ClC-2 Contributes to Native Chloride Secretion by a Human Intestinal Cell Line, Caco-2
2001; Elsevier BV; Volume: 276; Issue: 11 Linguagem: Inglês
10.1074/jbc.m006764200
ISSN1083-351X
AutoresRaha Mohammad-Panah, Katalin Gyömörey, Johanna M. Rommens, Monideepa Choudhury, Canhui Li, Yanchun Wang, Christine E. Bear,
Tópico(s)Helicobacter pylori-related gastroenterology studies
ResumoIt has been previously determined that ClC-2, a member of the ClC chloride channel superfamily, is expressed in certain epithelial tissues. These findings fueled speculation that ClC-2 can compensate for impaired chloride transport in epithelial tissues affected by cystic fibrosis and lacking the cystic fibrosis transmembrane conductance regulator. However, direct evidence linking ClC-2 channel expression to epithelial chloride secretion was lacking. In the present studies, we show that ClC-2 transcripts and protein are present endogenously in the Caco-2 cell line, a cell line that models the human small intestine. Using an antisense strategy we show that ClC-2 contributes to native chloride currents in Caco-2 cells measured by patch clamp electrophysiology. Antisense ClC-2-transfected monolayers of Caco-2 cells exhibited less chloride secretion (monitored as iodide efflux) than did mock transfected monolayers, providing the first direct molecular evidence that ClC-2 can contribute to chloride secretion by the human intestinal epithelium. Further, examination of ClC-2 localization by confocal microscopy revealed that ClC-2 contributes to secretion from a unique location in this epithelium, from the apical aspect of the tight junction complex. Hence, these studies provide the necessary rationale for considering ClC-2 as a possible therapeutic target for diseases affecting intestinal chloride secretion such as cystic fibrosis. It has been previously determined that ClC-2, a member of the ClC chloride channel superfamily, is expressed in certain epithelial tissues. These findings fueled speculation that ClC-2 can compensate for impaired chloride transport in epithelial tissues affected by cystic fibrosis and lacking the cystic fibrosis transmembrane conductance regulator. However, direct evidence linking ClC-2 channel expression to epithelial chloride secretion was lacking. In the present studies, we show that ClC-2 transcripts and protein are present endogenously in the Caco-2 cell line, a cell line that models the human small intestine. Using an antisense strategy we show that ClC-2 contributes to native chloride currents in Caco-2 cells measured by patch clamp electrophysiology. Antisense ClC-2-transfected monolayers of Caco-2 cells exhibited less chloride secretion (monitored as iodide efflux) than did mock transfected monolayers, providing the first direct molecular evidence that ClC-2 can contribute to chloride secretion by the human intestinal epithelium. Further, examination of ClC-2 localization by confocal microscopy revealed that ClC-2 contributes to secretion from a unique location in this epithelium, from the apical aspect of the tight junction complex. Hence, these studies provide the necessary rationale for considering ClC-2 as a possible therapeutic target for diseases affecting intestinal chloride secretion such as cystic fibrosis. cystic fibrosis CF transmembrane conductance regulator glutathioneS-transferase N-methyl-d-glutamine hypotonic shock kilobase(s) green fluorescence protein The physiological significance of ClC-2, a ubitquitously expressed member of the ClC family of chloride channels (1Thiemann A. Gründer S. Pusch M. Jentsch T. Nature. 1992; 356: 57-60Crossref PubMed Scopus (510) Google Scholar) is not fully understood. Based primarily on studies of native ClC-2 message and protein expression, roles for ClC-2 in neuronal and epithelial tissue have been proposed (2Staley K. Smith R. Schaack J. Wilcox C. Jentsch T. Neuron. 1996; 17: 543-551Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 3Jentsch T.J. Friedrich T. Schriever A. Yamada H. Pfluegers Arch. Eur. J. Physiol. 1999; 437: 783-795Crossref PubMed Scopus (297) Google Scholar). ClC-2 channel activity has been implicated in the regulation of neuronal responses to GABA-A receptor interaction (2Staley K. Smith R. Schaack J. Wilcox C. Jentsch T. Neuron. 1996; 17: 543-551Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). In non-neuronal cells, ClC-2 function has been linked to volume regulation, and in epithelial cells, it has been linked specifically to chloride secretion (3Jentsch T.J. Friedrich T. Schriever A. Yamada H. Pfluegers Arch. Eur. J. Physiol. 1999; 437: 783-795Crossref PubMed Scopus (297) Google Scholar). Immunolocalization studies of ClC-2 revealed that it is situated on the apical surface of airway epithelial cells in neonatal rat airways (4Murray C.B. Morales M.M. Flotte T.R. McGrath-Morrow S.A. Guggino W.B. Zeitlin P.L. Am. J. Respir. Cell Mol. Biol. 1995; 12: 597-604Crossref PubMed Scopus (125) Google Scholar). Subsequent Ussing chamber studies showed that luminal acidity promoted chloride secretion in neonatal airway cells via a cadmium-sensitive channel (5Blaisdell C.J. Edmonds R.D. Wang X.T. Guggino S. Zeitlin P.L. Am. J. Physiol. 2000; 278: L1248-L1255PubMed Google Scholar). These findings prompted us to suggest that ClC-2, a channel that exhibits these properties, may mediate chloride secretion in neonatal rat airways. In addition, Schwiebert et al. (6Schwiebert EM Cid-Soto LP Stafford D Carter M Blaisdell CJ Zeitlin PL Guggino WB Cutting GR Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3879-3884Crossref PubMed Scopus (129) Google Scholar) reported that ClC-2-like currents are present in airway epithelial cells derived from an adult patient with cystic fibrosis (CF)1 and suggested chloride transport via ClC-2 may be able to compensate for defective or absent CFTR chloride channels in the CF airway epithelium. Further, because ClC-2 message can be detected in intestinal epithelial tissue obtained from cftr-knockout mice, Joo et al. (7Joo N.S. Clarke L.L. Han B.H. Forte L.R. Kim H.D. Biochim. Biophys. Acta. 1999; 1446: 431-437Crossref PubMed Scopus (22) Google Scholar) also suggested that there is the potential for ClC-2 to provide a bypass pathway for chloride transport in CF affected intestines. Despite these intriguing observations, there was no direct molecular evidence to suggest that ClC-2 contributes to native chloride secretion. In the present work, we assessed the role of ClC-2 in chloride secretion in the Caco-2 cell line, a cell line that models the human small intestinal epithelium (8Rousset M. Laburther M. Pinto M. Chevalier G. Rouyer-Fessard C. Dussaulx E. Trugnan G. Boige N. Brun J.L. Zweibaum A. J. Cell. Physiol. 1985; 123: 377-385Crossref PubMed Scopus (125) Google Scholar, 9Sood 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). Using immunofluorescence and confocal microscopy, we confirmed that ClC-2 protein is endogenously expressed in the plasma membrane of Caco-2 cells. Further, we show that it is uniquely situated at the apical aspect of the tight junctions between cells in fully differentiated monolayers of these cells. Using an antisense strategy, we show that endogenously expressed ClC-2 mediates currents across the plasma membrane of single Caco-2 cells and, finally, that ClC-2 can contribute to native anion secretion across Caco-2 cell monolayers. 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.) containing 10% fetal calf serum, with 2 mm glutamine, 100 units penicillin G, and 100 μg/ml streptomycin sulfate at 37 °C in an atmosphere of 5% CO2, 95% air. For patch clamp studies, cells were used 1–2 days after plating onto 35-mm coverslips (Fisher). For Ussing chamber studies of chloride currents and assessment of ClC-2 localization by confocal microscopy, Caco-2 cells were seeded at high density (1 × 106 cells ml−1, 500 μl/filter) and grown to confluency on clear Snapwell (Costar) filters (pore size, 4 μm; diameter, 12 mm). Filters were cultured at 37 °C in an atmosphere of 5% CO2, 95% air, for 2 weeks with medium replacement every 2–3 days. The formation of an intact monolayer was assessed by measuring transepithelial resistance in Ussing chambers. Transepithelial resistance was calculated using Ohm's law, from measurements of the change in short circuit current measured (I sc, μA) upon passing 1 mV across the epithelium. Monolayers were considered acceptable when the transepithelial resistance exceeded 500 Ohms/cm2 and the transepithelial potential difference exceeded 2 mV. Alternatively, for iodide efflux studies and assessment of protein expression by Western blotting and confocal microscopy following transfection, Caco-2 cells were grown for 4–6 days on glass coverslips to achieve a differentiated phenotype, as documented by Sood et al. (9Sood 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). Total RNA was isolated from Caco-2 cell monolayers using the Trizol method as recommended by the supplier (Life Technologies, Inc.). RNA (2 μg) was analyzed on agarose gels (1%) containing 0.6 m formaldehyde and transferred to Hybond-N membranes (Amersham Pharmacia Biotech). Blots were cross-linked with UV radiation and hybridized with mouse-specific ClC-2 cDNA fragments radiolabeled by random priming (10Feinberg 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 −70 °C with one intensifying screen. Caco-2 cells, grown on 60-mm plastic dishes, were washed with phosphate-buffered saline containing 10 mm mono-dibasic mix, pH 6.8, and 150 mm NaCl, final pH 7.2, and incubated with 900 μl of lysis buffer containing 1% Triton X-100, 120 mm NaCl, 10 mm Tris, 25 mm KCl, 25 mm MgCl2, 1.8 mm CaCl2, and protease inhibitors leupeptin (10 μg/ml) and apotinin (10 μg/ml), 1 mm benzamidine, 10 μm E64, and 2 mm dithiothreitol. The cells were scraped off, and the suspension was mixed by vortexing. The supernatant was then centrifuged for 10 min at 4 °C at 48,000 × g to isolate a crude membrane preparation. Following protein assay of the supernatant, 50 μg of this preparation was analyzed by SDS-polyacrylamide gel electrophoresis (8% gel) using anti-ClC-2 antibody at a concentration of 2 μg/ml. This polyclonal antibody, as described previously, was generated against a GST fusion peptide containing amino acids 31–74 of rat ClC-2 (rClC-2 cDNA kindly provided by T. Jentsch). The ClC-2-specific antibody was immunopurified from a matrix of GST-N-peptide coupled on an activated agarose column as previously described (11Xiong H. Li C. Garami E. Wang Y. Ramjeesingh M. Galley K. Bear C.E. J. Membr. Biol. 1999; 167: 215-221Crossref PubMed Scopus (55) Google Scholar, 12Gyomorey K. Yeger H. Ackerley C. Garami E. Bear C.E. Am. J. Physiol. 2000; 279: C1787-C1794Crossref PubMed Google Scholar). The monoclonal antibody against β-actin (Sigma, anti-β-actin clone AC-74) was used at 1/1000 dilution. Immunoreactive protein was detected using the ECL system (Amersham Pharmacia Biotech). Immunofluorescence labeling was performed on Caco-2 cells grown on 35-mm circular coverslips or on clear 35-mm, 0.4-μm pore Snapwell (Corning Costar) filters. The pattern of ClC-2 labeling was identical regardless of the support employed. Cells were fixed with paraformaldehyde AM (4% in phosphate-buffered saline) and permeabilized with 0.5% Triton X-100 in phosphate-buffered saline. Cells were incubated for 0.5 h at 25 °C in 5% normal goat serum, 0.05% Triton X-100 in Tris-buffered saline containing 10 mm Tris-Cl and 150 mm NaCl with final pH 8, and then for 2.5 h with the polyclonal antibody against ClC-2 (30.7 μg/ml) or overnight in the refrigerator with the polyclonal antibody against ClC-3 (30 μg/ml) (Alomone Labs Ltd., Jerusalem, Israel). Then the cells were washed and incubated with Texas Red-conjugated or fluorescein isothiocyanate-conjugated anti-rabbit secondary antibody (0.02 mg/ml; Molecular Probes) and washed again before mounting. For colocalization studies of ClC-2 and the tight junction protein occludin, the above procedure was followed by additional incubation with the monoclonal anti-occludin antibody (0.002 mg/ml, Zymed Laboratories Inc., Missisauga, Canada) for 1 h and washed. Cells were then incubated with Texas Red-conjugated anti-mouse secondary antibody (0.02 mg/ml; Molecular Probes) and washed before mounting. For the competition studies, the anti-ClC-2 antibody was preincubated with 2-fold excess of the antigenic fusion peptide overnight at 4 °C before incubation. Slides were viewed on an Olympus Vanox AHBT3 microscope using epifluorescence, and images were captured using the Image Pro Plus program (Cybernetics, L.P.). For confocal microscopy, sections (each 0.7 μm in thickness) were viewed with a 100x objective a Leica TCS 4D microsope, and the images were captured using the SCANware 5.01 program. Caco-2 cell membrane currents were measured using conventional whole cell patch clamp technique (13Hamill O.P. Marty A. Neher E. Sakmann E. Sigworth F.J. Pfluegers Arch. Eur. J. Physiol. 1981; 191: 85-100Crossref Scopus (15172) Google Scholar). Patch clamp electrodes were prepared from borosilicate glass capillaries (outer diameter, 1.5 mm; inner diameter, 1.18 mm) with an inner filament (World Precision Instruments, Inc., Sarasota, FL) on a Narishige PP-83 patch electrode puller using the standard two-pull technique. The tip resistance was 3–5 mΩ when filled with pipette solution (see below for composition). Whole cell currents were measured using an Axopatch 200A patch clamp amplifier (Axon Instruments, Foster City, CA) and were filtered at 100 Hz with a 6-pore Bessel Filter. Sampling rate was 4 kHz for most data, and junction potentials were corrected. Voltage clamp protocols were generated using pCLAMP software (version 7, Axon Instruments) via a Pentium II computer interfaced with a 1200 series Digidata (Axon Instruments) The same software package was used both for data acquisition and analysis. Current-voltage relationships were determined in a stepwise clamp protocol. From a holding potential of −30 mV, voltage pulses of 3.0 s were applied from −160 to +40 mV in 20-mV increments. The bath solution contained 140 mm N-methyl-d-glutamine (NMDG) chloride, 2 mm MgCl2, 2 mm CaCl2, 5 mm HEPES, whereas the pipette solution contained 140 mm NMDG chloride, 2 mm MgCl2, 2 mm EGTA, and 5 mm HEPES. Both pipette and bath solutions were adjusted to pH 7.4 and 260 mOsm. In experiments in which the response to hypotonic shock was studied, the bath solution contained 110 mm NMDG chloride, 2 mmCaCl2, 2 mm MgCl2, 5 mmHEPES, pH 7.4, and the osmolarity was adjusted to 303 mOsm with sucrose. The hypotonic bath solution was made as above, maintaining equal ionic strength and pH, except that the osmolarity was adjusted to 228 mOsm with sucrose as assessed using a 5500 Vapor pressure osmometer (Wescor, Johns Scientific Inc.). Caco-2 cells were subjected to hypotonic shock using a gravity-fed superfusion system. The ClC-2 sense construct was made by directional cloning of the rat (r)ClC-2 open reading frame (kindly provided by T. Jentsch, Hamburg, Germany) with BamHI (5′) and EcoRI (3′) linkers into the BamHI andEcoRI restriction sites of the eukaryotic vector pCDNA 3.1 (+) (Promega, Madison, WI). The rat ClC-2 sequence shares 77% identity with the human sequence at the nucleotide level. The antisense ClC-2 construct was made by cloning the ClC-2 open reading frame into pCDNA 3.1(−) vector such that the reversed restriction sites on this vector would reverse the orientation of the open reading frame to create the antisense plasmid. The antisense murine ClC-4 construct (Clcn4, a gift from E. Rugarli, Milano, Italy) was made by cloning the ClC-4 open reading frame with BamHI (5′) andEcoRI (3′) into pCDNA 3.1(−). The murine ClC-4 construct shares 73% sequence identity with the human sequence. Caco-2 cells were microinjected with plasmids at day 1 after plating on glass coverslips for patch clamp experiments. In this procedure, the Eppendorf microinjector 5246 system, the micromanipulator 5171 system, and a Nikon Diaphot inverted microscope were used. Nuclear microinjection was performed with the Z (depth) limit option, using 0.3-s injection duration and 40–60 hPa injection pressure. Injection femtotips were pulled from borosilicate glass capillaries with an internal diameter of 0.5 ± 0.2 μm. Plasmids were diluted to a final concentration of 50 μg/ml for sense ClC-2 plasmids, 50 and 300 μg/ml for antisense ClC-2, and 300 μg/ml antisense ClC-4. The injection buffer contained in 50 mm HEPES, 50 mm NaOH, 40 mmNaCl, pH 7.4. Fluorescein isothiocyanate-labeled dextran (0.5%, Sigma) was also added to the injection medium to identify successfully microinjected cells. For transfection of Caco-2 cells with antisense ClC-2 DNA, the Lipofectin transfection protocol was followed (Life Technologies, Inc.). Briefly, ∼2 × 105 cells were seeded on 35-mm tissue culture plates in culture medium supplemented with serum. Cells were then incubated at 37 °C in a 5% CO2 incubator overnight to allow them to reach 80% confluency (∼106 cells/plate). Two solutions, one containing 2 μg of antisense ClC-2 cDNA (dissolved in serum-free medium) in 100 μl of OPTI-MEM I reduced serum medium and the second containing 20 μl of Lipofectin reagent in 100 μl of OPTI-MEM I reduced serum medium were mixed and incubated at room temperature for 45 min. Following addition of 800 μl of OPTI-MEM I reduced serum medium, this transfection mixture was applied to the cells (after washing them with serum-free medium). The cells were then incubated at 37 °C in a 5% CO2 incubator for 48 h, after which the transfection medium was replaced with normal medium containing 10% serum and antibiotics (as described above). 24 h later, cells were harvested for immunoblot analysis or studied by iodide efflux assay. Short circuit current measurements were performed on Caco-2 cells grown to confluency on Snapwell clear filters, with a surface area of 1.13 cm2 (Corning Costar). The average transepithelial resistance of the cells used was 2421 ± 357.2 Ω cm2. Filters were inserted into Ussing chambers and bathed in a buffer composed of 110.4 mm NaCl, 27.5 mm mannitol, 2.4 mmK2HPO4, 0.8 mmKH2PO4, 10 mm glucose, 10 mm HEPES, and 1 mm CaCl2, gassed with 95% O2 and heated to 37 °C. After 5–8 min of measuring basal current the apical solution was changed to one lacking (27.5 mm) mannitol for 20% hypotonic shock (HTS; 80% isotonicity; determined using a 5500 Vapor pressure osmometer, Wescor, Johns Scientific Inc.). Caco-2 cells grown on coverslips (80%) were transfected either with antisense ClC-2 plasmid in the pCDNA vector or with vector alone as described under "Experimental Procedures." Only monolayers possessing 1 × 106 cells after the entire transfection protocol were used for subsequent assays. The transfected cells were iodide loaded according to established methods (14Shen B.Q. Finkbeiner W.E. Wine J.J. Mrsny R.J. Widdicombe J.H. Am. J. Physiol. 1994; 266: L493-L501PubMed Google Scholar, 15Haws C.M. Nepomuceno I.B. Krouse M.E. Wakelee H. Law T. Xia Y. Nguyen H. Wine J.J. Am. J. Physiol. 1996; 270: C1544-C1555Crossref PubMed Google Scholar) using a 1-ml Ringers Nitrate loading buffer at pH 7.4 containing 136 mm NaI, 4 mm KNO3, 2 mm CaNO3·4H2O, 2 mmMgNO3·6H2O, 11 mm glucose, and 20 mm HEPES. The cells were incubated for 1 h at 37 °C in the presence of 5% CO2 in the above buffer. After this incubation period, the coverslips covered with iodide-loaded cells were washed for a total of 15 s in three separate baths containing Ringers nitrate efflux buffer (110 mm NaNO3, 4 mm KNO3, 2 mmCaNO3·4H2O, 2 mmMgNO3·6H2O, 11 mm glucose, and 20 mm HEPES, pH 7.4) with the osmolarity adjusted to 300 mOsm with added sucrose. After this washing period, efflux of cellular iodide was assessed continuously after placing the coverslip into an isotonic solution (the above Ringers Nitrate efflux buffer) or hypotonic solutions (osmolarity adjusted 228 mOsm). Iodide efflux (measured as a change in mV) was assessed over a 5-min period, using an iodide sensing electrode (Fisher). Changes in voltage were acquired using the FETCHEX data acquisition program (pCLAMP 6.04, Axon Inst.) and data analyzed using FETCHAN software. Patch clamp measurements are presented as the means ± S.E. Most statistical analyses were performed using the Student's unpaired test. Results obtained in Ussing chamber studies and in Patch clamp studies with hypotonic shock were analyzed using the Student paired test. Differences between two groups were considered significant with p values <0.05. ClC-2 mRNA in Caco-2 cells was detected by Northern blot analysis as a 4.6-kb transcript (Fig. 1 A). This size transcript plus a smaller transcript of ∼3.3 kb in size has been detected in several other tissues and cell lines, as well, including the colonic epithelial cell line T84 (6Schwiebert EM Cid-Soto LP Stafford D Carter M Blaisdell CJ Zeitlin PL Guggino WB Cutting GR Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3879-3884Crossref PubMed Scopus (129) Google Scholar). Immunoblots (Fig. 1 B) using a polyclonal antibody directed against ClC-2 (12Gyomorey K. Yeger H. Ackerley C. Garami E. Bear C.E. Am. J. Physiol. 2000; 279: C1787-C1794Crossref PubMed Google Scholar) showed that ClC-2, migrating as a 90–97-kDa protein, is expressed in Caco-2 cells. This signal was competed using the GST-ClC-2 fusion peptide against which the antibody was raised, not GST alone, indicating its specificity for ClC-2. Immunofluorescence labeling using the above antibody suggests that ClC-2 protein localizes to plasma membrane and/or submembranous vesicles in Caco-2 cells (Fig. 2). Further, this signal was specific because it was competed using the antigenic peptide described above. Whole cell patch clamp studies were performed to determine whether ClC-2 is functional in the membrane of Caco-2 cells. Because previous studies in heterologous systems showed that ClC-2 expression conferred chloride currents were activated by hyperpolarization and by hypotonic shock (1Thiemann A. Gründer S. Pusch M. Jentsch T. Nature. 1992; 356: 57-60Crossref PubMed Scopus (510) Google Scholar, 11Xiong H. Li C. Garami E. Wang Y. Ramjeesingh M. Galley K. Bear C.E. J. Membr. Biol. 1999; 167: 215-221Crossref PubMed Scopus (55) Google Scholar, 16Gründer S. Thiemann A. Pusch Jentsch T. Nature. 1992; 360: 759-762Crossref PubMed Scopus (361) Google Scholar), we assessed whether these manipulations could activate ClC-2 endogenously expressed in Caco-2 cell membranes by patch clamp electrophysiology. We functionally isolated anion-dependent currents by using intracellular (pipette) and extracellular (bath) solutions which contained NMDG chloride as the predominant salt. We chose a voltage step protocol that has been used in previously published studies of ClC-2 (11Xiong H. Li C. Garami E. Wang Y. Ramjeesingh M. Galley K. Bear C.E. J. Membr. Biol. 1999; 167: 215-221Crossref PubMed Scopus (55) Google Scholar, 16Gründer S. Thiemann A. Pusch Jentsch T. Nature. 1992; 360: 759-762Crossref PubMed Scopus (361) Google Scholar). Briefly, from a holding potential of −30 mV, the membrane potential was stepped by 20-mV increments from −160 to + 40 mV. Because ClC-2 currents have not been reported to be ATP-dependent and to minimize the contribution by the ATP-dependent, swelling-activated outwardly rectifying chloride channel, volume-sensitive organic osmolyte anion channel (17Bond T. Basavappa S Christensen M. Strange K. J. Gen. Phyiol. 1999; 113: 441-456Crossref PubMed Scopus (38) Google Scholar), MgATP was not included the patch pipette solutions. As shown in Fig. 3(A and C), currents typical of those previously associated with ClC-2 expression, i.e. showing activation with hyperpolarizing voltage steps and an inwardly rectifying current-voltage relationship, were detected in Caco-2 cells. At the hyperpolarized membrane potential of −160 mV, these currents had a magnitude of −37 pA/pF ± 0.96 (n = 8). These hyperpolarization-activated currents reversed close to the estimated equilibrium potential of chloride (E Cl = 0 in symmetrical NMDG chloride solutions (Fig. 3 C,circles). As shown in Fig. 3 (B and C), whole cell chloride currents in Caco-2 cell were stimulated by 25% hypotonic shock. We detected an increase in membrane currents from −38 pA/pF to −84 pA/pF within 3–5 min after bath dilution at −160 mV (Fig. 3). The mean current-voltage (I/V) curves of ClC-2 currents before and after application of hypotonic shock reversed close to the chloride equilibrium potential (+5.6 ± 0.47 mV) as shown in Fig.3 C. The average current at −160 mV following hypotonic shock (−65 ± 8 pA/pF) was elevated when compared with currents measured in isotonic condition (−32 ± 2 pA/pF) (p = 0.0208). The I/Vrelationship of the HTS-stimulated chloride currents was less inwardly rectifying than that observed in isotonic solutions (Fig.3 C). A similar change in the I/Vrelationship was observed with HTS in chloride currents specifically conferred by ClC-2 expression in Xenopus oocytes and Sf9 cells and has been attributed to an alteration in the inactivation gate of ClC-2 (11Xiong H. Li C. Garami E. Wang Y. Ramjeesingh M. Galley K. Bear C.E. J. Membr. Biol. 1999; 167: 215-221Crossref PubMed Scopus (55) Google Scholar, 16Gründer S. Thiemann A. Pusch Jentsch T. Nature. 1992; 360: 759-762Crossref PubMed Scopus (361) Google Scholar). These results indicate that native ClC-2 expression at the cell surface of Caco-2 cells is associated with appearance of chloride currents with activation and conductance properties similar to those conferred by ClC-2 expression in heterologous expression systems (1Thiemann A. Gründer S. Pusch M. Jentsch T. Nature. 1992; 356: 57-60Crossref PubMed Scopus (510) Google Scholar, 11Xiong H. Li C. Garami E. Wang Y. Ramjeesingh M. Galley K. Bear C.E. J. Membr. Biol. 1999; 167: 215-221Crossref PubMed Scopus (55) Google Scholar, 16Gründer S. Thiemann A. Pusch Jentsch T. Nature. 1992; 360: 759-762Crossref PubMed Scopus (361) Google Scholar, 18Jordt S.E. Jentsch T.J. EMBO J. 1997; 16: 1582-1592Crossref PubMed Scopus (207) Google Scholar). We used an antisense strategy to confirm that the above currents were mediated by ClC-2 because the pharmacological approach lacks specificity. First, we confirmed that transient transfection of ClC-2 antisense cDNA (see "Experimental Procedures") successfully reduced ClC-2 protein expression by Western analysis of cell lysates from ClC-2 antisense-transfected Caco-2 cells. Using the NIH Imaging Program, we found that there is a 70% decrease in ClC-2 protein quantity in antisense ClC-2 transfected Caco-2 cells relative to control (vector-alone transfected cells). We verified by assessing β-actin expression that differences in protein loading could not account for the decrease in ClC-2 expression in the antisense transfected cells (Fig. 4 A). Furthermore, we examined the effects of antisense ClC-2 transfection on immunolabeled ClC-2 detected by fluorescence confocal microscopy. DNA coding for green fluorescence protein (GFP) was cotransfected with antisense ClC-2 (or empty vector as a control) into Caco-2 cells to identify transfected cells (Fig. 4 B). We used an imaging program (Scion Corp.) to compare the ClC-2 immunofluorescence intensity in antisense ClC-2 and in vector transfected Caco-2 cells. We found that the fluorescence intensity of the signal (red) corresponding to membrane expression of ClC-2 was reduced by ∼75% in antisense ClC-2 transfected Caco-2 cells (24.9 units ± 4.3,n = 13, p < 0.0001) relative to the intensity of the ClC-2 signal in mock transfected cells (105.8 units ± 3, n = 10). Immunofluorescence corresponding to expression of ClC-3, a related family member, was not affected by antisense ClC-2 transfection (Fig. 4 C). The signal detected using this ClC-3 antibody in immunofluorescence studies can be competed using the antigenic peptide used to raise the antibody, confirming its specificity (19Shimada K. Li X. Xu G. Nonak D.E. Showalter L.A. Weinman S.A. Am. J. Physiol. 2000; 279: G269-G276Google Scholar). Fig. 4 C shows that the ClC-3 immunofluorescence (red) in antisense ClC-2 and GFP cotransfected Caco-2 cells (106.1 units ± 3.2, n= 20) was similar to that in vector and GFP cotransfected cells (105.9 units ± 2.2, n = 17, p = 0.97). Interestingly, our studies show that unlike ClC-2, immunoreactive ClC-3 appears to be primarily expressed in intracellular membranes, although there is signal detected at the cell surface in a subpopulation of cells. For patch clamp studies, we manipulated ClC-2 expression using intranuclear plasmid injection technique (20Shubeita H.E. Thorburn J.T. Chien K.R. Circulation. 1992; 85: 2238-2241Crossref Scopus (27) Google Scholar, 21Mohammad-Panah R. Demolombe S. Riochet D. Leblais V. Loussouarn G. Pollard H. Baro I. Escande D. Am. J. Physiol. 1998; 274: C310-C318Crossref PubMed Google Scholar), because this method permits control of plasmid copy number and hence has greater precision in manipulating the level of antisense expression. Fluorescein isothiocyanate-dextran was coinjected with the plasmid to permit identification of manipulated cells. We found that microinjection of antisense ClC-2 cDNA into Caco-2 cells decreased the ClC-2-like currents in a dose-dependent manner (Fig.5, A and B). The negative whole cell current measured at −160 mV decreased from −37 pA/pF ± 1 (n = 8) in uninjected cells to −26 ± 2 pA/pF (n = 5, p = 0.0003) and −12 ± 1 pA/pF (n = 10,p < 0.0001) in 50 and 300 μg/ml antisense ClC-2 cDNA injected cells, respectively (Fig. 5, A andB). To allow direct comparison of current amplitude in ClC-2 antisense injected and uninjected Caco-2 cells, we normalized these currents to the currents at −160 mV in uninjected cells (Fig.5 B). As shown in F
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