From the Medical Research Council Clinical Sciences
Centre, Imperial College School of Medicine, Hammersmith Hospital
Campus, Du Cane Rd., London W12 0NN, United Kingdom, the ¶ Cell
Signaling Unit, Department de Ciències Experimentals i de la
Salut, Universitat Pompeu Fabra, C/Dr Aiguader 80, Barcelona 08003, Spain, and the
Departamento de Biología Animal,
Universidad de la Laguna, Tenerife 38206, Spain
Received for publication, December 26, 2000, and in revised form, January 24, 2001
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ABSTRACT |
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Volume regulation is essential for normal
cell function. A key component of the cells' response to volume
changes is the activation of a channel, which elicits characteristic
chloride currents (ICl, Swell). The molecular
identity of this channel has been controversial. Most recently, ClC-3,
a protein highly homologous to the ClC-4 and ClC-5 channel proteins,
has been proposed as being responsible for ICl, Swell (1).
Subsequently, however, other reports have suggested that ClC-3 may
generate chloride currents with characteristics clearly distinct from
ICl, Swell. Significantly different tissue
distributions for ClC-3 have also been reported, and it has been
suggested that two isoforms of ClC-3 may be expressed with differing
functions. In this study we generated a series of cell lines expressing
variants of ClC-3 to rigorously address the question of whether or not
ClC-3 is responsible for ICl, Swell. The data demonstrate
that ClC-3 is not responsible for ICl, Swell and has no
role in regulatory volume decrease, furthermore, ClC-3 is not activated
by intracellular calcium and fails to elicit chloride currents under
any conditions tested. Expression of ClC-3 was shown to be relatively
tissue-specific, with high levels in the central nervous system and
kidney, and in contrast to previous reports, is essentially absent from
heart. This distribution is also inconsistent with the previous
proposed role in cell volume regulation.
The maintenance of a constant cell volume in the face of
fluctuating intra- and extracellular osmolarity is essential for normal
cell function. Following cell swelling upon exposure to hypotonic
solution, animal cells restore their volume toward its original value
by activation of channels and transporters in the plasma membrane: the
loss of K+ and Cl Although a cell swelling-activated chloride current
(ICl, Swell) required for RVD has been carefully
characterized in several cell types, the molecular identity of the
channel has not yet been established (3-5). This is primarily due to
three experimental limitations. First, the current is ubiquitous, such that cell lines exhibiting little or no current necessary for expression cloning are not available. Second, no specific, high affinity blockers of the current are known. Third, the magnitude and
the rate of activation of the currents are readily perturbed by both
endogenous and exogenous factors making quantitative analysis difficult. The latter issue is illustrated by the fact that
P-glycoprotein, proposed as a candidate for the cell swelling-activated
chloride channel, was subsequently shown to be a regulator of
endogenous channel activity (6, 7), whereas another candidate,
pICln, was also shown not to be the channel and its precise
role is still uncertain (8, 9). Another swelling-activated chloride
channel expressed in many cell types, ClC-2, has also been suggested as contributing to RVD (10). However ClC-2 generates a current with
biophysical and pharmacological characteristics that differ significantly from ICl, Swell (11-13), and in a human
intestinal epithelial cell line it has been shown that ClC-2 does not
contribute to RVD (13).
Recently, a series of studies has led to the suggestion that ClC-3 is
the swelling-activated chloride channel responsible for
ICl, Swell. The gene coding for ClC-3 was first cloned from rat by a homology-based cloning strategy; its predicted amino acid
sequence is similar to other ClC channels (14, 15). The human cDNA
was subsequently cloned and sequenced from fetal brain (16), and the
guinea pig version was cloned and sequenced from cardiac myocytes (1).
Expression of gpClC-3 was reported to increase significantly
ICl, Swell (1, 17). This view was supported by antisense
experiments where reduction of ClC-3 expression was reported to
decrease ICl, Swell (18).
Subsequently, the role of ClC-3 in ICl, Swell has been
challenged. Attempts to replicate experiments with gpClC-3 and hClC-3,
either in Xenopus oocytes or in mammalian cell lines (HEK293 and NIH3T3), were unsuccessful (19). In another study, expression of
rClC-3 in CHO cells was reported to generate a
Ca2+-sensitive chloride channel (15). Further complexity
was introduced by the suggestion that short and long versions of
ClC-3 could potentially be generated in vivo using two
different translation initiation sites, identical except for an
additional 58 amino acids at the N-terminal of the long version (20).
It was reported that expression of the short and long versions
generated, in Chinese hamster ovary-K1 cells, distinct currents that
are ClC-5-like and ICl, Swell-like, respectively (20),
although the ion selectivity of the ClC-5-like currents is reported
both as I The tissue distribution of ClC-3 is also ambiguous. Kawasaki et
al. (14) reported mRNA coding for ClC-3 mainly in brain and
kidney, but not in heart. In contrast, Shimada et al. (20) reported high levels of expression of ClC-3 protein in the liver, and
Britton et al. (22) high levels in heart. Furthermore, ClC-3 was reported in bovine non-pigmented ciliary cells to be localized mainly to the nucleus (18), yet to the canalicular membrane in
hepatocytes (20).
To resolve the controversy surrounding ClC-3 we generated cell lines
expressing the long and short versions of hClC-3-GFP, with hClC-5-GFP
as control, in HEK293 cells. Neither the long nor the short version of
hClC-3-GFP affected the swelling-activated currents significantly or
influenced cell volume regulation. Indeed, ClC-3 generated no
detectable chloride currents when overexpressed in response to change
in cellular Ca2+ concentrations. The findings presented
here exclude ClC-3 as the channel responsible for
ICl, Swell and cell volume regulation.
Generation of Cell Lines Expressing hClC-3-GFP and
hClC-5-GFP--
The original cDNA sequence coding for ClC-3
predicted a protein of 760 amino acids (the short version) (14).
Subsequently, an upstream ATG codon was identified, and a protein with
an additional 58 amino acids at the N terminus was predicted (the long
version) (20). It has been suggested that both versions may be
expressed (20). To study the localization of hClC-3, we generated cell lines expressing both the short and long versions fused to GFP at the C
terminus. It has previously been reported that GFP tags at the C
terminus of ClC channels do not impair function (23). As a control for
function, a cell line expressing hClC-3 with no GFP tag was also
generated. Plasmids containing overlapping cDNA fragments encoding
hClC-3 were obtained from Dr. Borsani (16). Appropriate restriction
fragments were excised from these plasmids, or generated from these
plasmids by PCR, and ligated together to generate a full-length
CLCN3 cDNA. The PCR primer at the 5'-end
(5'-AGTTGGAACGCTAGCCACCATGACAAATGGAGGCAGC-3') was designed to introduce
a consensus Kozak sequence and a unique NheI restriction
site upstream of the start codon. The full-length cDNA was inserted
as a NheI-XhoI fragment into the expression vector pCIneo (Promega) to generate the expression plasmid pCI-ClC-3. The cDNA was verified by sequencing. To generate the long version, the missing coding sequence was cloned from human primary lung fibroblasts by a reverse transcriptase reaction (Omniscript, Qiagen) using random hexamer primers (Roche Molecular Biochemicals), followed by PCR using primers specific for ClC-3: the upstream promoter was
5'-CGAGATAATGCTAGCCCACCATGGAGTCTGAGCAGCTGTTCCAT-3' and at the
downstream promoter 5'-AGAACTGTTAATGGATCCTCCATTTGTCAT-3'. The PCR
fragment was ligated as an NheI-BamHI fragment
into pCI-ClC-3, which had been modified by a PCR-based insertion of a
silent BamHI restriction site downstream of the short
version start codon. The resultant plasmid was designated
pCI-ClC-3long. To generate the mutant K579N, the Altered Sites
mutagenesis system (Promega) with an oligonucleotide containing the
desired mutation together with a silent AflII restriction
site (5'-GAAGCACACATCCGACTTAAGGGATACCCTTTCTTG-3') was used.
Plasmids encoding hClC-3-GFP fusion proteins were generated by a
two-step cloning procedure. First, the stop codon was substituted with
an XhoI site by PCR using primer
5'-CCTCATCTGTGACTCGAGGTTGAACATTATTG-3'. The EGFP-fragment from
the pIRES2-EGFP plasmid (CLONTECH) was then
inserted as a NcoI-NotI fragment into plasmid
pCI-ClC-3 after digestion with XhoI and NotI,
using two short linker oligonucleotides to mediate ligation between the
XhoI and the NcoI ends (TCGAGTCTAGAGCCAC and
CATGGTGGCTCTAGAC). The plasmid encoding hClC-5-GFP was a generous gift
of Prof. T. Jentsch, Hamburg, and was based on the same pCIneo vector
and EGFP gene as the hClC-3 fusions generated here.
HEK293 (human embryonic kidney-transformed cells (24)) cell lines,
expressing the various hClC-3, hClC-3-GFP, and hClC-5-GFP proteins,
were generated by electroporating with linearized plasmids. To generate
a cell line expressing short hClC-3 without a GFP tag, HEK293 cells
were cotransfected with pGreenLantern-1, which encodes GFP (Life
Technologies, Inc.), and the pCI-ClC-3-expressing hClC-3 at a ratio of
1:3. Clones were selected for resistance to 800 µg/ml G418 and by
fluorescence-activated cell sorting. The "greenest" clones were
then manually selected and maintained in Dulbecco's modified Eagle's
medium, 10% fetal bovine serum, and 500 µg/ml G418. After subsequent
screening for GFP fluorescence and G418 resistance, clones were tested
for hClC-3 expression by immunoblotting.
Antibody Production--
A polyclonal antibody against ClC-3,
called D1, was produced by injecting a synthetic peptide
MTNGGSINSSTHLLD (corresponding to amino acids 59-73 of the long
version of hClC-3; amino acids 1-15 of the short version) coupled to
bovine serum albumin, together with Freund's adjuvant, into rabbits at
four time points over a 5-month period (Regal Group Ltd., UK). This
peptide was selected because it is specific for ClC-3, and we showed
that the antibody D1 does not cross-react with overexpressed ClC-4 or
ClC-5 in Western blots (data not shown). Sera from bled animals were
affinity-purified using N-hydroxysuccinimide-activated
Sepharose (Amersham Pharmacia Biotech) coated with the immunogenic
peptide. The eluates were analyzed by SDS-PAGE and Coomassie Blue
staining. The IgG-containing fractions were then dialyzed and
concentrated using spin columns (Millipore) with a cut-off size of 70 kDa.
The commercial antibody against ClC-3, Anti-ClC-3, was obtained from
Alomone and was raised against residues 592 to 661 of short ClC-3. The
antibody against GFP, JL-8, was a monoclonal antibody from
CLONTECH.
Confocal Immunofluorescence Imaging--
Cells, grown at low
density on glass coverslips coated with poly-L-lysine, were
fixed with 4% formaldehyde in phosphate-buffered saline (PBS) with
calcium and magnesium: 8.0 g/liter NaCl, 1.15 g/liter
Na2HPO4, 0.2 g/liter KCl, 0.2 g/liter
KH2PO4, 0.13 g/liter CaCl2-2H2O, 0.1 g/liter
MgCl2·6H2O) for 30 min at room temperature. Cell nuclei were stained with propidium iodide after treatment with
RNase A (Sigma) (100 µg/ml) for 3 min at 37 °C. The coverslips were then mounted onto glass slides with mounting oil containing anti-fading agent (Vectashield, Vector). Images were taken by a
MicroRadiance Bio-Rad confocal microscopy through a 63× planapochromat 1.4 numerical aperture objective. The GFP signal was detected by
excitation with the 488-nm line of an argon laser through a HQ515/30
emission filter. The signal from propidium iodide in the same cell
section was detected by excitation using the 543-nm line of a
helium-neon laser through an E600LP emission filter.
Biotinylation of Membrane Proteins--
Cells grown on
poly-L-lysine-coated 75-cm2 flasks or 35-mm
dishes to 80% confluency were incubated in 5 ml of reducing buffer (50 mM dithiothreitol, 1 mM EDTA in PBS) for 5 min
at room temperature. The cells were then washed twice with 5 ml of 1 mM EDTA in PBS. Cells were labeled by incubation for 30 min
at room temperature with 1 ml of biotin maleimide (Molecular Probes)
dissolved in DMSO and diluted to 1 mM in EDTA buffer. The
reaction was quenched by washing twice with 2 ml of 5 mM
dithiothreitol in PBS and twice with PBS plus 1 mM EDTA.
The labeled cells were lysed by addition of 1 ml of lysis buffer
containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl,
1% Nonidet P-40 plus complete mini protease inhibitors (Roche
Molecular Biochemicals). 1 mg of protein was incubated with 100 µl of
prewashed immobilized Ultralink neutravidin (Pierce) and gently rotated
overnight at 4 °C. The slurry was recovered by centrifugation,
washed twice (by resuspension and centrifugation) with 0.5 ml of lysis
buffer, twice with 0.5 ml of high salt buffer (0.5 M NaCl,
25 mM Tris-HCl, pH 7.5, 1% Nonidet P-40 in
dH2O), and once with 0.5 ml of 50 mM Tris-HCl
(pH 7.5). The bound, biotinylated proteins were eluted by incubating
the washed slurry with 100 µl of Laemmli sample buffer (50 mM Tris HCl, pH 6.8, 10% glycerol, 5%
Protein Preparation and Western Blotting--
Cell extracts for
Western blotting were prepared by detaching cells with PBS containing 5 mM EDTA followed by lysis buffer (50 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40 and
complete mini protease inhibitors; Roche Molecular Biochemicals). The
lysate was sonicated to shear the DNA.
Proteins from tissues were obtained from whole organs removed from
laboratory mice (C57/Bl6) immediately after killing and snap-frozen in
liquid nitrogen. Frozen organs were stored at
For electrophoresis, samples were mixed with equal amounts of Laemmli
sample buffer and incubated at room temperature for 10 min. Proteins
were separated by SDS-PAGE on 7.5% polyacrylamide gels at 15-40 V/cm
for ~1.5 h in a Bio-Rad chamber. The separated proteins were then
transferred to polyvinylidene difluoride membranes (0.45-µm pore
size, Immobilon-P, Millipore) using a semi-dry blotting apparatus
(Anachem) as described by the manufacturer. After blocking with PBS
containing 4% dried skimmed milk and 0.02% Tween 20 for at least 30 min, the membranes were incubated overnight at 4 °C with diluted
antiserum (1/1000, JL-8 CLONTECH; 1/200 Anti-CLC-3, Alomone and 1/100 D1) in the blocking buffer. Membranes were washed three times for 15 min with PBS containing 0.02% Tween 20, incubated with the horseradish peroxidase-conjugated secondary antibody (anti-mouse or anti-rabbit-IgG, as appropriate; Dako) for 1 h at
room temperature, washed three times for 15 min each, and finally the
horseradish peroxidase signal detected using the ECL chemiluminescence system (Amersham Pharmacia Biotech).
Electrophysiology--
Chloride currents were measured in whole
cell recording mode of the patch-clamp technique as described
previously (7). To prevent voltage offset when bath Cl Cell Volume Measurements--
Cells were grown on
poly-L-lysine-coated coverslips and loaded, before the
experiment, for 5 min with 2.5 µM Calcein-AM (Molecular Probes) in isotonic solution (70 mM NaCl, 5 mM
KCl, 0.5 mM MgCl2, 2 mM
CaCl2, 5.5 mM glucose, 10 mM Hepes
buffer, pH 7.4, and osmolarity was adjusted to 320 mOsm with mannitol).
After extensive washing with isotonic solution not containing
calcein-AM, cells were transferred to a perfusion chamber set on the
stage of the microscope. Experiments were performed at room
temperature. Cells were imaged by a MicroRadiance Bio-Rad confocal
microscopy through a Plan-Neofluor 40 × 1.3 numerical aperture
oil immersion objective (Zeiss), and the dye was excited with a 488-nm
line of an argon ion laser. The emitted fluorescence was collected
through a QH500LP filter. An optical section passing through the cells
was acquired every 30 s, and the average fluorescence signal from
a representative cellular area was plotted along the time. To calibrate
the fluorescence signal, brief exposures to 15% hypotonic and 15%
hypertonic solutions, obtained by adjusting the amount of mannitol,
were used (25). The cells were then perfused, for at least 5 min, with
an isotonic solution containing 10 µg/ml Gramicidin (Sigma) and where
NaCl was substituted with 70 mM NMDGCl and then challenged
with a 40% hypotonic solution, obtained by reduction of mannitol
concentration. The signal was then analyzed and converted in volume
measurement and the percentage of RVD was calculated (6, 25). The
fluorescence signal due to the GFP tag, present in the cells expressing
ClC-3-GFP, was negligible in comparison to the signal due to calcein
and was shown to be non-osmotically sensitive, so it could readily be accounted for in the calibration of the signal.
Generation of Cell Lines Expressing hClC-3--
To facilitate
study of ClC-3 and its subcellular location, stable cell lines
expressing short hClC-3-GFP and long hClC-3-GFP, and short
hClC-3(N579K)-GFP and long ClC-3(N579K)-GFP, were generated in HEK293
cells (see "Experimental Procedures"). It has previously been shown
for other ClC channels that a C-terminal GFP tag does not affect
function (23). As controls, cell lines expressing hClC-5-GFP (which is
known to localize to the plasma membrane and to endosomal compartments
(26, 27)), and short hClC-3 without a GFP tag, were also generated.
Fig. 1 shows Western blots, probed with
anti-GFP antibody (panel a) or antibodies against ClC-3
(panels b and c), demonstrating expression of the
appropriate proteins in these cell lines. Note that the fusion proteins
remain intact with the GFP tag at the C terminus.
Localization of ClC-3-GFP--
To localize the ClC-3-GFP fusion
proteins, the cell lines were studied by confocal microscopy. Fig.
2 (top row of panels) shows
three optical sections taken along the z axis of a typical cell expressing short hClC-3-GFP. The signal in green
corresponds to the ClC-3-GFP fusion protein, and the red
signal corresponds to the cell nucleus (DNA stained with propidium
iodide). The majority of the hClC-3 protein was localized to an
organelle in proximity to the nucleus, presumably the Golgi, and to a
variety of differently sized intracellular vesicles scattered
throughout the cytoplasm. However, a proportion of the protein was
located in a rim around the periphery of the cell, presumably the
plasma membrane. A very similar pattern of localization was also
observed for short hClC-3 bearing the mutation N579K (middle row
of panels) and for hClC-5-GFP (lower row of panels).
The cells shown were representative of the large majority of the cells
within a single clone and of several independent clones expressing each
GFP fusion protein (data not shown).
ClC-3 Is Present on the Plasma Membrane--
The data above
suggest that a proportion of the ClC-3-GFP fusion protein is localized
to the plasma membrane. However, because of the limited horizontal
resolution of confocal microscopy, the data do not exclude the
possibility that the protein is located in vesicles just beneath the
plasma membrane. To demonstrate that the ClC-3-GFP fusion proteins are
inserted in the plasma membrane, cells were labeled with the
membrane-impermeant, thiol-reactive reagent biotin-maleimide (28). The
biotinylated proteins were then isolated using immobilized neutravidin,
and any GFP fusion proteins in the biotinylated protein fraction were
detected by SDS-PAGE and Western blotting using anti-GFP antibodies. In
the absence of biotinylation, no proteins were bound by the resin, showing that the labeling is specific (data not shown). A significant proportion of each of the GFP fusion proteins tested was accessible to
biotin maleimide in intact cells, consistent with a plasma membrane
location (Fig. 3, top panel
a). Under these conditions, an intracellular control antigen known
to be located just beneath the plasma membrane, the Ras pathway
protein SHC (29) was not labeled, demonstrating that the biotin
maleimide does not permeate the membrane (Fig. 3, bottom panel
a). When cell membranes were permeabilized with saponin (Fig.
3b), there was, as expected, a significant increase in
labeling of ClC-3-GFP proteins (top panel b), and SHC was
now also labeled (bottom panel b). Similar results were
obtained also with the clone expressing the native ClC-3 protein (data
not shown). Thus, in the HEK cell lines a significant proportion of
both the hClC-3 and hClC-5-GFP fusion proteins is located in the plasma
membrane.
hClC-3 Is Not a Swelling-activated Channel--
Having established
that the hClC-3GFP fusion proteins were present in the plasma membrane,
cell swelling-activated chloride currents were studied under the whole
cell configuration of the patch clamp technique. Cells were maintained
at the holding potential of 0 mV and stimulated by a standard protocol
from
Although data with other ClC channels show that a GFP tag at the C
terminus has no effect on channel properties (23), we also confirmed
that no difference in current magnitude or current characteristics were
observed between HEK293 cells expressing ClC-3 and ClC-3-GFP fusions
(Figs. 4 and 5).
Previous studies (1) have suggested that the N579K mutation of ClC-3
suppresses outward rectification of the current and changes its halide
selectivity. Expression of hClC-3(N579K)-GFP induced no change in
either the magnitude of swelling-activated chloride currents (Fig.
5c) or its outward rectification (Fig. 4). The anion
selectivity of currents recorded in cells expressing the mutated
protein (Fig. 6) did not change when
compared with the currents recorded from cells expressing the wild type
protein: for all cell lines tested the halide permeability was:
I
In conclusion, neither the magnitude, rate of activation, nor
characteristics of swelling-activated currents in HEK293 cells were
affected by overexpression of any of the versions of hClC-3 tested.
This is despite the fact that we demonstrated that the proteins were
localized to the plasma membrane and that similar expression of a
closely related protein, hClC-5 (see below), generates characteristic
currents. These data provide strong evidence that ClC-3 as expressed
has no channel activity.
Expression of ClC-3 Does Not Affect RVD--
To further test the
involvement of ClC-3 in volume regulation, we measured RVD following
exposure to hypo-osmotic solution. The measurement of volume change was
performed by acquiring an optical section from single cells loaded with
the fluorescent dye calcein. Changes in intensity of fluorescence are
proportional to changes in the concentration of the dye, which are
strictly dependent on changes in cell water volume, allowing the
measurement of relative changes in cell volume (6, 25). Unexpectedly, a
significant proportion of both hClC-3-GFP and the parental HEK293 cells, did not show RVD. The non-responsive cells readily converted calcein-AM to calcein, an indication of active esterases, and did not
show leak of the dye indicating an intact plasma membrane. We do not
have an explanation for the heterogeneity of response, although it was
not ClC-3-dependent. The cells that did exhibit cell volume
regulation showed no significant difference in RVD due to the
expression of ClC-3 (Fig. 7B).
Thus, ClC-3 does not appear to be involved in cell volume
regulation.
hClC-5 Is Not Activated by Cell Swelling--
hClC-5 is closely
related to ClC-3, and a current associated with ClC-5 expression has
been recorded previously in Xenopus oocytes and in
transiently transfected HEK293 cells (19). We therefore used hClC-5 as
a control. Like the ClC-3-GFP cells, in the hClC-5-GFP cell line a
similar proportion of the fusion protein was located in the plasma
membrane (see above). However, in contrast to ClC-3-expressing cells, a
typical strong outwardly rectifying current was measured in
ClC-5-expressing cells in isotonic conditions (Fig.
8A), with an anion conductance
at positive potentials: Cl Changes in Intracellular Calcium Do Not Activate hClC-3--
The
data above provide strong evidence against the hypothesis that ClC-3 is
responsible for cell swelling-activated chloride currents, and also
show that (unlike ClC-5) if ClC-3 is a chloride channel at all, it is
not constitutively active under the recording conditions used in this
study. The other chloride channels that have been characterized in
epithelial cells are activated by change in intracellular
Ca2+. We therefore studied cells expressing hClC-3 in
response to elevations of intracellular Ca2+ using the
calcium ionophore A23187 (Fig.
9B). This stimulus elicited,
in both ClC-3-expressing HEK293 and untransfected HEK293 cells (Fig.
9A), currents with properties similar to well-characterized epithelial Ca2+-activated chloride channels (31-33). These
currents are easily distinguishable in kinetic characteristics from the
cell-swelling activated current. Thus, hClC-3 does not appear to encode
a calcium-activated chloride channel.
Tissue Distribution of ClC-3--
Regulatory volume decrease is a
general property of almost all cell types (2, 34). Similarly, a typical
ICl, Swell has been identified in most cell types studied
(4, 5). Therefore, any candidate protein for ICl, Swell
might be expected to have a widespread tissue distribution. We examined
expression of ClC-3 by Western blotting in a wide array of murine
tissues using an antibody against the N terminus of ClC-3 (D1).
Although the D1 antibody, like the Alomone antibody, is not absolutely specific for ClC-3, comparison of the two antibodies allowed us to
unambiguously identify protein bands that correspond to ClC-3. It is
important to note that ClC-3 runs as multiple bands in SDS gels. This
may reflect the predicted long and short versions, because D1
recognizes both versions (Fig. 1), or may reflect different post-translation modifications. Very high levels of ClC-3 were detected
in kidney and the central nervous system. However, for all other
tissues examined (thymus, lung, liver and spleen, heart, skeletal
muscle, upper and lower intestine, and testis) little or no signal was
detected (Fig. 10A). The
absence from heart is in contrast to a previous report (22) (Fig.
10B, right panel). To resolve this apparent
discrepancy we also probed the blot with Alomone anti-ClC-3 antibody.
This antibody (Alomone) did detect a band in cardiac tissue (Fig.
10B, left panel), but because this was not
detected by antibody D1 it must reflect a lack of specificity of the
Alomone antibody rather than the presence of ClC-3 in the tissue. In
conclusion, the distribution of ClC-3 is consistent with a specific
role of ClC-3 in kidney and central nervous system, rather than with a
more general role in RVD.
Cell volume regulation is an important property of all cells, and
characteristic cell swelling-activated chloride currents play an
important role in RVD in most cell types. Despite intensive study, the
molecular identity of the channel(s) underlying ICl, Swell has been elusive. Recently, ClC-3 has been proposed as a candidate for
ICl, Swell with a role in cell volume regulation (1, 17,
18, 22). However, this conclusion has been questioned (19) and further
complicated by the identification of two potential isoforms, a short
and long version of ClC-3 (20). In this study we have rigorously
assessed whether ClC-3 is responsible for ICl, Swell and
conclude that ClC-3 is neither responsible for ICl, Swell nor plays a role in RVD.
Cell lines overexpressing hClC-3 were generated. The use of GFP fusions
and extracellular biotinylation were used to demonstrate that a
significant proportion of ClC-3 was inserted in the plasma membrane.
Neither the short nor long form of ClC-3, when overexpressed at the
surface of HEK293 cells, had any significant influence on
ICl, Swell or on cell volume regulation. Furthermore, overexpression of a mutant ClC-3, N579K, that has previously been reported to change both the anion selectivity and the rectification of
ICl, Swell (1) had no effect on the characteristics of the
swelling-activated currents. As a positive control, cell lines
generated in parallel over-expressing ClC-5 exhibited characteristic ClC-5-like currents. Finally, we generated antibodies against ClC-3 and
showed that the protein is expressed in a highly tissue-specific fashion, a result incompatible with a general role in RVD or in underlying ICl, Swell.
We conclude that the previous confusion about the role of ClC-3 is
likely due to two experimental difficulties. First, we have shown that
the commonly used commercially antibody (Alomone) is not specific to
ClC-3 and cross-reacts with other cellular proteins of similar apparent
molecular weight. The generation of a new antibody against ClC-3 has
also allowed us to clarify the tissue distribution. It appears that
ClC-3 is relatively restricted in its distribution, being expressed
primarily in kidney and the brain. Second, most or all mammalian cells
possess swelling-activated chloride channels: the magnitude of these
"background" currents can vary substantially from cell to cell,
and, perhaps more importantly, their magnitude is sensitive to
manipulations such as transfection and cell confluency (data not
shown). Thus, unless a very large number of cells is studied in a
controlled and rigorous fashion, and no selection of cells with high
current is involved, misleading conclusions can be drawn. Indeed, in
preliminary studies using transient transfections we demonstrated
higher currents in cells expressing ClC-3 (35), yet, on more detailed
analysis, it became apparent that this effect was a result of
experimental manipulation of the cells rather than expression of ClC-3 itself.
If ClC-3 is not responsible for ICl, Swell and has no role
in RVD, what is its cellular role? Expression of GFP fusion proteins
showed the majority of hClC-3 is located in Golgi or intracellular
vesicles. In contrast to a published report (18), no ClC-3 was
localized to the nucleus: This previous result was likely an
unfortunate consequence of lack of antibody specificity. A similar
pattern of subcellular expression was seen for ClC-5-GFP, a protein
known to be located and functional in intracellular vesicles in the
kidney (26, 27). This suggests that ClC-3 may also have a role in
intracellular membranes.
When expressed at the cell surface, neither the long or
short version of ClC-3 generated chloride currents (whereas ClC-5 as a
positive control did), and no current was elicited in response to
changes in intracellular calcium or cell volume. A report that short
ClC-3 generates a ClC-5-like current with a selectivity of
Cl
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ions and organic osmolytes,
followed by obligatory loss of water, leads to regulatory volume
decrease (RVD)1 (2).
> Cl
and Cl
> I
(21).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, 2% SDS, 0.05% bromphenol blue) for 10 min at
90 °C and analyzed by Western blotting.
80 °C until further
processing. To homogenize, the frozen organs were first pulverized with
pestle and mortar in liquid nitrogen, and then resuspended in ice-cold
lysis buffer with protease inhibitors (described above) and treated for
30 s using an Ultra-Turrax homogenizer. Where necessary, proteins
were deglycosylated by resuspending the cell lysate in a 50 mM sodium phosphate buffer (pH 7.5) and with 5 units/µl
PNGase F (New England BioLabs) at 37 °C for 1 h
with occasional gentle mixing. To digest DNA, Benzonase (Sigma Chemical
Co.) was added to a final concentration of 5 units/µl and incubated
at 37 °C for 5 min prior to addition of Laemmli sample buffer.
concentration was changed, the bath reference electrode was connected through an agar bridge that maintains a constant Cl
concentration in the immediate vicinity of the Ag/AgCl electrode. The
extracellular (bathing) isotonic solution contained 100 mM NMDGCl, 0.5 mM MgCl2, 1.3 mM
CaCl2, 10 mM HEPES titrated to pH 7.4 with
Tris. The osmolarity was corrected to 300 mOsm with mannitol. The
extracellular hypotonic solution, used to elicit swelling-activated currents, had the same composition as the isotonic solution except that
the osmolarity was corrected with mannitol to 220 mOsm. Extracellular solutions with chloride substitution were obtained by replacing 100 mM NMDGCl with 100 mM of the respective anion
salts (NaI, NaF, or NaBr), and osmolarity was adjusted with mannitol to
220 or 300 mOsm for hypotonic or isotonic solutions, respectively. The
intracellular (pipette) solution was 100 mM NMDGCl, 1.2 mM MgCl2, 1 mM EGTA, 10 mM HEPES, 2 mM ATP titrated to pH 7.4 with Tris, and osmolarity was adjusted to 280 mOsm with mannitol. Data are
expressed as mean ± S.E. (n, number of cells).
Statistical analyses were performed by non-paired t test;
statistical significance was accepted for p < 0.05(*)
or p < 0.01(**).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Western blots demonstrating overexpression of
hClC-3. Total cell proteins were separated by SDS-PAGE and
analyzed by Western blotting using the JL-8 monoclonal antibody against
GFP (a), the anti-ClC-3 antibody D1 raised for this study
(c), or the Alomone anti-ClC-3 antibody (b). 100 µg of total protein were loaded per lane. Lane 1, HEK293
cells; lane 2, HEK293-short hClC-3-GFP cells; lane
3, HEK293-long hClC-3-GFP cells; lane 4, HEK293-short
hClC-3-GFP cells after deglycosylation; lane 5, HEK293-long
hClC-3-GFP cells after deglycosylation; lane 6,
HEK293-shortClC-3 cells; lane 7, HEK293-short ClC-3-GFP
cells.
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Fig. 2.
Confocal images of cell lines expressing
ClC-3-GFP and ClC-5-GFP fusion proteins. Three
representative confocal optical sections (top,
middle, and bottom) taken along the z
axis of cells expressing short hClC-3-GFP, short hClC-3(N579K)-GFP, and
hClC-5-GFP. Green fluorescence corresponds to the GFP fusion
proteins and red the propidium iodide staining of DNA. The
majority of each of the GFP fusion proteins is localized to a large
organelle close to the nucleus, presumably the Golgi, to small vesicles
throughout the cytoplasm, and to the plasma membrane.
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Fig. 3.
Biotin labeling of plasma membrane GFP fusion
proteins. Cells were reacted with biotin maleimide without
(a) and with (b) permeabilization of the plasma
membrane. The amount of sample loaded per lane corresponds to 200 µg
of protein of the initial protein extract in panels a and 50 µg in panels b. Lane 1, short hClC-3-GFP;
lane 2, short hClC-3(N579K)-GFP; lane 3, long
hClC-3-GFP; lane 4, hClC-5-GFP. The top set of
panels shows biotinylated proteins detected with an anti-GFP
antibody, detecting the ClC-GFP fusion protein: The GFP fusion proteins
are accessible to biotin maleimide before and after permeabilization.
The bottom set of panels shows biotinylated proteins
detected with an anti-SHC antibody: This intracellular protein is only
biotinylated once the membrane is permeabilized demonstrating that the
non-permeabilized cells were intact and that, therefore, the
biotinylation of the ClC-GFP fusions reflects their insertion in the
plasma membrane.
80 mV to +120 mV in 40-mV steps. Fig.
4 shows representative current traces in
isotonic solution (upper panels), and 7 min after exposure to a 30% hypotonic solution (lower panels) at which time
the currents had reached steady state (Fig.
5a). Very low currents were
recorded in isotonic conditions (Fig. 5b) except for cells
expressing hClC-5. Following hypo-osmotic shock, all cell lines,
including the parental HEK293 cells, exhibited a similar current,
showing moderate outward rectification, fast activation, and time and
voltage inactivation at +120 mV (Fig. 4, bottom panel). No
statistically significant difference in either the magnitude (Fig.
5c) or rate of activation of ICl, Swell was
observed between the different lines. Osmotically induced currents were
blocked by addition of 10 µM tamoxifen, an inhibitor of
swelling-activated chloride channel (30) (data not shown).
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Fig. 4.
Swelling-activated chloride currents in
ClC-3-expressing HEK293 cells. Representative chloride currents
in iso-osmotic (top set of panels) and 7 min after
exposure to 30% hypo-osmotic (bottom set of panels)
solutions are shown. Cells were stimulated with square voltage
pulses from 80 mV to +120 mV in 40-mV steps from a 0-mV holding
potential. a, control HEK-293 cells; b,
HEK293-short ClC-3-GFP; c, HEK293-long
ClC-3-GFP; d, HEK293-short ClC-3(N597K)-GFP;
e, HEK293-long ClC-3(N597K)-GFP; f, HEK293-short
CLC-3 (no GFP fusion).
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Fig. 5.
Mean chloride currents in ClC-3 and
ClC-5-expressing cells. The mean steady-state peak chloride
currents for the indicated cell types at +120 mV are shown.
a, the rates of chloride current activation were measured
from 1 min prior to exposure to hypo-osmotic solution up to 10 min
following hypo-osmotic solution and are plotted as a fraction of the
maximal steady-state currents at +120 mV. The data were fitted with a
sigmoidal function. No significant difference was observed between the
rates of activation of any of the cell lines generated. The data shown
here are representative of only two cell lines, expressing short
ClC3-GFP and ClC-5-GFP, for clarity. b and c, the
mean currents in iso-osmotic (panel b, black
bars) and after 7 min of exposure to 30% hypo-osmotic solution
(steady state) (panel c, open bars) are shown. In
panel c the open circles show the individual
current readings obtained for different cells, demonstrating the
heterogeneity between individual cells. Note the differences in pA/pF
scales between b and c. No significant difference
in currents between cell types were observed except for ClC-5
expressing cells under iso-osmotic conditions (**). These currents
showed characteristics of previously described ClC-5 currents (see also
Fig. 8). The number of cells recorded for each cell line (n)
were: HEK293, n = 9; short hClC-3-GFP,
n = 9; long hClC-3-GFP, n = 5; short
N579K hClC-3-GFP, n = 5; short hClC-3,
n = 3; hClC-5-GFP, n = 9.
> Br
> Cl
> F
(Fig. 6).
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Fig. 6.
Anion permeability sequences for cells
expressing hClC-3-GFP variants. Cells were stimulated with a ramp
protocol from 80 mV to +80 mV lasting 1 s in the presence of
different halides as indicated.
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Fig. 7.
RVD is unaffected by expression of
ClC-3. Cell volume recovery (RVD) following exposure to
a 40% hypo-osmotic solution was measured. A, a
representative time course for relative change in cell volume
(Vt/Vo) for an HEK293 cell. B, mean values for
%RVD calculated from cell volume changes in single cells after 5-min
exposure to hypo-osmotic conditions. Control HEK293 cells,
n = 22; ClC-3-GFP-expressing cells, n = 15. No significant difference was measured.
= Br
> I
> F
(Fig. 8C). This is
similar to currents previously reported for ClC-5, and different in
activation, rectification, and ion selectivity from the endogenous
swelling-activated currents seen in parental HEK293 cells and in cells
expressing hClC-3. Exposure of hClC-5-expressing cells to hypo-osmotic
solution elicited a typical swelling-activated current
indistinguishable in magnitude and anion selectivity from the
endogenous currents of HEK293 control cells or hClC-3-GFP-expressing cells (Fig. 8, B and D). These data demonstrate
that, unlike hClC-3, hClC-5 is constitutively active under the
conditions used and elicits characteristic currents very different from
the cell swelling-activated currents seen in HEK293 cells. These data
provide a positive control for the absence of currents seen when
expressing ClC-3 and also demonstrate that addition of a GFP tag to
ClC-5 has no significant effect on current magnitude or
characteristics.
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Fig. 8.
ClC-5 currents in cells overexpressing
hClC-5-GFP. Chloride currents in cells expressing ClC-5-GFP were
measured. A, the family of currents in isotonic conditions
elicited by square pulses, ranging from 80 mV to +120 mV in 40-mV
steps from a holding potential of
40 mV. B,
swelling-activated currents recorded after 7-min exposure to 30%
hypo-osmotic solution. C, anion selectivity
(Cl
= Br
> I
> F
) recorded for currents in isotonic conditions.
D, anion replacement experiments recorded under hypo-osmotic
conditions using a ramp protocol identical to that used in Fig.
6.
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Fig. 9.
Calcium-activated chloride currents in ClC-3
expressing cells. Currents were activated by perfusion with the
calcium ionophore A23187 (10 µM) in control HEK 293 cells
(A) and in cells expressing short hClC-3-GFP (B).
The top panels show the chloride currents in iso-osmotic
media, and the lower panels show the currents after exposure
to the calcium ionophore. Cells were held at 0 mV and pulsed from 80
mV to +120 mV in 40-mV steps.
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Fig. 10.
Expression of endogenous ClC-3 in mouse
tissues. A, Western blot of mouse tissues probed with
anti-ClC-3 antiserum D1. 100 µg of protein was loaded in each lane.
The mobility of molecular mass markers is indicated. (heart, skeletal
muscle, upper and lower intestine, and testis are not shown). The same
protein bands were also detected using the Alomone antibody
demonstrating they do correspond to ClC-3 (data not shown).
B, ClC-3 is not expressed in murine heart. The blot was
probed with the Alomone anti-ClC-3 antibody (left panel) and
the anti-ClC-3 polyclonal antibody D1 (right panel). The
band of around 90 kDa in heart detected with the Alomone antibody is
not detected using the D1 antibody, demonstrating that it is a
consequence of a non-specific interaction.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
> I
(21) is countered by a second
report from the same team, which suggests that the short version
generates currents with the opposite selectivity (I
> Cl
) (20). We observe no such currents generated by either
the long or short version. Thus, the conditions or accessory proteins required to activate ClC-3 are unknown. Either way, these results are
incompatible with ClC-3 being responsible for known chloride currents
at the plasma membrane and are consistent with an intracellular role.
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ACKNOWLEDGEMENT |
---|
We are grateful to Thomas Jentsch for providing the ClC-5 fusion plasmid.
![]() |
Addendum |
---|
When this article was under review Stobrawa et al. (36) showed that disruption of the Clcn3 gene does not impair swelling-activated currents.
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FOOTNOTES |
---|
* This work was supported in part by the Medical Research Council.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.
§ Supported by European Union grant and by the German Academic Exchange Service.
To whom correspondence should be addressed: Tel.:
44-20-8383-8270; Fax: 44-20-8383-8337; E-mail:
a.sardini@csc.mrc.ac.uk.
Published, JBC Papers in Press, February 22, 2001, DOI 10.1074/jbc.M011667200
** Supported by the TMR Marie Curie Research Training Grant.
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ABBREVIATIONS |
---|
The abbreviations used are: RVD, regulatory volume decrease; GFP, green fluorescence protein; PCR, polymerase chain reaction; EGFP, enhanced green fluorescence protein; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; NMDGCl, N-methyl-D-glucamine chloride.
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