Mohammad-Panah R. Ackerley C. Rommens J. Choudhury M. Wang Y. Bear C.E. J. Biol. Chem. 2002; 277: 566-574Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), raising the possibility that ClC-4 may participate with ClC-5 in regulating the function of this organelle. The primary goal of the present project was to assess the role of ClC-4, relative to ClC-5, in endosomal trafficking in epithelial cells and to determine the mechanism underlying this putative function. We show for the first time that the specific depletion of ClC-4 reduces the rate of transferrin receptor recycling and that this reduction is associated with a defect in endosomal acidification. Hence, normal endosomal trafficking in epithelial cells may require the functional expression of ClC-4 as well as ClC-5. Constructs—The antisense murine ClC-4 was generated as described previously (26Mohammad-Panah R. Ackerley C. Rommens J. Choudhury M. Wang Y. Bear C.E. J. Biol. Chem. 2002; 277: 566-574Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Similarly, the antisense human cDNA ClC-5 (ClC-5 cDNA was a gift from Dr. T. J. Jentsch) was generated by cloning the ClC-5 open reading frame with BamHI (5′) and EcoRI (3′) linkers on the forward and reverse primers, respectively. As described previously, the resulting PCR fragment was subcloned into pcDNA3.1 (–) to create the antisense plasmid. The HA 1The abbreviations used are: HA, hemagglutinin; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; Tfn, transferrin; GST, glutathione S-transferase; PBS, phosphate-buffered saline; BSA, bovine serum albumin; CHO, Chinese hamster ovary; Rhd, rhodamine. tag was inserted onto the amino terminus of hClC-5 with the help of BamHI-ATG-HA-hClC-5 oligonucleotide (+) and hClC-5 EcoRIstop (–)oligonucleotides. The subsequent PCR product was then subcloned into pCDNA 3.1 (+) with the BamHI and EcoRI linkers for eukaryotic expression. Pfu enzyme (Stratagene) was used for PCR, and sequencing was done with three different oligonucleotides that spanned the hClC-5 open reading frame. Tissues—Kidney tissues were obtained from adult male rats (Wistar) fed a standard diet. For immunofluorescence staining, we infused fluorescein isothiocyanate (FITC)-labeled dextran, 10 kDa (1.75 mg/100 g body weight; Molecular Probes, Leiden, The Netherlands), dissolved in 0.5 ml of 0.9% NaCl, into the penile vein of a rat over a period of 30 s as described previously (27Obermuller N. Kranzlin B. Blum W.F. Gretz N. Witzgall R. Am. J. Physiol. 2001; 280: F244-F253PubMed Google Scholar). Eight minutes after the injection of FITC-dextran, the infrarenal abdominal aorta was cannulated, and the rats were subjected to perfusion-fixation for subsequent visualization of FITC-dextran. Rats were perfused with 2% paraformaldehyde in phosphate-buffered saline (PBS) for 3 min and subsequently with a 18% sucrose solution in PBS for another 3 min. Kidneys were removed and cut along the longitudinal axis and incubated in a 18% sucrose/PBS solution, containing 0.02% sodium azide for 24 h at 4 °C. Tissues were then frozen in liquid nitrogen and stored at –80 °C until use. Immunoblotting—Expression of ClC-4 and ClC-5 protein in Caco-2, CHO cells, and rat tissue was determined by immunoblotting as described previously (28Mohammad-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, 29Gyomorey K. Rozmahel R. Bear C.E. Pediatr. Res. 2000; 48: 731-734Crossref PubMed Scopus (17) Google Scholar). The anti-ClC-4 polyclonal antibody was generated against a GST fusion peptide containing amino acid residues 1–52 of mouse ClC-4. The antiserum was pre-absorbed to a GST-coupled matrix to remove anti-GST antibodies. The signal recognized by anti-ClC-4 antibody is specific for ClC-4 as it is competed by the fusion peptide of the amino terminus of ClC-4 but not a fusion peptide containing the amino terminus of the closely related channel protein ClC-5 (26Mohammad-Panah R. Ackerley C. Rommens J. Choudhury M. Wang Y. Bear C.E. J. Biol. Chem. 2002; 277: 566-574Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Furthermore, immunoreactive protein, migrating as a 90-kDa polypeptide in Western blots, is absent in immunoblots of tissue obtained from Clcn4 null mice. The anti-ClC-5 polyclonal antibody (a gift of Dr. T. Jentsch, Hamburg, Germany) was generated against a synthetic peptide (CKSRDRDRHREITNKS) representing a part of the amino terminus of ClC-5. This antibody was originally characterized and shown to be specific by Gunther et al. (16Gunther 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). Immunoreactive protein was detected using the ECL system (Amersham Biosciences). Culture and Transfection of Cells—Caco-2, CHO, and LLCPK1 cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were transfected using Lipofectin (Invitrogen), as described previously (26Mohammad-Panah R. Ackerley C. Rommens J. Choudhury M. Wang Y. Bear C.E. J. Biol. Chem. 2002; 277: 566-574Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). For immunofluorescence studies, cells were used 1 day following transfection. Transferrin-Biotin Endocytosis Assay—Receptor-mediated endocytosis was measured by assaying the uptake of biotinylated human transferrin (Molecular Probes, Leislen, The Netherlands) by Caco2 cells. Cells were seeded onto 55-mm Petri dishes and grown to 50% confluency prior to transfection. 42 h post-transfection, cells were first serum-starved for 1 h and then incubated in serum-free media supplemented with 50 μg/ml biotinylated-transferrin for 20 min at 37 °C. Cells were then washed twice with cold PBS, pH 4.5, to remove surface-associated transferrin-biotin. Cells were lysed using SDS sample buffer and subjected to SDS-PAGE analysis. Internalized transferrin was detected using Extravidin (Sigma) and quantitated by band densitometry analysis using ImageJ software. Co-immunoprecipitations—CHO cells and kidney tissues were lysed in modified RIPA buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 0.05% SDS, 1% Triton X-100, and protease inhibitors: 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mm benzamidine, E64 10 μm, and 2 mm dithiothreitol). Antibodies against ClC-4 or mouse influenza hemagglutinin (HA, Babco, Richmond, CA) were added to the supernatant, and the mixtures were incubated at 4 °C overnight. Protein G-Sepharose (Amersham Biosciences) beads were incubated with the antigen/antibody mixture at 4 °C for 2 h. Protein-antibody-bead complexes were washed with detergent-free RIPA buffer. The final washed pellet were eluted with the Laemmli buffer at 65 °C for 15 min. Eluant was subjected to 10 mm tris(2-carboxyethyl)phosphine treatment to reduce antibody disulfide bands and analyzed for the presence of ClC-4 and ClC-5 proteins. Patch Clamp Electrophysiology—Caco-2 cell membrane currents were measured using conventional whole cell patch clamp technique as described (26Mohammad-Panah R. Ackerley C. Rommens J. Choudhury M. Wang Y. Bear C.E. J. Biol. Chem. 2002; 277: 566-574Abstract Full Text Full Text PDF PubMed Scopus (49) 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): 140 N-methyl-d-glutamine chloride, 2 MgCl2, 2 CaCl2, 5 HEPES, whereas the pipette solution contained (in mm) 140 N-methyl-d-glutamine-Cl, 2 MgCl2, 2 EGTA, and 5 HEPES. Both pipette and bath solutions were adjusted to pH 7.4. The tip resistance was 3–5 megohms when filled with the pipette solution. Endosomal pH measurements—Endosomal pH was determined using fluorescence ratio imaging as described (30Hackam D.J. Rotstein O.D. Zhang W.J. Demaurex N. Woodside M. Tsai O. Grinstein S. J. Biol. Chem. 1997; 272: 29810-29820Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 31Jankowski A. Scott C.C. Grinstein S. J. Biol. Chem. 2002; 277: 6059-6066Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Caco cells plated on coverslips were co-transfected with enhanced blue fluorescence protein and either vector control or ClC-4 or ClC-5 antisense and serum-starved overnight. Endosomes were loaded with 150 μg/ml FITC-transferrin in sodium-rich saline (140 mm NaCl, 5 mm KCl, 5 mm glucose, 1 mm CaCl2, 1 mm MgCl2, 10 mm HEPES, pH 7.4) for 30 min. Coverslips were washed and pulsed briefly with PBS (pH 5.0) to remove surface-bound FITC-transferrin before transferring to Leiden chambers for analysis. Transfected cells were identified using UV filters using a ×40 oil-immersion objective on a thermostatted Leiden holder of a Zeiss IM-35 microscope. Internalized FITC-transferrin was then excited at alternating wavelengths of 490 (700 ms) and 440 nm (100 ms) using a Sutter filter wheel. The fluorescent light was directed to a 535-nm emission filter placed before a cooled CCD camera used for fluorescent detection using 8 × 8 binning. Image acquisition was controlled using the Metafluor software (Universal Imaging Corp.). Images were captured at 2-min intervals. The fluorescence ratio versus pH was calibrated by equilibrating the cells in K+-rich medium (140 mm KCl, 10 mm NaCl, 5 mm glucose, 1 mm CaCl2, 1 mm MgCl2, 10 mm HEPES, pH 7.4) adjusted to varying pH values (ranging from 7.3 to 5.7) containing 10 μm of nigericin and monensin. Endosomes were defined as regions of interest, and pixel intensity values were determined for resting endosomes and defined calibration pH acquisitions following background subtraction for both 490 (pH-dependent) and 440 nm (pH-independent). Calibration curves of the fluorescence ratio (490:440) were then plotted against pH, and endosomal pH values were extrapolated from the curve. Immunofluorescence—Immunofluorescence labeling was performed on 5-μm cryosections of rat kidney tissues and on Caco-2, CHO, COS-7, and LLCPK1 cells as described previously (28Mohammad-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). The following primary antibodies were used: rabbit anti-ClC-5 (1:150), rabbit anti-ClC-4 antibody (1:200), and mouse HA antibody (1:1000, Babco, Richmond, CA). Cy3- or Cy5-conjugated or FITC-conjugated anti-rabbit or anti-mouse secondary antibodies (1:1000, Molecular Probes, Leiden, The Netherlands) were used for immunodetection. To study the localization of ClC-4 and ClC-5 proteins relative to transferrin-positive compartments, cells were serum-starved for 1 h at 37 °C in free serum medium (Invitrogen) and then loaded with 50 μg/ml transferrin conjugated to iron and tetramethylrhodamine (transferrin-Fe2+-Rhd, Molecular Probes, Leiden, The Netherlands) for 60 min. Following fixation, cells were labeled with ClC-4 or ClC-5 antibodies for subsequent immunolocalization. For kinetic analyses of transferrin internalization in control and ClC-4- and ClC-5-depleted (antisense-treated) cells, cells were serum-starved as above and then pulse-labeled for 2.5, 5, 10, 20, 40, and 60 min with 50 μg/ml transferrin-Fe2+-Rhd. To examine recycling, cells were pulse-labeled with 50 μg/ml transferrin-Fe2+-Rhd for 1 h and then washed and chase for 5, 20, 40, 60, and 80 min in medium containing 10% fetal calf serum. At the end of each labeling and chase interval, cells were washed and then fixed in cold 4% paraformaldehyde. Slides were viewed with a ×100 objective on a Carl Zeiss LSM 510 equipped with an Axiovert 100 confocal microscope. Quantification of Immunofluorescence—GFP-transfected cells were analyzed with respect to ClC channel-specific immunofluorescence (labeled with Cy3-conjugated secondary antibodies) or rhodamine-transferrin. Although cross-talk of the fluorophores into the wrong detectors was negligible, GFP fluorescence was specifically subtracted prior to Cy3 or rhodamine immunofluorescence measurements. All images were acquired using the palette function of LSM510 confocal acquisition software, which allows verification that the fluorescence intensity is in the linear range. The linear range is defined as 0 units = white and 255 units = black. The Cy3 or rhodamine-specific immunofluorescence was converted to a gray scale image, and the background (determined from the mean intensity detected in cell nuclei) was subtracted prior to quantitation. Mean pixel intensity of the gray scale image was measured (Scion Image software) for the outlined region of the whole cell or for the perinuclear region (by superpositioning a box at 8 locations around the nucleus). Data were plotted, and the half-times for transferrin internalization or recycling were determined by fitting logarithmic or monoexponential decay functions, respectively, to data acquired at different time points using Origin software (Microcal Origin). Electron Microscopy—CHO cells co-transfected with both Rab5a-GFP and HA-ClC-5 cDNA were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 m phosphate buffer, pH 7.4. They were then harvested with a rubber policeman, drawn off the culture dish, and lightly centrifuged into a mm3 pellet in a microcentrifuge tube. They were fixed for an additional 4 h and washed in PBS containing 20 mm azide and stored at 4 °C until further processing. Prior to cryo-ultramicrotomy the cells were infiltrated with a solution of 10% gelatin in PBS at 37 °C, allowed to gel at 4 °C, and infused with 2.3 m sucrose overnight. Blocks of gelatin containing cells were attached to aluminum pins, frozen in liquid nitrogen, and cut at –95 °C on a diamond knife ∼100 nm thick using a Leica Ultracut R with a cryo-chamber (Leica Canada, Willowdale, Ontario, Canada). Sections were transferred to Formvar-coated grids in a drop of molten sucrose, and the aldehyde residues were blocked with a solution of PBS containing 0.15% glycine and 0.5% BSA. Samples were rinsed several times with PBS with just BSA prior to incubation in a polyclonal rabbit antibody against ClC-4 for 1 h. Grids were then washed in PBS/BSA thoroughly and incubated for another hour in goat anti-rabbit IgG 10-nm gold complexes (Amersham Biosciences). Again after rinses in PBS/BSA samples were incubated for 1 h in a monoclonal antibody against GFP. This was followed by more washes and another 1 h of incubation in goat anti-mouse IgG 5-nm gold complexes (Amersham Biosciences). Grids were thoroughly washed with PBS followed by distilled water. They were then stabilized in a thin film of methylcellulose containing 0.2% uranyl acetate. Controls included the omission of the primary antisera or the IgG gold complexes. Specimens were examined and images acquired with a JEOL JEM transmission electron microscope (JEOL, Peabody, MA) equipped with a digital camera (AMT Corp. Danvers, MA). Statistics—Statistical analyses, analysis of variance followed by Bonferoni's non-paired"t"test, were conducted using Prism software, and p values of 0.05 or less were considered significant. ClC-4 Is Expressed in the Renal Proximal Tubule—Previously, we showed that a polyclonal, affinity-purified antibody directed against the amino terminus of ClC-4 labels a 90–97-kDa protein in immunoblots of rat brain tissue. This band was competed using the amino terminus of ClC-4, and not the amino terminus of the related protein ClC-5, attesting to its specificity (26Mohammad-Panah R. Ackerley C. Rommens J. Choudhury M. Wang Y. Bear C.E. J. Biol. Chem. 2002; 277: 566-574Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). In Fig. 1A, we show that only this band is detected in whole mouse brain and is completely absent in immunoblots performed using brain tissue obtained from two Clcn4 null mice, providing compelling evidence in support of the specificity of this antibody. Clcn4 null mice were generated by breeding two strains of mice, Mus spretus and C57BL/6J, as described previously by Rugarli et al. (32Rugarli E.I. Adler D.A. Borsani G. Tsuchiya K. Franco B. Hauge X. Disteche C. Chapman V. Ballabio A. Nat. Genet. 1995; 10: 466-471Crossref PubMed Scopus (73) Google Scholar). Clcn4 is located on the X chromosome in the M. spretus strain and on chromosome 7 in the C57BL/6J strain permitting the generation of a population of male mice devoid of Clcn4 through backcrossing C57CL/6J × M. spetus F1 females with M. spretus males. To date, however, only two Clcn4 null mice have been produced during 6 months due the difficulty involved in breeding these two strains. Therefore, the following in vivo studies of ClC-4 function were performed in rats or using an antisense strategy in established intestinal and renal cell lines. In Fig. 1B, we show that ClC-4 protein is expressed as a 90–97-kDa protein in immunoblots of total rat kidney lysates. Confocal micrographs (Fig. 1C) reveal that ClC-4-specific immunofluorescence is expressed in the epithelium of the proximal tubule. The apical aspect of the epithelium of the proximal tubule was defined visually by in vivo injection of fluorescein-labeled dextran (FITC-dextran), known to be internalized by this tissue via fluid phase endocytosis. As described in previously published studies (27Obermuller N. Kranzlin B. Blum W.F. Gretz N. Witzgall R. Am. J. Physiol. 2001; 280: F244-F253PubMed Google Scholar), 8 min after injection, FITC-dextran will have been filtered through the glomerulus and internalized across the apical membrane of the epithelium of the proximal tubule by endocytosis. After this perfusion time, the kidneys were fixed in situ and flash-frozen. Confocal micrographs of frozen sections confirmed that FITC-dextran (27Obermuller N. Kranzlin B. Blum W.F. Gretz N. Witzgall R. Am. J. Physiol. 2001; 280: F244-F253PubMed Google Scholar) can be detected close to the apical membrane and in sub-apical vesicles of the proximal renal tubule epithelium. These same sections were labeled using the ClC-4 antibody described above, and we observed that the pattern of ClC-4-specific immunofluorescence overlapped with that of FITC-dextran (Fig. 1C). These findings are similar to those reported for the related protein ClC-5 (16Gunther 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) and suggest that both channels are expressed in the proximal tubule, probably in the apical membrane and sub-apical membrane vesicles. Expression of ClC-4 or ClC-5 Channels Can Be Specifically Depleted in Caco-2 Cells Using an Antisense Strategy—We used an antisense strategy to determine the relative functional expression of ClC-4 and ClC-5 endogenously expressed in epithelial cells. As both channel proteins are endogenously expressed in the Caco-2 cell line (26Mohammad-Panah R. Ackerley C. Rommens J. Choudhury M. Wa
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