Departments of 1 Medicine, 2 Neurology, 3 Pediatrics, and 4 Cell Biology, University of Colorado Health Sciences Center, Denver, Colorado 80262
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ABSTRACT |
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Membrane Cl channels play an important role in
cell volume homeostasis and regulation of volume-sensitive cell
transport and metabolism. Heterologous expression of ClC-2 channel cDNA leads to the appearance of swelling-activated Cl
currents, consistent with a role in cell volume regulation. Since channel properties in heterologous models are potentially modified by
cellular background, we evaluated whether endogenous ClC-2 proteins are
functionally important in cell volume regulation. As shown by whole
cell patch clamp techniques in rat HTC hepatoma cells, cell volume
increases stimulated inwardly rectifying Cl
currents when
non-ClC-2 currents were blocked by DIDS (100 µM). A cDNA closely
homologous with rat brain ClC-2 was isolated from HTC cells; identical
sequence was demonstrated for ClC-2 cDNAs in primary rat hepatocytes
and cholangiocytes. ClC-2 mRNA and membrane protein expression was
demonstrated by in situ hybridization, immunocytochemistry, and Western
blot. Intracellular delivery of antibodies to an essential regulatory
domain of ClC-2 decreased ClC-2-dependent currents expressed in HEK-293
cells. In HTC cells, the same antibodies prevented activation of
endogenous Cl
currents by cell volume increases or
exposure to the purinergic receptor agonist ATP and delayed HTC cell
volume recovery from swelling. These studies provide further evidence
that mammalian ClC-2 channel proteins are functional and suggest that
in HTC cells they contribute to physiological changes in membrane
Cl
permeability and cell volume homeostasis.
hepatocyte; purinergic receptors; liver
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INTRODUCTION |
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CELL VOLUME IS A DYNAMIC
PARAMETER that is closely coupled to physiological changes in
solute transport, intracellular metabolism, and membrane ion
permeability. In most cells, increases in volume are followed after a
delay by increases in membrane K+ and Cl
permeability of 20-fold or more. The resulting efflux of ions represents a critical adaptive response that favors passive water loss
and restoration of cell volume toward basal values (1). Volume recovery is usually incomplete, however, and emerging evidence suggests that small residual differences from baseline act as a signal
that directly influences a broad range of cellular processes, including
gene expression, kinase activation, metabolism, and membrane transport
(15). Consequently, molecular identification of the
channels involved represents an important focus for defining the
mechanisms that link changes in cell function to hormonal and other
pathways that alter the cellular hydration state.
Recently, complementary DNAs encoding multiple members of the ClC
family of voltage-gated Cl channels have been identified.
ClC-2 transcripts are distributed broadly in most mammalian tissues,
including secretory epithelia such as lung, kidney, and liver
(20, 21, 35). Membrane hyperpolarization, hypotonic
exposure, and extracellular acidity have been shown to activate
inwardly rectifying Cl
-selective currents following
expression of ClC-2 proteins in different model systems (2, 12,
14, 18, 27, 32, 40). Although heterologous expression of ClC-2
enhances cell volume recovery from swelling (10, 40), the
biophysical properties of these currents are distinct from the
outwardly rectifying, volume-sensitive currents typical of most
mammalian cells (8, 25, 31, 33, 37, 39). Thus the role of
native ClC-2 channels as endogenous regulators of cell volume has not
been firmly established.
In liver epithelial cells, Cl current activation during
volume increases is regulated by a sensitive autocrine signaling
pathway involving release of the purinergic agonist ATP into the
extracellular space and subsequent stimulation of P2 receptors coupled
to membrane Cl
channels (23, 38). To assess
whether natively expressed Cl
channels encoded by ClC-2
contribute to volume-sensitive changes in membrane Cl
permeability in liver epithelia, rat liver ClC-2 cDNA, mRNA, and
membrane-associated protein were identified. Selective inhibition of
ClC-2 proteins by intracellular delivery with antibodies to an
essential regulatory domain 1) decreased heterologous ClC-2 currents in HEK-293 cells, 2) inhibited activation of native
currents in HTC hepatoma cells during increases in cell volume and P2
receptor stimulation, and 3) delayed HTC cell volume
recovery from swelling. These findings are consistent with the concept
that endogenous ClC-2 channels are functionally active and contribute
to volume-sensitive changes in membrane Cl
permeability.
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MATERIALS AND METHODS |
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Cells and solutions.
Most studies were performed using HTC rat hepatoma cells, which possess
metabolic pathways similar to those found in primary hepatocytes.
Previous studies indicate that recovery from HTC cell swelling depends
on activation of separate K+- and
Cl-selective whole cell currents (3). HTC
cells were grown at 37°C in 5% CO2-95% air atmosphere
in MEM containing 5% fetal calf serum, 2 mM L-Gln, 100 IU/ml penicillin, and 100 µg/ml streptomycin (GIBCO BRL). For
physiological studies, culture medium was replaced with a standard
isotonic extracellular buffer, which contained (in mM) 140 NaCl, 4 KCl,
1 KH2PO4, 2 MgCl2, 1 CaCl2, 10 glucose, and 10 HEPES (pH 7.4 with NaOH).
ClC-2 cDNA isolation.
An amplified HTC cell cDNA library (Superscript Lambda system, GIBCO
BRL; provided by N. Lomri, University of California at San Francisco)
was screened with a 477-bp oligonucleotide cDNA probe spanning the
D12-D13 loop of rat brain ClC-2 (gift of J. Cuppoletti and D. Malinowska, University of Cincinnati). In addition, cDNAs were
synthesized from HTC cell, primary rat hepatocyte, and normal rat
cholangiocyte mRNA using the high- fidelity Pfu DNA
polymerase (Stratagene). cDNAs were sequenced by the chain termination
method using [-35S]dATP and T7 DNA polymerase
(Sequenase version 2.0; Amersham Life Science), and by the 373A DNA
Sequencer (Taq DyeDeoxy terminator cycle sequencing kit; Applied
Biosystems) using the AmpliTaq polymerase.
In situ hybridization. For both in situ hybridization and immunocytochemistry, HTC cells on plastic coverslips (Starstedt) were fixed with 4% paraformaldehyde for ~12 h. In situ hybridization was performed using digoxigenin-labeled sense and antisense riboprobes directed against the amino terminal domain of ClC-2 as previously described (29).
ClC-2 antibody development and immunocytochemistry. Rabbits were immunized by standard protocols with a purified glutathione-S-transferase (GST)-ClC-2 fusion protein containing amino acids 20-68 in frame with pGEX-2T (Pharmacia). The peptide target region includes the putative essential and modulatory domains previously characterized; deletions within these regions have been shown to induce open phenotypes that are either insensitive (essential domain) or sensitive (modulatory domain) to volume changes (12). Serum was depleted of GST, and antibodies were purified by affinity chromatography using a cyanogen bromide-activated Sepharose 4b affinity matrix. Antibody staining was detected by the avidin-biotin-peroxidase method as previously described (32). For control studies, cells were coincubated with antibodies and either purified GST or GST-ClC-2 at 100 µg/ml. These antibodies were used for in situ, Western blot, whole cell patch clamp, and cell volume experiments.
Western blot analysis.
Total membrane fractions were separated from the cytosolic fraction by
subjecting the cell lysate to high-speed centrifugation. Briefly, cells
were disrupted by agitating the cells in ice-cold 1 mM
NaHCO3 for 30 min. After light homogenization, the
homogenate was cleared of nuclei and large debris by centrifuging at
10,000 g for 15 min. The supernatant was subjected to
centrifugation at 190,000 g for 45 min. The pellet was
solubilized in 5× PAGE buffer (5% SDS, 25% sucrose, 50 mM Tris, 5 mM
EDTA, 5% -mercaptoethanol, and protease inhibitor cocktail). The
supernatant was lyophilized and solubilized in an equal volume to the
pellet sample. Equivolume membrane and cytosol samples were assayed for
ClC-2 content by Western blotting, as previously described
(6); antibody dilutions were 1:500 (primary) and
1:20,000 (secondary). Samples enriched in plasma membranes were
isolated by sucrose gradient centrifugation (5). Briefly,
HTC cells were disrupted and homogenized as described above. The
homogenate was then centrifuged at 27,000 g for 15 min. The
pellet was resuspended in 65% sucrose and adjusted to 44% using a
refractometer. This sample was overlaid with 41% sucrose and 38%
sucrose and centrifuged at 190,000 g for 70 min, and the plasma membrane fraction was recovered from atop the 38% step. The
recovered plasma membrane fraction was then solubilized in 5× PAGE
buffer and used for immunoblotting for ClC-2 and
Na+-K+-ATPase.
Heterologous expression of ClC-2 in HEK-293 cells. Transfection of immortalized human embryonic kidney cells (HEK-293 cell line) grown at low density was accomplished by standard calcium phosphate precipitation methods with some modification (13, 26). In brief, cells were grown at 37°C and 5% CO2 on coverslips in multiwell (no. 24) plates containing DMEM supplemented with fetal bovine serum (1:10), ampicillin (0.1 mg/ml), and gentamicin (0.04 µg/ml). Precipitated DNA (500 ng/well) was added to achieve a final concentration of 2-5 ng/µl. Cotransfection was accomplished by adding equal amounts of the ClC-2 and green fluorescent protein (GFP) plasmid DNA for a total of 500 ng/well. For GFP-only controls, equal amounts of GFP and nonspecific salmon sperm DNA was added to achieve the same relative DNA ratios. The plasmid (pAC-CMV) containing the full- length coding region for ClC-2 or a construct containing the coding sequence for GFP (pEGFP-N1; Clontech), both under the control of the cytomegalovirus immediate-early promoter (CMVIE), were used in these experiments. Cells were allowed to grow overnight, after which the culture medium was replaced with standard supplemented DMEM (see above) and then grown for another 24-48 h before experimentation. Cells expressing GFP were assumed to have been transfected with ClC-2. GFP expression was observed within 12-16 h of transfection and appeared stable in culture for as long as 4 days. Individual GFP-expressing cells were identified for patch clamp analysis.
Analysis of Cl currents: whole cell
patch clamp recording.
Whole cell currents were measured using patch clamp recording
techniques as previously described (38). Cell volume
increases were induced by exposure to a buffer containing 20% less
NaCl or by addition of 50 mM sucrose to the pipette solution as
indicated. The standard pipette (intracellular) solution contained (in
mM) 130 KCl, 10 NaCl, 2 MgCl2, 10 HEPES/KOH (pH ~ 7.2), and free Ca2+ adjusted to ~100 nM (0.5 CaCl2, 1 EGTA) with a total Cl
of 145 mM
(4). Non-ClC-2 currents were inhibited by addition of DIDS
(final concentration 100 µM; Calbiochem). Membrane Cl
currents during cytosolic dialysis with ClC-2 antibodies (0.005 µg/ml
final concentration in the pipette solution) were compared with control
currents using 1) standard pipette solutions devoid of
antibodies, 2) heat-inactivated ClC-2 antibodies (100°C
for 30 min, 0.005 µg/ml), 3) polyclonal rabbit antibodies
to
-galactosidase (5 µg/ml, 5 Prime
3 Prime), and 4)
antibody buffer without ClC-2 antibodies containing 0.0001% sodium
azide. Recordings were made beginning ~15 min after achieving the
whole cell configuration using an Axopatch ID amplifier (Axon
Instruments, Foster City, CA). Current-voltage relationships were
measured by 400-ms steps to test potentials between
120 mV and +100
mV in 20-mV increments or other protocols as indicated. In the whole
cell configuration, Vp corresponds to the membrane
potential, and upward deflections of the current trace indicate outward
membrane current. Whole cell currents (pA) and current density (pA/pF)
refer to measurements at Vp
80 mV
(EK) to minimize any contribution of
K+ currents, as previously described (38).
Measurement of cell volume. Microinjection of individual HTC cells was performed using previously described methods (30). Ten percent of the volume of the injection solution (48 mM K2HPO4, 14 mM NH2PO4, and 45 mM KH2PO4) was replaced with 0.1 mg/ml of either ClC-2 or polyclonal rabbit glial fibrillary acidic protein antibodies (DAKO) in PBS. Rhodamine dextran (Molecular Probes) was included at a final concentration of 1 mg/ml so that injected cells could be identified and imaged. Injection pressures were adjusted to produce a minimal detectable change in cellular refringence, and any cells demonstrating irregular profiles suggestive of cell damage or fluorescence localized to nucleus were excluded from analysis. After injection, cells were allowed to recover for 45 min in L-15 media (Life Technologies) at room temperature. Perfusion studies were performed in a chamber (500-µl volume) at a flow rate of 4-5 ml/min; solution was removed across the width of the chamber by capillary action to facilitate laminar flow. Images were captured using a Leica DMIRB inverted microscope equipped with epifluorescence and a Cohu charge-coupled device interfaced to a Power Macintosh 7100. In individual experiments, five measurements per cell were made at each time point and subsequently averaged, and injected cells and noninjected (control) cells were measured within the same microscopic field to accurately represent cells under identical conditions within the culture dish. Cell swelling was induced by switching the perfusate to hypotonic buffer (25% less osmolarity by reducing NaCl). An observer blinded to experimental subsets made all measurements, and findings were confirmed by analyzing multiple microscopic fields.
Statistics. Pooled data are presented as means ± SE, where n represents the number of cells for patch clamp and video planimetry studies. Statistical comparisons of patch clamp measurements were made using the paired or unpaired t-test and of cell area measurements using ANOVA with Fischer's post hoc analysis where appropriate. P < 0.05 was considered significant.
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RESULTS |
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Isolation of a rat liver ClC-2 cDNA.
Whole cell patch clamp studies indicate that both primary liver
epithelial cells and cell lines exhibit volume-sensitive
Cl currents (ICl-Swell) where
increases in cell volume enhance Cl
permeability 30-fold
or more (3, 16, 19). They do not, however, exhibit the
voltage-activated, inwardly rectifying currents typical of ClC-2
present in heterologous models. To assess whether ClC-2 is expressed in
liver cells, ClC-2 channel transcripts were identified in a
representative cell model, HTC rat hepatoma cells, by RT-PCR (data not
shown). Subsequently, screening of an amplified HTC cDNA library with a
477-bp oligonucleotide probe corresponding to the D12-D13 region
of ClC-2 led to the isolation of seven identical ClC-2 cDNAs, each
2,987 bp in length. Sequence analysis of both strands indicates that
the liver ClC-2 (
RR) cDNA is identical to bp 172-3206 of the
ClC-2 cDNA previously isolated from rat brain, starting just after the
first putative initiation codon, except for a single substitution of G
to A at position 3202 within the 3'-untranslated region
(35). Primary cDNA isolates lacked the bp 1693-1740
segment within the D11 domain of the brain homologue. This in-frame
deletion was determined to be a library artifact since PCR of HTC cell
cDNA using primers flanking this region always produced the undeleted
but not the deleted product (>80 reactions) with an identical sequence
to that of rat brain ClC-2. In addition, RT-PCR of the 5'-untranslated
region demonstrated identity to bp 148-171 of the rat brain cDNA,
including the putative initiation codon at 168-171.
Detection of ClC-2 transcripts and protein in liver
epithelial cells.
The cellular expression of ClC-2 mRNA was evaluated. With the use of in
situ hybridization, RNA signals were readily apparent in individual HTC
cells exposed to antisense, but not sense, amino terminal-specific
riboprobes, indicating the presence of ClC-2 mRNA (Fig.
1, A and B). To
assess the cellular expression of ClC-2 channel proteins,
immunocytochemistry was performed using affinity-purified polyclonal
rabbit antibodies directed against a unique cytoplasmic amino terminal
region of ClC-2. This sequence is not present in other ClC family
members. As shown in Fig. 1C, ClC-2 proteins were present
and signal was detected both internally and in the periphery of each
cell. In control studies, staining was specifically blocked by
coincubation with GST-ClC-2 fusion proteins but not by GST alone (Fig.
1D), indicating selective recognition of ClC-2 proteins.
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Endogenous ClC-2 is distributed in the plasma
membrane.
Western blotting was used to characterize the cellular distribution of
ClC-2 proteins. ClC-2 antibodies recognized ~100 kDa proteins in HTC
cells (data not shown). Subsequently, plasma membrane distribution was
determined by cellular fractionation and plasma membrane isolation.
Initial fractionation showed that ClC-2 was almost exclusively present
within the membrane fraction, with little or no ClC-2 found within the
cytosolic fraction (Fig. 2). After
further enrichment of the plasma membrane by sucrose density separation, ClC-2 and Na+-K+-ATPase were both
present in the plasma membrane fraction (Fig. 2). These results suggest
that endogenous ClC-2 is expressed in the plasma membrane of HTC cells.
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Identification of DIDS-resistant currents in
HTC cells.
Since the properties of ClC-2 channels are different from those of
swelling-activated currents in HTC cells, additional studies were
performed in the presence of DIDS (100 µM) to block non-ClC-2 currents (35). Cell volume increases were produced by
addition of sucrose (50 mM) to the pipette solution to ensure a
standard transmembrane osmolar gradient, and currents were measured at test potentials between 160 mV and +60 mV or
120 mV to +100 mV in
20-mV increments as indicated. Under control conditions, swelling-activated currents reversed at
1 ± 4 mV, values not different from the Cl
reversal potential of 0 mV, and
were outwardly rectifying (n = 9), as described
previously (3). In the presence of DIDS, a residual
current was still detectable (Fig. 3).
Currents at
80 mV decreased from
544 ± 122 pA
(n = 9) to
341 ± 15 pA (n = 3, not significant) in the absence vs. presence of DIDS, and currents at
+80 mV decreased from 897 ± 212 pA to 238 ± 25 pA (P < 0.01) in the absence vs. presence of DIDS. The
DIDS-resistant current was inwardly rectifying and accounted for
~60% of total current over the physiological range of liver cell
potentials between
20 and
60 mV. These findings suggest that a
portion of the swelling-activated Cl
conductance in HTC
cells has properties consistent with the conductance associated with
ClC-2 channels.
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Expression of ClC-2 in HEK-293 cells.
The polyclonal rabbit antibodies were developed to recognize a target
peptide antigen corresponding to amino acids 20-68 of the
translated rat brain ClC-2 protein. Since this region has been shown to
contribute directly to channel gating and is not found in other members
in the ClC family (12, 32), binding would be anticipated
to alter ClC-2 channel function in a selective and specific manner. To
determine whether these antibodies inhibit channel function, ClC-2 was
expressed in HEK-293 cells, which do not have endogenous ClC-2. Cells
were transfected with ClC-2-containing plasmids in conjunction with
plasmids containing GFP using standard calcium phosphate transfection
methods. Subsequently, individual fluorescent cells were selected for
patch clamp studies, and the results are shown in Fig.
4. Compared with control cells
expressing GFP alone, expression of ClC-2/GFP was associated with a
large increase in anion permeability. Current amplitude measured in ClC-2/GFP-transfected cells was 1,320 ± 230 pA at
80 mV and
3,540 ± 600 pA at
160 mV (n = 7), which is
>300- and 70-fold greater than
4 ± 10 and
50 ± 20 pA
measured in GFP controls (n = 5, P < 0.001). ClC-2 currents were characterized by inward rectification and
activation at hyperpolarizing potentials. As shown in Fig. 4,
intracellular dialysis with ClC-2 antibodies (final concentration 0.005 µg/ml added to pipette solution) inhibited currents in
ClC-2-expressing cells by ~75%. Currents at
160 mV decreased from
3,539 ± 595 pA in the absence of antibody (n = 7) to
884 ± 195 pA (n = 6, P < 0.01) in the presence of antibody. In contrast, cytoplasmic delivery of
antibodies in a similar fashion had no effect on currents in
GFP-expressing control cells lacking ClC-2 (
48 ± 19 pA at
160
mV, n = 3). Thus ClC-2 amino terminus-specific antibodies effectively inhibit ClC-2 protein function. These findings are consistent with recent methods applied using ClC-2 antibodies directed against a similar peptide regions to inhibit ClC-2 channels expressed in Sf9 insect cells (40).
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Intracellular delivery of ClC-2 antibodies inhibits
swelling- and ATP-dependent
Cl currents.
These findings suggest that inhibition of currents by intracellular
delivery of these antibodies might provide insights into the function
of endogenous ClC-2 channels. Consequently, the effects of
intracellular dialysis with ClC-2 antibodies on basal,
swelling-activated, and ATP-sensitive whole cell Cl
currents in HTC cells were assessed (Fig.
5). Intracellular
delivery of ClC-2 antibodies had no effect on basal currents but
markedly prevented activation of ICl-Swell
during exposure to hypotonic buffer (20% less NaCl) in all studies
(n = 6, P < 0.01). In contrast, the
amplitude of swelling-activated currents in cells perfused with
heat-inactivated ClC-2 antibodies (n = 8), a similar
dilution of the ClC-2 antibody buffer (n = 4), and
polyclonal rabbit antibodies to an unrelated protein
-galactosidase
(n = 6) was not different from untreated controls. In
other studies, addition of the P2 receptor agonist ATP (10 µM) to
isotonic bath solutions, which reproducibly stimulates Cl
currents in HTC cells (9) and is essential for cell volume recovery from swelling (38), failed to induce significant
currents in cells perfused with ClC-2 antibodies (n = 3, P < 0.02). These findings indicate that antibodies,
which selectively target the cytoplasmic amino terminus of ClC-2
channels, inhibit volume- and ATP-dependent channel opening in rat
hepatoma cells.
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Functional ClC-2 channels contribute to cell volume
homeostasis.
In epithelial cells, outward movement of Cl,
K+, and other organic osmolytes, including amino acids,
polyols, and/or methylamines, contributes to recovery of cell volume
following swelling (1, 3, 16, 34). To evaluate the
relative contribution of ClC-2 vs. other channel types to HTC cell
volume homeostasis, intact individual cells were microinjected with
ClC-2 antibodies to specifically inhibit native channel function (Fig.
6). In control experiments, hypotonic
exposure (25% less osmolarity) induced a rapid increase in the
relative area of noninjected cells (10.3 ± 1.0% increase at 1 min compared with basal values; n = 8). Subsequently,
relative area decreased, reaching basal values by 8 min despite the
continued presence of hypotonic buffer. Neither the magnitude of cell
swelling nor recovery of cell area were different in cells within the
same field injected with control antibodies (to glial fibrillary acidic protein, 0.1 µg/ml; n = 8), indicating that injection
alone did not alter cellular ability to recover during hypotonic
exposure. In contrast, injection of cells with ClC-2 antibodies
markedly attenuated volume regulation. As shown in Fig. 6, swelling was more rapid in ClC-2 antibody-injected cells (0.1 µg/ml) by 30 s
of hypotonic challenge (% area increase 7.6 ± 1.2 and 3.7 ± 0.7 for injected and noninjected cells, respectively;
n = 8 for each, P = 0.02). Although
noninjected cell area recovered rapidly toward baseline by 8 min, there
was no significant recovery in cells injected with antibodies to ClC-2
over the period analyzed (% area increase at 8 min: 9.2 ± 1.8 and 0.2 ± 1.0 for injected and noninjected cells, respectively;
P = 0.0006). Thus selective inhibition of ClC-2 channel
function prevents recovery of cell volume during hypotonic exposure,
suggesting that endogenous ClC-2 channels represent an important
volume-sensitive anion efflux pathway in liver epithelial cells.
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DISCUSSION |
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In mammalian tissues, cell volume is maintained within a narrow
physiological range by adaptive mechanisms that permit rapid and
precise changes in membrane K+, Cl, and
organic osmolyte permeability. The present studies of HTC cells provide
evidence that ClC-2 Cl
channels are functionally
important in this process and therefore represent a potential site for
modulation of volume-sensitive changes in cell transport and metabolism.
ClC-2 transcripts are nearly ubiquitous, and functional channels have been expressed and characterized in multiple model systems, including Xenopus oocytes, Sf9 insect cells, and mammalian cells (2, 10, 27, 32, 35, 40). In concert with original observations in oocytes, heterologously expressed ClC-2 currents are activated by membrane hyperpolarization and increases in cell volume and are resistant to the anion channel blocker DIDS (35). In neuronal models, a role for ClC-2 channels in preventing neuronal excitability and paradoxical GABAA-mediated excitation has also been proposed (32). In addition, ClC-2 currents expressed in Xenopus oocytes and airway epithelial cells respond to extracellular acidity (14, 27). Despite these different stimuli, ClC-2 currents in these different models are characterized by typical inward rectification, activation at depolarizing potentials, and resistance to DIDS. Although ClC-2 channels expressed in Xenopus oocytes and Sf9 insect cells have been shown to contribute to regulatory volume decrease (10, 40), the biophysical properties of these currents are different from swelling-activated anion currents described in most epithelial cells.
Liver cells are subject to substantial volume stresses as a result of high transport and metabolic capacities and exposure to large changes in the solute and hormonal composition of portal blood (11). HTC cells derived from rat hepatoma regulate their volume during osmolar challenges by activation of outwardly rectifying anion-selective currents that are characteristic of most mammalian cells. Despite the dissimilarities between volume-activated currents in native cells and ClC-2 currents in heterologous models, three complementary observations support a role for ClC-2 channels in this process.
First, both molecular and immunocytochemical evidence indicate that
ClC-2 channel mRNA and protein are expressed in liver epithelia. The
cDNA cloned from an HTC cell library encodes a full-length protein that
is identical to the rat brain ClC-2 channel previously characterized.
The coding region of the HTC ClC-2 cDNA is completely homologous to
cDNAs synthesized from primary rat hepatocytes and cholangiocytes and
thus represents a rat liver epithelial ClC-2 channel. In HTC cells, RNA
transcripts were readily detectable by in situ hybridization, and
immunocytochemical staining using antibodies to a unique amino terminal
domain demonstrated cellular ClC-2 protein expression. With the use of
these same antibodies, ClC-2 proteins of ~100 kDa were detected by
Western blot in HTC cell membranes. In addition, ClC-2 proteins are
detected in the same membrane fraction as
Na+-K+-ATPase, consistent with the plasma
membrane localization anticipated for a contributing role in the
regulation of membrane Cl permeability.
Second, in the presence of DIDS to block non-ClC-2 channels
(7), volume-activated Cl currents are still
detectable in HTC cells, accounting for ~60% of current at
physiological potentials. The inward rectification and DIDS resistance
are consistent with the properties anticipated for functional ClC-2 channels.
Third, intracellular dialysis with antibodies raised against a unique
sequence in the essential regulatory amino terminus of ClC-2 results in
inhibition of volume-activated currents in HTC cells. The efficacy of
these antibodies was first demonstrated by their inhibition of currents
associated with ClC-2 expression in HEK-293 cells. Consequently, the
same antibodies were used as a tool to determine the potential
contribution of ClC-2 channels to whole cell currents in HTC cells.
Intracellular delivery of ClC-2 antibodies produced an inhibition of
Cl current activation during both cell volume increases
and purinergic stimulation by extracellular ATP. These effects are
likely to reflect selective inhibition of native ClC-2 channel proteins since 1) ClC-2 antibodies at similar concentrations were
effective inhibitors of ClC-2 currents overexpressed in HEK-293 cells;
2) intracellular dialysis with heat-inactivated ClC-2
antibodies, unrelated antibodies, and buffer solutions did not inhibit
current activation; and 3) ClC-2 antibodies recognize
appropriate proteins in plasma membrane fractions, and immunostaining
is specifically blocked by coincubation with the peptide antigen.
This approach is similar to that of Xiong et al. (40), in which cytosolic perfusion with antibodies to the same regulatory region inhibited ClC-2 currents expressed in Sf9 insect cells. The strategy is based on extensive characterization of the molecular structure and function of the ClC-2 proteins by Jentsch and coworkers (12, 14). Mutations within a putative "essential" amino terminal region, which extends for ~18 amino acids starting at leucine 21, and within the cytoplasmic D7-D8 loop, induce constitutively open channels that are volume insensitive. Whether these domains regulate channel gating by a ball-and-receptor model analogous to that proposed for some K+ channels or by interaction with other proteins is still speculative. Since the antibodies used in the present studies were targeted to the amino-terminal region of interest, they may prevent the conformational changes in the channel protein associated with cell volume increases that are necessary for pore accessibility. Although further studies are required, our results are compatible with previous experimental evidence that the cytoplasmic amino-terminal domain is directly involved in regulation of channel gating.
The degree of inhibition caused by these antibodies was surprising. Although HTC cells express ClC-2-like, DIDS-resistant currents, these currents account for only a portion of the volume-sensitive conductance. Moreover, the outwardly rectifying, volume-sensitive currents observed in the absence of blockers differ from currents associated with heterologous expression of ClC-2 channels. The explanation for these discrepancies is not readily apparent. However, the differences are not likely to be a reflection of alterations in pore or gating regions intrinsic to ClC-2 since the predicted channel proteins are structurally identical.
Another possibility is that the volume-sensitive currents result from an association of ClC-2 with other as yet unidentified pore-forming or channel-associated proteins. Indeed, ClC channels, such as ClC-1 and ClC-2, are capable of forming heteromultimers with novel biophysical properties (17). Transcripts of other ClC channels are detectable in liver epithelial cells (ClC-3, -6, and -7; data not shown) (24). Expression of ClC-3 in NIH/3T3 cells induces outwardly rectified volume-sensitive anion currents, (7) but coassociation with ClC-2 proteins as functional channels has not been demonstrated. Moreover, studies by Shimada et al. (28) indicate that native ClC-3 proteins in hepatocytes are primarily intracellular or localized to the smaller apical (canalicular) domain, consistent with a role in secretion but not cell volume regulation. The methods used for these studies cannot resolve these issues. However, our approach is the first designed to specifically target endogenous ClC-2 proteins in the absence of overexpression, the latter of which would be expected to favor the formation of ClC-2 homomultimers. Thus determination of the significance of these findings will require application of similar techniques to other model systems.
Despite these biophysical limitations, a strong point in favor of a
functional role for ClC-2 proteins is that microinjection of intact
cells with ClC-2 antibodies prevented cell volume recovery from
swelling. Thus, in addition to directly modulating membrane Cl permeability, endogenous ClC-2 channels are likely to
play a specific role in cell volume regulation. These findings are
compatible with previous observations that overexpression of ClC-2 in
Xenopus oocytes and Sf9 insect cells enhances regulatory
volume decrease. The repertoire of channels that regulate cell volume
is likely to vary from tissue to tissue, with channels unrelated to
ClC-2 contributing to volume-sensitive Cl
efflux in other
cell types. ClC-3 channels, for example, represent viable candidates
for ICl-Swell (7). Evidence that
pICln and the P-glycoprotein products of
mdr genes modulate volume-sensitive Cl
currents in some cell models implies that another level of regulation may involve tissue-specific expression of different channels or channel-associated proteins (22, 36).
In summary, these findings in a model mammalian cell expressing native
ClC-2 proteins support a role for ClC-2 channels as an effector of
volume-sensitive changes in membrane Cl permeability and
an important contributor to cell volume homeostasis. ClC-2 channel
opening is also coupled to purinergic receptor stimulation by
extracellular ATP, which represents a novel mechanism for channel activation. Consequently, modulation of ClC-2 channel expression or
gating represents a potential target for the regulation of volume-dependent cellular transport and metabolism.
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ACKNOWLEDGEMENTS |
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We would like to acknowledge J. Cuppoletti and D. Malinowska (University of Cincinnati) for their gracious help in the initiation of the liver ClC-2 cloning project and J. Schaack (UCHSC) for his advice and assistance with cell transfection experiments.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Kidney Diseases Grants DK-46082, DK-43278, and K08-DK-02539-01 and by the Waterman Fund for Liver Research.
Address for reprint requests and other correspondence: G. Fitz, Campus Box B158, Rm. 6412, Univ. of Colorado Health Sciences Center, 4200 East Ninth Ave., Denver, CO 80262 (E-mail: greg.fitz{at}uchsc.edu).
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.
Received 11 August 1999; accepted in final form 9 October 2000.
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