1 Centro de Estudios Científicos, Valdivia, and 2 Instituto de Histología y Patología, Universidad Austral de Chile, Valdivia, Chile
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ABSTRACT |
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The principal function of the colon in fluid
homeostasis is the absorption of NaCl and water. Apical membrane
Na+ channels, Na+/H+ and
Cl/HCO
is unknown. We have
previously demonstrated the presence of the ClC-2 transcript in the
guinea pig intestine. Now we explore in more detail, the tissue and
cellular distribution of chloride channel ClC-2 in the distal colon by
in situ hybridization and immunohistochemistry. The patch-clamp
technique was used to characterize Cl
currents in
isolated surface epithelial cells from guinea pig distal colon and
these were compared with those mediated by recombinant guinea pig
(gp)ClC-2. ClC-2 mRNA and protein were found in the surface epithelium
of the distal colon. Immunolocalization revealed that, in addition to
some intracellular labeling, ClC-2 was present in the basolateral
membranes but absent from the apical pole of colonocytes. Isolated
surface epithelial cells exhibited hyperpolarization-activated chloride
currents showing a Cl
> I
permeability and
Cd2+ sensitivity. These characteristics, as well as some
details of the kinetics of activation and deactivation, were very
similar to those of recombinant gpClC-2 measured in parallel
experiments. The presence of active ClC-2 type currents in surface
colonic epithelium, coupled to a basolateral location for ClC-2 in the distal colon, suggests a role for ClC-2 channel in mediating
basolateral membrane exit of Cl
as an essential step in a
NaCl absorption process.
hyperpolarization-activated chloride currents; in situ hybridization; immunohistochemistry
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INTRODUCTION |
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SMALL AND LARGE
INTESTINES are sites of abundant fluid transport in mammalian
species. In humans, ~10 liters/day of fluid are absorbed in these two
segments of gut, most of it being absorbed in the small bowel with
~1,500 ml crossing the ileocecal valve. The colon extracts most of
this fluid, leaving only 100-200 ml of water per day. The colon is
also capable of increasing its absorptive capacity two- to threefold
when the small bowel losses are increased. The result of ion transport
is excretion of a stool containing <5 mM Na+, 2 mM
Cl, and 9 mM K+. This highly efficient
transport of large amounts of salt and water is due to polarized
colonic epithelial cells equipped with a number of ion channels,
carriers, and pumps, located either on the luminal or basolateral
aspects of the membrane. Several of these have been identified at
the molecular level (26).
It has generally been accepted that secretion and absorption in the colon are spatially separated in the intestine surface crypt axis. Absorptive processes are located in surface epithelial cells, whereas the secretion would be a property of crypt epithelial cell (20). This model might require some modification, because there is recent evidence that NaCl absorption might also occur in colonic crypts (16). Crypt cells express abundant CFTR chloride channels located in the apical membrane of the colonocytes. This constitutes the rate-limiting step in the transepithelial translocation of sodium chloride under the action of secretagogues (17).
In distal colon epithelium, depending on species or salt status, the
transport of NaCl is due to electroneutral absorption by luminal
Na+/H+ and
Cl/HCO
channel rat colonic crypts (11) and an outwardly
rectifying DIDS-sensitive Cl
channel of 20-90 pS
conductance in mouse crypts (29). No molecular counterpart
has been proposed for these channels.
ClC-2 belongs to the most numerous family of chloride channel proteins discovered so far. This ClC family consists of nine different mammalian members with sequence identity varying between 30 and 90% (21). ClC-2 is widely expressed in mammalian tissues, but its physiological role has not yet been ascertained. It has been suggested to be important in certain neurons, where ClC-2 is thought to be implicated in the control of intracellular chloride to regulate the effects the GABAA receptor action (38). ClC-2 has also been identified in the human colonic T84 epithelial cell line (5) where it was proposed to participate in fluid secretion not associated with the cAMP-dependent CFTR chloride channel (14). Similar speculations have been raised for other epithelial cells (4, 33, 35), but a function in transepithelial transport has not been clearly defined. Studies of ClC-2-deficient mice have recently shown a severe degeneration of the retina and the testes, which might be related to a deficient control of their ionic environment by the blood-testes and blood-retina barriers (3).
ClC-2 shows low activity under resting conditions but opens slowly on hyperpolarization (39). When expressed in amphibian oocytes, ClC-2 can be activated by hypotonic cell swelling (18). This is consistent with a role in regulatory volume adjustments, but evidence against such function has also been reported (1). It is of great interest to understand the possible physiological role of ClC-2 and particularly its function in determining the membrane conductance of epithelial and other cells. The possibility of altering the gating of ClC-2 is relevant given its potential as an alternative to CFTR in the impaired secretory state of cystic fibrosis patients (30, 35).
We (6) previously demonstrated the presence of the ClC-2
transcript in the guinea pig small intestine and colon and speculated that it might play a role in transepithelial transport. Other groups
have explored the possible function of ClC-2 in the epithelium of the
gastrointestinal tract. Sherry et al. (36) reported the presence of ClC-2 in the canalicular membrane of gastric parietal cells
where it would play a role in HCl secretion. Lipecka et al.
(27) have used immunohistochemical techniques to suggest that the location of ClC-2 is predominantly basolateral in rat colon,
but intracellular in human intestine, where it is seen mainly at a
cytosolic supranuclear region. Gyömörey et al.
(19) have used murine small intestine to show that ClC-2
is expressed at the tight junction between epithelial cells where it
colocalizes with zonula occludens-1 protein. Despite this
unexpected finding, functional data suggest that a ClC-2-like channel
activity contributes to Cl secretion, compatible with a
function as apical membrane conductance. A similar tight junctional
location for ClC-2 has been reported by Mohammad-Panah et al.
(30) in Caco-2 cells. In this colonic carcinoma cell line,
they demonstrate hypotonicity-induced anion secretion (measured with
I
), which is partially inhibited using a ClC-2 antisense approach.
The aim of this work is to explore, in more detail, the presence of
ClC-2 in the colon by in situ hibridization and immunohistochemistry and to obtain evidence of its function by electrophysiological means.
The results indicate that ClC-2 mRNA is preferentially expressed in
surface epithelium of guinea pig distal colon and is absent from the
crypt compartment. A similar result was obtained by immunocytochemistry
that, in addition, suggests the ClC-2 is absent from the apical aspect
of colonocytes but expressed at the basolateral membrane. Isolation of
surface epithelial cells followed by patch-clamp study shows currents
with the hallmarks of recombinant guinea pig (gp)ClC-2. This
includes Cl > I
permeability, sensitivity
to Cd2+ and kinetics of opening and closing similar to that
seen with recombinant gpClC-2. Cellular location and subcellular
distribution of ClC-2 in guinea pig colon suggest a function for this
channel in the NaCl absorption process.
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MATERIALS AND METHODS |
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Cell and tissue isolation. Male guinea pigs obtained from the Instituto de Salud Pública (Santiago, Chile) and weighing 200-400 g were used throughout the study. All experiments were done according to international regulations for animal care and were approved by the Bioethics Committee of the Centro de Estudios Cientificos, Valdivia, Chile. Surface colonocytes were isolated by a modification of previously published methods (10). Food was withheld for 48 h before animals were killed by using ketamine overdose. A segment of distal colon ~8 cm in length was rinsed with warmed 0.9% sodium saline. The segment was filled with solution A containing (in mM) 44 K2HPO4, 7 K2SO4, 10 sodium citrate, 180 glucose, and 10 HEPES, pH 7.4. The segment was tied at both ends under moderate pressure and incubated in solution A at 37°C for 10 min. The intraluminal solution was then changed by solution B (in mM): 44 K2HPO4, 7 K2SO4 , 10 Na2EDTA, 180 glucose, 0.5 DTT, and 10 mM HEPES, pH 7.4 and incubated for 3 min at 37°C. Surface epithelial cells were released by gentle mechanical disruption and recovered from the luminal solution. After isolation, the colonocytes were washed and suspended in solution A and maintained at 4°C. It has been demonstrated that the cell fraction used here is devoid of dividing cells as judged by bromodeoxiuridine labeling (10). In addition, the cells used for patch-clamp retained the characteristic "figure-of-eight" morphology of highly differentiated surface cells that is also observed in villus cells from small intestine (31). No release of crypts, which are easily identifiable morphologically, occurred during the rather mild procedure used here.
In situ hibridization. Nonradioactive in situ hybridization was carried out using digoxigenin-labeled riboprobes. Two pairs of antisense and sense probes of 250 and 300 bp, respectively, were synthesized from gpClC-2 -3' and -5' untranslated ends (GenBank accession no. AF113529). After plasmid linearization, in vitro transcription was performed with T7 and T3 RNA polymerases according to a previously published protocol (9). Cryosections (7-µm-thick) obtained from colon segments were fixed in 4% paraformaldehyde for 30 min at room temperature. The endogenous peroxidase was inactivated with H2O2 at 0.3% in methanol. Subsequently, the sections were permeabilized with 0.2% Triton X-100 in PBS and acetylated using 0.1 M triethanolamine, pH 8.0, plus 0.25% acetic anhydride. After 3-h prehybridization, the sections were incubated at 65°C overnight in a solution containing 50% formamide and a final mix of probes concentration of 0.2 ng/µl. After the hybridization, the samples were treated with 40 µg/ml RNAase at 37°C for 45 min. After washing with 2× SSC and 0.1× SSC for 15 min at 65°C twice each, the endogenous biotin activity was blocked (Biotin blocking reagent; DAKO). The slides were then incubated with an horseradish peroxidase-coupled anti-digoxigenin antibody diluted 1:100 for 45 min at room temperature. Sections were incubated with biotinyl-tyramide and with streptavidin-horseradish peroxidase solution following the manufacturer's instructions (GenPoint kit; DAKO). Peroxidase was developed using a diaminobenzidine-substrate-chromogen system, and methyl green was used for counterstaining.
Immunohistochemistry. Samples obtained from guinea pig colon were fixed in Bouin's fluid or periodate-lysine paraformaldehyde at room temperature for 24-48 h (28). The tissue blocks were dehydrated in a graded series of ethanol and embedded in Histosec (Merck). Sections (5-µm-thick) were mounted on glass slides previously coated with polylysine (Sigma-Aldrich). Tissue sections were dewaxed with xylene, rehydrated through a graded series of ethanol and treated with absolute methanol and 1% hydrogen peroxide to block endogenous pseudoperoxidase activity. After being rinsed several times in 0.05 M Tris · HCl buffer, pH 7.8, the sections were incubated overnight with an anti-peptide antibody raised in rabbits against the synthetic peptide RSRHGLPREGTPSDSDDKC corresponding to residues 888-906 of rat ClC-2 (1:100 to 1:400) (Alomone Labs, Israel). Once incubation was completed, the sections were rinsed three times for 5 min each with 0.05 M Tris · HCl buffer and incubated with a biotinylated anti-rabbit antibody and streptavidin-peroxidase (Kit LSAB+; DAKO), for 15 min each. Peroxidase was developed for 5 min using a commercial liquid diaminobenzidine-substrate-chromogen system (DAKO). All incubations were carried out at 22°C in a water bath that was used as a moist chamber. When immunostaining was completed, the sections were rinsed with distilled water and contrasted with Harris hematoxylin for 30 s. Finally, the sections were dehydrated in ethanol, cleared with xylene, and mounted using Canada balsam. In addition, acetone-fixed frozen sections were also used, and the bound anti-ClC-2 antibody was detected by a fluorescein-labeled anti-rabbit (Fab')2 immunoglobulin (DAKO). Sections were analyzed by laser confocal microscopy. Controls of the immunostaining procedure included omission of the specific antibody, replacement by nonimmune rabbit serum and incubation with the specific antibody in the presence of an excess of the same peptide used for immunization (25 to 50 µg/ml).
Electrophysiological studies.
The experiments using fresh isolated colonocytes were performed in
cells at room temperature in 35-mm diameter polylysine-treated plastic
petri dishes mounted directly on the stage of an inverted microscope.
The bath solution contained (in mM) 140 NaCl, 2 CaCl2, 1 MgCl2, 22 sucrose, 10 HEPES pH 7.4. Alternatively, a 16 mM
Cl solution was made by equimolar replacement with
gluconate or I
. The pipette solution (35 mM
Cl
) contained (in mM) 100 Na gluconate, 33 CsCl, 1 MgCl2, 2 EGTA, 1 ATP, and 10 HEPES, pH 7.4. The gpClC-2
plasmid used in the electrophysiological studies is in an expression
vector under the control of the cytomegalovirus promoter
(pCR3.1; Invitrogen). HEK-293 cells used for transient transfections
were grown in DMEM/F-12 supplemented with 10% fetal bovine serum at
37°C in a 5% CO2 humidified incubator. At 60-80% confluence, the cells were cotransfected with 1.5 µg of total expression plasmids for gpClC-2 and
H3 in a 3:1 ratio using
Lipofectamine Plus (Life Technologies). Expression of CD-8 antigen was
used as a means to identify effectively transfected cells within the dish (22). After 24-48 h, the cells were incubated
briefly with microspheres coated with an antibody against the CD8
antigen (Dynabeads). The experiments were performed in bead-decorated
cells at room temperature. Solutions were as described for isolated
colonocytes experiments.
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RESULTS |
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Distribution of gpClC-2 transcript in distal colon epithelium.
The location of ClC-2 transcript distal colon epithelium was determined
by in situ hybridization with digoxigenin-labeled riboprobes. Figure
1, A and B shows
distal colon cryosections hybridizing antisense and sense gpClC-2
riboprobes, respectively. Prominent staining with the antisense probe
was seen only on surface epithelium, whereas there was no apparent
staining over crypts or nonepithelial tissue (Fig. 1A). No
background staining was observed in control sections hybridized with
the sense probe (Fig. 1B). Figure 1, C and
D, shows higher magnification pictures confirming the
presence of prominent staining of surface epithelium colonocytes (Fig.
1C) with absence of staining over crypt epithelial cells (Fig. 1D). Similar results were obtained with proximal colon
cryosections (not shown).
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Location of gpClC-2 by immunohistochemistry.
The distribution of ClC-2 in guinea pig distal colon was studied by
immunoperoxidase and immunofluorescence. Figure
2B shows a low magnification
view of a distal colon section reacted with anti-ClC-2 antibody and
revealed with a peroxidase reaction. There was strong immunostaining
over surface epithelium with no staining over the crypt region. In
contrast with the transcript distribution, there was also staining
present in the muscularis mucosae. Figure 2, A and
C, shows higher magnification views of surface cells in
which subcellular distribution of ClC-2 was examined. There was diffuse
immunolabeling over what appears to be the cytosolic compartment, with
a defined staining delineating the limit between the cells and also at
the basal pole of surface colonocytes (arrows in Fig. 2, A
and C). The label was absent from nuclei and also from the
apical border of the cells (arrows in Fig. 2D). A control section demonstrates that preincubation with the antigenic peptide blocked immunolabeling (Fig. 2E). No immunostaining was
observed in goblet cells in the vicinity of the colonocytes.
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Hiperpolarization-activated chloride currents in isolated
colonocytes.
The evidence presented above suggests that ClC-2 is expressed in
surface epithelial cells in guinea pig distal colon. To verify whether
currents consistent with this expression were present in these cells,
surface colonocytes were isolated by established methods
(10) and assayed by patch-clamp. Initial experiments without ATP in the pipette gave occasional hyperpolarization-activated currents that were consistently found after inclusion of the
nucleotide. Figure 4B shows a
family of currents elicited in an isolated colonocyte by the voltage
protocol shown in Fig. 4A. As described before for
recombinant gpClC-2, there were small currents at positive or
moderately negative potentials, but larger currents activated slowly
with strong hyperpolarization. In 20 separate cells, the mean current
observed at 140 mV was
520 ± 87 pA. A postpulse to 40 mV
shows typical slow deactivation of tail currents. Figure 4C
shows that replacement by gluconate of all but 16 mM Cl
in the bathing medium had little effect on inward currents. This replacement, however, substantially decreased outward currents observed
at positive main pulses and tail currents, consistent with them being
due to Cl
influx. Figure 4D shows currents in
high and low Cl
obtained after activation to
140 mV in
response to a 100-ms voltage ramp taking the potential to 40 mV. The
relationship was almost linear with a reversal potential of
approximately
40 mV [equilibrium potential for chloride
(ECl) =
37 mV]. On partial replacement of
Cl
with gluconate, there was a displacement of the
reversal potential to a more positive value and a general decrease in
current with a tendency to inward rectification. This result is similar
to that obtained with recombinant gpClC-2 as illustrated in the
current-voltage relations shown in Fig. 4E.
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DISCUSSION |
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The colonic epithelium has secretory and absorptive functions
(26). Absorptive transport consists mainly of short-chain fatty acid transport and NaCl absorption accompanied by water, thus
allowing for only minor losses of water in the feces, depending on the
species. Secretory processes are set into action by the so-called
secretagogues that through second messengers, such as cAMP or
Ca2+, promote the secretion of NaCl and water. The step
governing the rate of secretion is the output of Cl from
epithelial cells, where it is accumulated above electrochemical equilibrium. The permeability pathway for Cl
exit is
provided by CFTR, a ClC activated by phosphorylation (17).
CFTR is defective in cystic fibrosis, thus leading to reduced salt and
fluid secretion. There is, therefore, a great deal of interest in
finding possible alternative pathways for Cl
exit that
could be activated to replace the defective conductance. A candidate
that has been repeatedly proposed to play such a role is the inwardly
rectifying ClC-2. Currents that could presumably be associated with
ClC-2 expression have been detected in T84 and Caco-2 cells
(14, 30), and in the Caco-2 line, it has been proposed to
mediate anion secretion not associated with CFTR function. ClC-2-like
currents have been reported in other cells capable of fluid secretion
such as pancreatic acinar cells (4), parotid acinar cells
(33), and mandibular duct gland cells (25), but their function in transepithelial transport has not been clearly defined. Assigning currents to a molecular counterpart, however, can be
difficult as has been shown recently for hyperpolarization-activated Cl
currents of choroid plexus that have some resemblance
with ClC-2 except for an I
> Cl
permeability (23). These have now been reported to remain
unchanged in a ClC-2 knockout mouse and cannot, therefore, be
attributed to this channel (37). In contrast, loss of
hyperpolarization-activated Cl
current in salivary acinar
cells from Clcn2 knockout mice has firmly established the
correspondence of the current and ClC-2 (32).
Absorption of NaCl and water in colonic epithelium can be electrogenic
or electroneutral (26). The first is mediated by ENaC
located in the apical membrane and providing the entry step for
Na+ that is followed by pump-mediated efflux at the
basolateral membrane. For electroneutral absorption, the entry step is
provided by parallel exchangers of Na+/H+ and
Cl/HCO
conductive pathway in the basolateral membrane has
been proposed for both electrogenic and neutral NaCl transport, as
shown in the models discussed by Kunzelmann and Mall (26)
in Figs. 2 and 3 of their review. The only Cl
conductance
that has been described in basolateral membranes of colonocytes is a
volume-regulated, outwardly rectified conductance of isolated crypts
(11). Outwardly rectified channels with
I
> Cl
permeability of small
intestinal villus enterocytes have been proposed to be basolateral and
involved in Cl
absorption (31). The
molecular identity of the channels is unknown.
In the present work, we present data demonstrating the presence of ClC-2 in the colonic epithelium both at the transcript and protein level. In addition, we demonstrate a functional activity in isolated colonocytes consistent with the presence of active ClC-2 in the epithelium. We argue that ClC-2 might be a good candidate to be involved in NaCl absorption across the epithelium by providing the required exit pathway at the basolateral membrane of absorptive cells.
NaCl absorption and secretion in colonic epithelium has classically
been assumed to be the property of separate compartments. The surface
epithelium would be mainly absorptive, whereas the crypts would be the
site of secretion (26). This contention had a rather
convincing functional demonstration in now classical experiments
showing secretion of fluid emerging from crypt mouths (41). Such a compartmentalization also agrees well with
the tissue distribution of the membrane transport systems known to contribute to these processes. The Na+/H+
exchanger is typically associated with apical membranes of epithelia, and in the colon, it is expressed in surface epithelial cells (2). The ENaC is also expressed in the apical membrane of
surface colonocytes (13). CFTR, the
phosphorylation-regulated Cl channel, is mainly expressed
in the crypt epithelium (40). Despite this evidence,
controversy remains as to this strict separation of functions. There
are reports of NaCl absorption in the crypts (16) and of
secretion in surface epithelium (24). In the present paper, we demonstrate that ClC-2 transcript is exclusively expressed in
the surface epithelium. If present in the crypt, ClC-2 is only in the
upper reaches of these glands. The same distribution is observed when
using an anti-ClC-2 antibody in immunohistochemical analyses. The
immunoreactivity is present in the surface epithelium as a diffuse
labeling within the cells and as a more defined label delineating the
basolateral aspect of colonocytes. Previous intestinal immunohistochemical studies show intracellular, but not nuclear, staining and a suggested basolateral location for ClC-2 in rat colon,
but only intracellular in human large intestine (27). Studies with murine small intestine show that ClC-2 expressed at the
tight junction between epithelial cells exclusively (19). Our results are inconsistent with an apical membrane location, which
would rule out a role for ClC-2 in a secretory process. We cannot be
sure about the diffuse cytoplasmic immunolabeling observed here. If it
reflects ClC-2 expression, one could speculate that cytoplasmic
immunolabeling reveals an intracellular pool of channels that might be
targeted to the membrane under appropriate stimulus. A similar
speculation has been proposed to explain the intracellular location of
ClC-2 in the human colon (27). Clear immunolabel
delineation of the basolateral aspect of the cells observed in the
present work could be due to the presence of the channel protein in
that membrane domain. If this interpretation were correct, it would
lead to the prediction that functional channel activity compatible with
ClC-2 expression should be observed in colonocytes. We tested this by
direct comparison of ion currents in surface epithelial cells isolated
from guinea pig distal colon and those generated by transfection of the
recombinant channel into HEK-293 cells.
Whole cell patch-clamp studies in surface epithelial cells from guinea
pig distal colon demonstrated the presence of Cl currents
that activated slowly in response to hyperpolarization. This gave rise
to inwardly rectifying currents strongly reminiscent of recombinant
gpClC-2. Unlike what is seen with the recombinant channel, small
outward Cl
current was also observed, perhaps suggesting
a degree of contribution of channels other than ClC-2. Comparison of
the kinetics of activation of Cl
colonocyte current with
that generated by gpClC-2 expression, however, gives an astonishing
agreement. This strongly suggests that, at least at these
hyperpolarized potentials, the contribution of a ClC-2 current is
predominant. For deactivation, the kinetics is closer to that seen for
a splice variant of gpClC-2 (gpClC-2
77-89), which is
characterized by faster deactivation kinetics. Interestingly, this
variant is best represented in guinea pig colonic epithelium (6). Experiments of partial replacement of extracellular
Cl
with gluconate in colonocytes and in cells
overexpressing gpClC-2 are also in agreement. In addition to the
difference in permeability, there is evidence for modulation of the
gating by the Cl
concentration suggested by the observed
decrease in inward current in both types of experiments.
Two other pieces of evidence suggest that the Cl currents
recorded in isolated colonocytes are the expression of ClC-2 channels present in their membranes. The first is their sensitivity to relatively low concentration of Cd2+, which has been
demonstrated in rat ClC-2 (7). This divalent cation is
without any effect on the widely distributed Cl
channels
activated by increases in cell volume (1).
Cd2+ blocks the Cl
currents of guinea pig
colonocytes in a very similar fashion as it does the overexpressed
gpClC-2. Perhaps more importantly, ClC-2 has a permeability sequence in
which PCl is greater than PI (15,
39). As shown here for gpClC-2, I
also inhibits
the current markedly. These characteristics are reproduced in the
Cl
currents in colonocytes. The functional data, together
with the transcript and protein analyses are, therefore, consistent
with the presence of active ClC-2 channels in surface epithelial cells from distal colon.
Is the conductance mediated by ClC-2 compatible with electroneutral
Cl absorption? To answer this question would require
knowledge of the transepithelial flux of Cl
mediated via
the operation of the Na+/H+ and
Cl
/HCO
flux has been measured in guinea pig
colon (8). Under resting conditions the colon epithelium
shows a small secretory Cl
flux, which is increased
fivefold by theophylline. This occurs on the background of a relatively
constant mucosa-to-serosa flux of ~3
µeq · cm
2 · h
1, which we
may assume to be due to electroneutral Cl
absorption in
this short-circuited epithelium. ClC-2 current at
60 mV, deemed a
physiological potential, is ~40 pA in the cell illustrated in Fig.
4B, and in five separate experiments, it was 49 ± 21 pA. If one assumes that there are 2 × 106 cells per
cm2, with surface ClC-2-expressing cells forming virtually
a planar continuous epithelial layer, a flux of ~4
µeq · cm
2 · h
1 is
obtained. This is in agreement with measured mucosa-to-serosa Cl
flux and would suggest that ClC-2 conductance might be
enough to account for absorption of this anion.
The ClC-2-like current described here in surface colonocytes has not
been studied in guinea pig crypts. It does not appear, however, to be
present in rat or mouse crypt colonocytes. As reviewed by Schultheiss
and Diener (34), rat crypt cells possess a slightly outwardly rectified basolateral Cl conductance, besides
that regulated by cAMP and that corresponding to CFTR. Rectification
properties, single channel conductance, and anion selectivity separate
this anion permeability pathway from ClC-2. Outwardly rectified
Cl
channels are the only anion channels detected in a
study of the basolateral membrane of mouse colonic crypts
(29). Again selectivity and other properties are clearly
different from those of ClC-2.
In summary, we demonstrate that ClC-2 mRNA and protein are located in
the surface epithelium of the distal colon. Immunolocalization reveals
that, in addition to some intracellular labeling, ClC-2 is present in
the basolateral membranes but absent from the apical pole of
colonocytes. Isolated surface epithelial cells exhibited hyperpolarization-activated chloride currents showing
voltage-dependence, a Cl > I
permeability
and Cd2+ sensitivity indistinguishable from those measured
in parallel experiments with recombinant gpClC-2. The presence of
active ClC-2-mediated currents in surface colonic epithelium, coupled
to a basolateral location for ClC-2 in the distal colon,
suggests a role for ClC-2 channel as the basolateral membrane exit
pathway for Cl
as part of the NaCl absorption process.
Confirmation or otherwise of these speculations will require the study
of transepithelial Cl
fluxes and their sensitivity to
ClC-2 inhibitors and regulation by changes in cell volume and
extracellular pH.
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ACKNOWLEDGEMENTS |
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We are grateful to Inés Siegmund, Pamela Ehrenfeld, and José Sarmiento for generous help during this work.
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FOOTNOTES |
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This study was funded by Fondecyt Grants 1990939 and 1000622 and an Equipment Grant from Fundación Andes. Support to the Centro de Estudios Científicos (CECS) from Empresas CMPC is also acknowledged. F. V. Sepúlveda was an International Research Scholar of the Howard Hughes Medical Institute and a Fellow of the J. S. Guggenheim Foundation. CECS is a Millennium Science Institute.
Address for reprint requests and other correspondence: L. P. Cid, Centro de Estudios Científicos, Avenida Arturo Prat 514, Casilla 1469, Valdivia, Chile (E-mail: pcid{at}cecs.cl).
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/ajpgi.00158.2002
Received 26 April 2002; accepted in final form 23 May 2002.
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