1 Institut National de la Santé et de la Recherche Médicale (INSERM), Unité 467, Faculté de Médecine Necker-Enfants Malades, 75015 Paris; 2 INSERM, Unité 410, Institut Fédératif de Recherche Xavier Bichat, 75018 Paris; 3 INSERM, Unité 468, Hôpital Henri Mondor, 94010 Créteil; and 4 Centre National de la Recherche Scientifique, Unité Propre de Recherche 1524, Hôpital Saint Vincent de Paul, 75674 Paris, France
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ABSTRACT |
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The ubiquitous ClC-2 Cl
channel is thought to contribute to epithelial Cl
secretion, but the distribution of the ClC-2 protein in human epithelia
has not been investigated. We have studied the distribution of ClC-2 in
adult human and rat intestine and airways by immunoblotting and
confocal microscopy. In the rat, ClC-2 was present in the lateral
membranes of villus enterocytes and was predominant at the basolateral
membranes of luminal colon enterocytes. The expression pattern of ClC-2
in the human intestine differed significantly, because ClC-2 was mainly
detected in a supranuclear compartment of colon cells. We found
significant expression of ClC-2 at the apex of ciliated cells in both
rat and human airways. These results show that the distribution of
ClC-2 in airways is consistent with participation of ClC-2 channels in
Cl
secretion and indicate that extrapolation of results
from studies of ClC-2 function in rat intestine to human intestine is
not straightforward.
small intestine; colon; ciliated cells; immunohistochemistry; antibody
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INTRODUCTION |
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THE SECRETION
of Cl is critical for fluid and electrolyte transport
across epithelia. The essential part of Cl
secretion is
driven by the cystic fibrosis (CF) transmembrane conductance regulator
(CFTR) located in the apical membranes of secretory epithelia, such as
those of the intestine and airways (1). Impaired or
enhanced Cl
secretion results in diseases such as CF and
secretory diarrhea (4, 5). The manifestations of CF result
from a failure to secrete sufficient Cl
, so that mucosal
surfaces are not adequately hydrated. There are, however, other,
non-CFTR apical membrane Cl
channels that participate in
epithelial Cl
secretion. In the pulmonary epithelium,
Cl
secretion occurs through apical
Ca2+-regulated Cl
channels (14).
Although the presence of Ca2+-dependent Cl
channels in the apical membranes of enterocytes has not yet been confirmed, several findings point to the existence of a similar conductive pathway (4). Recent studies suggest that the
ClC-2 Cl
channel, a member of the voltage-gated
Cl
channel family, may be involved in Cl
secretion in rat fetal airway epithelial cells (7, 43) and in the small intestine from an unexpected location at the tight junction complex between epithelial cells (29, 41).
Northern blot analysis revealed that ClC-2 is widely expressed in
mammalian tissues and in a variety of cultured cell lines (51). The expression of ClC-2 mRNA in Xenopus
laevis oocytes induces currents that are slowly activated by
strong hyperpolarization (51), a low extracellular pH
(33), and hypotonicity (27). Similar currents
are present in native cells, including epithelial cells and neurons
(32). The biophysical properties and distribution pattern
of ClC-2 have led to the suggestion that it helps regulate cell volume
(23, 27, 58) and control intracellular Cl
concentration (46), in addition to its possible role in
Cl
secretion. However, these suggestions must be
confirmed, because a recent report indicated that ClC-2-deficient mice
do not show spontaneous seizures or major changes in intestinal and
lung anatomy and that the main abnormalities are severely
degenerated retina and seminiferous tubules (8). The
physiological role of ClC-2 in tissues other than the retina and testes
may be taken by other proteins, and the question remains as to whether
ClC-2 provides an alternative pathway for Cl
secretion in CF.
Epithelial ClC-2 distribution has so far been investigated only in rodents (29, 42, 45) and in a human intestinal cell line (41). The present study was therefore carried out to identify and locate the ClC-2 protein in human and rat tissues with a new polyclonal antibody (pAb-218) generated against a 16-amino acid sequence [amino acids (AA) 847-862] within the COOH-terminal region of ClC-2. The peptide sequence was selected because of its amphipathy and possible antigenicity, because the amino acids in the rat and human peptides are identical, and because there are no conserved amino acids in other ClC proteins. A different anti-ClC-2 antibody (Clcn2), raised against the COOH terminus of ClC-2 (AA 888-906), became commercially available while our work was in progress. We therefore used both antibodies for comparative analysis of ClC-2 tissue distribution.
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MATERIALS AND METHODS |
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Rat and Human Tissues
Lung and intestinal tissues were taken from 8-wk-old male rats (Sprague-Dawley) fed a standard diet. Samples of human lung were obtained at surgery from patients undergoing resection for lung carcinoma (Department of Pathology, Hôpital Laennec, Paris, France). Surgical specimens of normal human intestinal tissue were obtained from the Department of Pathology, Hôpital Necker-Enfants Malades (Paris, France). Only resected regions considered to be histologically normal were analyzed. Nasal samples were obtained from healthy adult volunteers. The human tissues were analyzed after obtaining informed consent and approval by the Institutional Ethics Committee of Hôpital Necker-Enfants Malades.Cell Culture and Transient Expression of ClC-2 in HeLa Cells
T84 cells were cultured as previously described (22). Briefly, cells were grown at 37°C with 5% CO2 in Dulbecco's modified Eagle's medium (DMEM)-Ham's F-12 medium (1:1 vol/vol; Life Technologies) containing 10% fetal calf serum, 2 mM glutamine, 100 IU/ml penicillin, and 170 µg/ml streptomycin. For immunocytochemistry, cells were cultured for 48 h at low density on thin glass coverslips.HeLa cells were grown in 35-mm culture dishes at 37°C with 5% CO2 in DMEM (Glutamax; Life Technologies) containing 10% fetal calf serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Subconfluent cells were transfected with LipofectAMINE Plus reagent (Life Technologies) according to the manufacturer's instructions. Rat ClC-2 cDNA (kindly provided by Prof. T. Jentsch, Hamburg University, Hamburg, Germany) was inserted into the expression plasmid pTracer (Invitrogen), a vector designed for the visual detection of transfected mammalian cells by green fluorescent protein (GFP). All experiments were performed 48 h after transfection.
HEK293 (tsA) cells stably expressing ClC-3 (Ref. 30; kindly provided by Drs. P. Huang and D. J. Nelson, University of Chicago, Chicago, IL) and HEK293 cells stably expressing ClC-5 (Ref. 55; kindly provided by Dr. A. Sardini, Imperial College School of Medicine, London, UK) were cultured as previously described.
Antibody Generation
Polyclonal antibodies were generated in New Zealand White rabbits against a peptide corresponding to AA 847-862 (AIEGSVTAQGVKVRPP) of the COOH-terminal region of rat ClC-2 coupled to KLH (AgroBio). The peptide was selected after analysis of sequences as previously published (24, 31). Briefly, the sequence amphipathy was analyzed with the algorithm of Eisenberg, and a hydrophobic cluster analysis was used to identify a fragment with a high probability to lie at the protein-water interface. The selected fragment had the same sequence in the rat and human ClC-2 isoforms. The sequence of the peptide was screened to ensure that it was not homologous to that of any other member of the ClC family, minimizing the cross-reactivity of anti-ClC-2 antibody. No homology was found by using the protein data bank SWISSPROT. The serum obtained from the final bleed was affinity-purified on a peptide column prepared by covalently linking the peptide to N-hydroxysuccinimide-activated Sepharose (NHS-activated HiTrap, Amersham Pharmacia Biotech). The antibodies were eluted from the column, dialyzed, concentrated using Centricon 10 (Amicon), and frozen atMembrane Preparation
Young male Sprague-Dawley rats (5-8 wk old) were killed by decapitation, and their brain, lung, small intestine, and colon were rapidly removed. All subsequent manipulations were carried out on ice. The intestinal epithelium was scraped off, and all the tissues were homogenized in a glass homogenizer in 10 volumes of sucrose buffer (250 mM sucrose, 20 mM HEPES, pH 7.4) containing protease inhibitors (protease inhibitor cocktail; Boehringer Mannheim). Tissue homogenates were centrifuged at 1,500 g for 10 min to remove cell debris. The supernatant was centrifuged at 17,000 g for 40 min, and the resulting supernatant was ultracentrifuged at 100,000 g for 1 h. The final pellet was suspended in buffer containing (in mM) 10 KCl, 1.5 MgCl2, and 10 Tris · HCl, pH 7.4. Protein concentration was determined by a modified Lowry method (40). The proteins (8-15 mg/ml) were then frozen and stored atMicrosomes were prepared from transfected HeLa cells as described
previously (18). Briefly, the cells were scraped off into ice-cold PBS, lysed in buffer containing (in mM) 10 KCl, 1.5 MgCl2, and 10 mM Tris · HCl, pH 7.4, supplemented
with protease inhibitor cocktail, and homogenized with a glass
homogenizer. The homogenate was centrifuged at 200 g for 10 min to remove unbroken cells, and the resulting supernatant was
ultracentrifuged at 100,000 g for 1 h. The final pellet
containing the plasma membranes was suspended in lysis buffer, and the
protein concentration was measured with the modified Lowry procedure.
The proteins (5-10 mg/ml) were frozen and stored at 80°C.
35S Metabolic Labeling and Immunoprecipitation
Transiently transfected HeLa cells were labeled with Redivue Pro-mix L-[35S] in vitro labeling mix, containing 35S-labeled L-methionine and 35S-labeled L-cysteine (150 µCi/ml for 3 h; Amersham Pharmacia Biotech). Cells were then lysed, and the proteins were dissolved in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris · HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, and 0.1% SDS) containing protease inhibitor cocktail. Cell lysates were precleared by incubation with 10% Pansorbin suspension (Calbiochem) for 30 min. Staphylococcus aureus cells were pelleted (12,000 g, 5 min), and the resulting supernatant (500 µl) was incubated with 1 µg of purified anti-ClC-2 antibody pAb-218 for 1 h. The samples were washed, denatured in SDS sample buffer, separated on 7.5% SDS-PAGE gel, and visualized with a PhosphoImager (Amersham Pharmacia Biotech). Controls were prepared by the same procedure except that the antibody incubation step was omitted.Immunoblotting
Rat membrane proteins (400 µg), protein extracts (200 µg) from transiently transfected HeLa cells, or protein extracts (600 µg) from HEK293 cells stably transfected with human (h)ClC-3 or hClC-5 were suspended in RIPA buffer and processed for immunoprecipitation as described in 35S Metabolic Labeling and Immunoprecipitation. Precipitated proteins were separated on 7.5% SDS-PAGE and transferred to nitrocellulose filters (Bio-Rad). Free binding sites were blocked with 1% non-fat dry milk-1% bovine serum albumin (BSA)-0.05% Tween 20 in Tris-buffered saline (TBS; in mM: 10 Tris · HCl, 150 NaCl, pH 8.0), and the membranes were probed with pAb-218 (diluted 1:5,000) or Clcn2 antibody purchased from Alomone Labs (diluted 1:250) and incubated overnight at 4°C. Nitrocellulose membranes were washed three times with TBS-T (0.05% Tween 20 in TBS) and incubated with goat anti-rabbit IgG(H+L) horseradish peroxidase (HRP)-conjugated (diluted 1:5,000; Biosys). Precipitated proteins were detected by incubating the nitrocellulose filters in ECL Plus reagent (Amersham Pharmacia Biotech) according to the manufacturer's instructions and exposing them to Kodak X-ray film.Immunohistochemical Procedures
Cells and cryosections of rat and human tissues were fixed in cold acetone for 10 min at 4°C and stored atNegative controls were routinely performed in parallel by omitting the primary antibody or by peptide competition. For the latter, ClC-2 antibodies in the working dilutions used were preincubated for 1 h at room temperature with a fivefold excess of the corresponding peptide antigens. Specific staining and negative controls were photographed under identical conditions. All final images were prepared with Paint Shop Pro and Adobe Illustrator software.
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RESULTS |
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Generation and Characterization of Anti-ClC-2 Antibodies
The specificity of the two antibodies, pAb-218 and Clcn2, was determined with immunoprecipitation and immunocytochemistry. Western blot analysis of extracts from HeLa cells transiently transfected with ClC-2 cDNA revealed that pAb-218 antibody did not detect ClC-2 protein, presumably because of a weak interaction with the SDS-denatured protein. Thus the specificity of anti-ClC-2 antibody was first assessed by immunoprecipitation of metabolically labeled HeLa cells (Fig. 1A). A 90- to 97-kDa protein band, corresponding to the predicted molecular mass of ClC-2, was detected (Fig. 1A, lane 4), whereas no band was seen in mock-transfected cells (lane 3); nonspecific IgGs purified from preimmune serum did not detect this band (lane 2). The background was due to nonspecific binding of 35S-methionine/cysteine-labeled proteins to the protein A beads, as confirmed by omitting the antibody (Fig. 1A, lane 1). pAb-218-immunoprecipitated proteins could also be detected with anti-ClC-2 antibodies. As shown in Fig. 1B, the pAb-218-immunoprecipitated ClC-2 protein was recognized by both pAb-218 (lane 2) and Clcn2 (lane 3).
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The suitability of both antibodies for immunocytochemistry was tested in HeLa cells transiently transfected with ClC-2 cDNA (Fig. 1C). Visualization of GFP fluorescence in living cells before immunoprocessing confirmed that only transfected cells were labeled by the anti ClC-2 antibodies. As described in MATERIALS AND METHODS, GFP-positive cells were identified and photographed before immunoprocessing (Fig. 1C, a and d). The same cells were photographed after staining with pAb-218 (Fig. 1C, c) or Clcn2 (Fig. 1C, e). The GFP fluorescence almost completely disappeared during cell fixation, as illustrated in Fig. 1C, b, and thus could not overlap with the green fluorescence of the secondary antibody. Confocal images obtained in x-y and x-z planes (Fig. 1C, f-i) showed that ClC-2 labeling was not restricted to the plasma membrane, which can be expected with an overexpressing system.
Although the ClC-2 antibodies used in the present study were raised against epitopes on the basis of no similarity to other ClC isoforms, we tested a possible cross-reactivity of the ClC-2 antibodies with two other isoforms, ClC-3, which is broadly expressed (32), and ClC-5, which, besides its predominant expression in kidney cells, has been demonstrated to be present in rat intestinal cells (52).
In immunoprecipitation experiments, a slight cross-reactivity to ClC-3 and ClC-5 was detected, but only when high concentrations of total protein were used (concentrations were 3-fold higher than those used for ClC-2 detection). Clcn2 antibody immunoprecipitated proteins of ~85 (Fig. 1D, left) and 100 (Fig. 1E, left) kDa, consistent with the predicted mass of hClC-3 and of hClC-5 tagged with GFP protein, respectively. pAb-218 antibody did not immunoprecipitate ClC-3 protein (Fig. 1D, lane 2) but detected the ClC-3 immunoprecipitated by Clcn2 antibody (Fig. 1D, lane 1). A slight cross-reactivity to ClC-5 protein was observed with pAb-218 (Fig. 1E, lane 2).
On the other hand, immunostaining of HEK293 cells stably expressing ClC-3 or ClC-5 was negative with pAb-218 (Fig. 1, D, a and E, a, for ClC-3 and ClC-5, respectively) and with Clcn2 (Fig. 1, D, b and E, b, for ClC-3 and ClC-5, respectively).
Expression of ClC-2 in Rat Tissues
The ClC-2 in rat tissues was analyzed by immunoprecipitation followed by immunoblotting with pAb-218, because tissue samples cannot be metabolically labeled. We first assessed the specificity of pAb-218 in rat brain membranes because ClC-2 is known to be abundant in this tissue (15). As expected, pAb-218 recognized a single 90- to 97-kDa protein band (Fig. 1F, lane 1). A protein of ~97 kDa was also immunoprecipitated from membrane preparations of rat colon, small intestine, and lung and detected with pAb-218 (Fig. 1F, lanes 2-4) or with Clcn2 antibody (not shown).Immunohistochemical Localization of ClC-2 in Rat and Human Tissues
All the tissues shown were fixed in acetone because this gave optimal fluorescent signals. Tissues fixed in paraformaldehyde were unsuitable for cytoimmunodetection of the protein, perhaps because the epitope involves lysine and arginine, two amino residues that are modified by these fixatives. Ethanol-fixed tissues showed no labeling.Rat colon and small intestine.
Both antibodies predominantly stained luminal enterocytes in the rat
colon (Fig. 2A, a
and c), whereas preabsorption of the antibodies with their
corresponding peptide antigens resulted in no staining (Fig.
2A, b and not shown). The staining with pAb-218 appeared to be mainly on the basolateral membranes and to a lesser extent in the cytoplasm (Fig. 2A, d). Similar
results were obtained with Clcn2, but with lower resolution of the
basolateral membranes and more diffuse cytoplasmic labeling (Fig.
2A, g). ClC-2 staining was weaker in the cells
lining the crypts and was observed only in the upper two-thirds of the
crypts. The basolateral membranes were faintly labeled by the pAb-218
antibody (Fig. 2A, e), but little or no staining
was detected with Clcn2 (Fig. 2A,
f).
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Human colon and small intestine.
There was significant ClC-2 immunostaining in the enterocytes lining
the luminal surface of the human colon, but the subcellular staining
pattern differed from that observed in the rat. The expression of ClC-2
was not present at the basolateral membranes, but pAb-218 mainly
stained a cytosolic supranuclear region (Fig.
3a), and a discrete, punctate
staining was detected at the apical pole (Fig. 3, a,
f, and h). Omission of the primary antibody or
application of preabsorbed antibody yielded no detectable signal (Fig.
3b). Staining with Clcn2 was similar but less intense (Fig.
3c). Intracellular ClC-2 was also detected with pAb-218 in
the upper two-thirds of the crypts (Fig. 3d), but cells at
the base of the crypts were not stained (Fig. 3e). The
punctate apical staining pattern observed with both antibodies was
reminiscent of ClC-2 staining at the tight junction complex, previously
described in the murine small intestine (29). However, we
did not observe colocalization of ClC-2 with the tight junction protein
ZO-1, indicating a different subcellular expression at or near the
apical membrane (Fig. 3f). Figure 3, g and
h, shows labeling of the lateral membranes by an antibody
against the adhesion protein E-cadherin. Hence, the absence of ClC-2
expression at the basolateral membranes as observed in rat colonic
cells could not simply be explained by degradation of the tissue.
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Rat and human airway epithelium.
ClC-2 staining with the pAb-218 antibody was mainly localized in the
apex of the ciliated cells in the rat bronchial epithelium (Fig.
4A,
a and b). The punctate labeling of the
peribronchial connective tissue was not specific, because it was also
present in control sections (Fig. 4A, c). The
Clcn2 antibody stained the same structures of the ciliated cells,
but the signal was much weaker (Fig. 4A, d).
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DISCUSSION |
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In this work we have analyzed the pattern of ClC-2 distribution in intestinal and airway epithelia. The main tool used was a new polyclonal antibody, pAb-218, raised against a COOH-terminal sequence (AA 847-862) that is identical in the rat and human ClC-2. Of note, the rat sequence (AA 888-906) used to raise the other antibody tested, Clcn2, differs by two amino acids from the human ClC-2. Clcn2 detected the denatured protein (11), whereas pAb-218 antibody preferentially recognized the native conformation of ClC-2. Neither pAb-218 nor Clcn2 recognized ClC-3 and ClC-5 isoforms in immunocytochemistry experiments. In immunoprecipitation experiments, a slight cross-reactivity with the two ClC isoforms was observed for both anti-ClC-2 antibodies but only when immunoprecipitation was performed with high amounts of ClC-3 or ClC-5.
There were few differences in the subcellular localization of ClC-2 labeling with the two antibodies; however, within the same cellular structures, staining with pAb-218 was more accentuated.
Intestine Epithelia
In the rat, both antibodies stained the villus cells of the small intestine and the surface enterocytes of the colon. It has been reported that the expression of ClC-2 in the rat intestine is downregulated during late gestation (42), but Gyömörey et al. (29) detected ClC-2 in the small intestine of 6- to 8-wk-old mice. Although we did not compare fetal and adult tissues, our results agree with this latter study and further demonstrate that ClC-2 protein is significantly expressed in intestinal tissues of adult rats. However, the subcellular distribution of ClC-2 reported here differs from that reported for the mouse (29). Those authors used an antibody generated against a NH2-terminal epitope and detected ClC-2 predominantly at the tight junction complex between the enterocytes of the small intestine (29). The discrepancy between this restricted distribution and the expression all along the lateral membranes found by us may be explained by different patterns of ClC-2 gene expression in the rat and mouse or by differences in the sensitivities of the antibodies used. We detected an intense fluorescent signal with pAb-218 at an antibody concentration 50-fold lower than that used in the mouse sections, but, in agreement with the preliminary observations of Gyömörey et al., we found a high level of ClC-2 expression at the basolateral membranes of the rat colon luminal cells. The labeling by both antibodies was found at the lateral or basolateral membranes; the labeling by Clcn2, but not by pAb-218, also extended to apical material in the villus cells or intracellular material in the luminal colon cells. Because pAb-218 did not stain these subcellular compartments, it remains difficult to conclude whether Clcn2 was more sensitive for detection of ClC-2 in the rat intestine or whether it additionally bound to cross-reactive material. Because Clcn2 antibody did not stain ClC-3- and ClC-5-expressing HEK cells, it is unlikely that the cross-reactive material corresponds to these isoforms, which can be expressed in the gut (32, 52).Our results reveal important differences between the subcellular distribution of ClC-2 in the rat and human intestine. The protein was found in surface colon enterocytes in both species, but ClC-2 immunolabeling was mainly present in an apically oriented perinuclear compartment, associated with discrete and punctate ClC-2 labeling at the apex of human colon cells. This pattern may indicate accumulation of the protein in the Golgi apparatus or in a distinct compartment involved in the polarity-dependent targeting of proteins (53). This is in keeping with our recent suggestion (3) that the number of ClC-2 channels in the membrane of human intestinal T84 cells may be regulated by vesicular transport. In addition, the intracellular localization of ClC-2 may point to a role in pH regulation in intracellular compartments, as demonstrated for other members of the ClC gene family (28, 36, 48).
The reason for the different distributions of ClC-2 expression in rat and human luminal colon cells is unknown. The overall amino acid sequences of the rat and human isoforms are 93% identical but differ in a putative protein kinase (PKA) consensus site in the COOH-terminal region of the human isoform, which is absent from the rat ClC-2 protein (13, 51). There are several examples of phosphorylation by PKA acting as a mechanism for protein targeting (2, 34, 54). Perhaps this phosphorylation site contributes to the different localization of the ClC-2 protein in human intestinal tissues, which would suggest different functions.
The lack of ClC-2 staining at the membranes of human crypt colonocytes
apparently contradicts functional data demonstrating ClC-2 currents in
two human intestinal cell lines, T84 cells (22), which are
representative of secretory crypt cells (19), and Caco-2
cells (41), which have been used to model absorptive as
well as secretory functions (6, 25). These results may suggest that the density of ClC-2 channels is lower in native and
normal crypt cells. However, it should be noted that that the amplitude
of the ClC-2 current in T84 cells varies from one cell to another,
being smaller than 100 pA at
120 mV in most cells
(22). The variable pattern of ClC-2 labeling in T84 cells demonstrated here is thus compatible with the various current amplitudes reported previously and with the absence or very low expression of ClC-2 in native crypt cell membranes.
In the rat, the high level of basolateral expression of ClC-2 channels
in absorptive cells of the villi and colon is more consistent with a
role for these channels in NaCl absorption than secretion.
Electroneutral NaCl absorption is the primary absorption process in
surface cells of the rat colon and enterocytes of the villi, via the
coupling of Na+/H+ exchanger (NHE)2/3 and
Cl/anion exchange. The luminal uptake of substrates for
Na+-coupled transport during absorption causes the cell to
swell, leading to volume regulatory activation of K+ and
Cl
channels at the basolateral membrane. The activation
of these channels not only limits cell swelling but maintains the
electrical driving force for continuous transport (44).
Thus, in accord with their osmotic sensitivity, ClC-2 channels at the
basolateral membrane may contribute to cell volume regulation and
Cl
exit during NaCl absorption. It is also interesting
that intestinal epithelial cells maintain pH microclimates both outside
and inside their plasma membranes and that the lateral intercellular
spaces and external basal domains are slightly acidic (pH ~6.7),
whereas their intracellular counterparts are more alkaline (12,
39). These pH gradients may be necessary for activation of NHEs
and Na+ absorption (39). Whether ClC-2 plays a
role in regulating the pH and ionic composition of these
microenvironments remains to be determined, but its location in
basolateral membranes fits well with its activation by extracellular
acidification or intracellular alkalinization.
In contrast to the rat, the presence of ClC-2 in the apex of human
luminal colonocytes, although relatively low, suggests that the channel
may contribute to Cl backleak in the secretory direction,
hence opposing the net transepithelial Cl
absorption,
which is predominant at this site.
Airways
ClC-2 is present in ciliated cells and probably in alveolar type II pneumocytes of rat and human adult airways. These findings disagree with previous reports showing a marked downregulation of ClC-2 mRNA and protein in the rat lung after birth (43). A recent study also showed that the amount of ClC-2 mRNA in the human pulmonary epithelium declines by the end of pregnancy and remains relatively low at all postnatal times (37). As discussed above, detection of the protein in adult tissues may be due to the great sensitivity of the pAb-218 antibody.The distribution of ClC-2 along the luminal membrane of developing
airways may be taken as evidence that ClC-2 contributes to
Cl secretion (43), which is the dominant ion
transport in the fetal lung (16, 59). It has been also
proposed that the relative acidic pH of fetal lung fluid may lead to
its activation (7). However, a ClC-like
hyperpolarization-activated Cl
current,
Ihyp-act, whose sensitivity to extracellular pH
differs from that of ClC-2, has been identified in adult murine nasal ciliated cells (50). Ihyp-act is
fully active over the pH range 5.4-9 and inhibited only when
extracellular pH is reduced to 5.0. This sensitivity resembles that of
members of the second branch of the ClC Cl
channel family
(21). The current-voltage relationship of
Ihyp-act also differs from those of ClC-2 and
other endogenous or expressed ClC channels. Because ClC-2, ClC-3, and
ClC-6 mRNAs have been described in tracheal epithelia
(37), Ihyp-act and the protein detected in the ciliated cells may represent a functional
heterooligomeric channel with different properties (38).
We found that ClC-2 expression in the ciliated cells was not restricted
to the apical membrane, which is further evidence that the number of
channels in the plasma membrane may be regulated by vesicle
trafficking. It is well established that the airway surface epithelium
essentially absorbs NaCl under normal conditions after birth. Although
the mechanisms of NaCl absorption by airway and intestine epithelia
differ, it remains intriguing that ClC-2 channels are mainly present in
those cells involved in fluid absorption. This raises the question of
their role in the regulation of this process. The presence of the
anionic exchanger DRA in the trachea (56) and the need for
an apical mechanism for removing HCO
ClC-2 as Alternative Chloride Pathway in CF
Our findings that there is no ClC-2 in human submucosal glands or in secretory enterocytes of the deep crypts, which are the major sites of CFTR expression (20, 49) may call into question the relevance of ClC-2 activation in bypassing defective CFTR to ensure sufficient Cl ![]() |
ACKNOWLEDGEMENTS |
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The authors thank Dr. Paul Fritsch for helpful discussions and comments. The English text was edited by Owen Parks. The authors thank Professors D. Jan, N. Labrousse, and C. Danel for obtaining human tissue samples.
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FOOTNOTES |
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This work was supported by the Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, and Association Vaincre la Mucoviscidose. J. Lipecka was supported by a fellowship from the French Ministry of Foreign Affairs, Association Vaincre la Mucoviscidose, and Fondation pour la Recherche Médicale. M. Bali was supported by a fellowship from Centre Volvic pour la Recherche sur les Oligo-éléments and Fondation pour la Recherche Médicale.
Address for reprint requests and other correspondence: A. Edelman, Institut National de la Santé et de la Recherche Médicale, Unité 467, Faculté de Médecine Necker-Enfants Malades, 75015 Paris, France (E-mail: edelman{at}necker.fr).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpcell.00291.2001
Received 26 June 2001; accepted in final form 16 November 2001.
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