From the ¶ Programme in Structural Biology, Research
Institute, Hospital for Sick Children, Toronto, Ontario M5G 2X8, and
Departments of Physiology and
Pathology,
University of Toronto, Toronto, Ontario M5S 1A8, Canada
Received for publication, September 25, 2002, and in revised form, February 21, 2003
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ABSTRACT |
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The ClC-2 chloride channel has been
implicated in essential physiological functions. Analyses of ClC-2
knock-out mice suggest that ClC-2 expression in retinal pigment
epithelia and Sertoli cells normally supports the viability of
photoreceptor cells and male germ cells, respectively. Further, other
studies suggest that ClC-2 expression in neurons may modify inhibitory
synaptic transmission via the ClC-2 is a member of the ClC family of chloride channels (1).
Expression of recombinant ClC-2 in Xenopus oocytes (2) and
in mammalian cells (3) confers the expression of a hyperpolarization and swelling-activated chloride conductance path exhibiting an inwardly
rectifying current-voltage relationship. Recent studies in which ClC-2
expression was disrupted in model organisms support the claim that
ClC-2 directly mediates this chloride conductance. For example, a
chloride conductance path with the above biophysical properties was
detected in studies of salivary gland cells obtained from normal mice
whereas this function was absent in salivary gland cells obtained from
ClC-2 knock-out mice (4). Similarly, depletion of the
Caenorhabditis elegans ortholog of ClC-2, CLH-3, in
maturing oocytes by RNA interference leads to the complete abrogation
of the native, inwardly rectifying chloride conductance (5).
The physiological consequences of disrupting ClC-2 expression in these
organisms implicate a role for this channel at the cell surface in
modifying extracellular microenvironments or cell-cell communication
(5, 6). For example, ClC-2-deficient mice are blind, and the males are
infertile. These defects have been attributed to altered chloride
conductance across the plasma membrane of retinal pigment epithelium
and Sertoli cells, affecting the microenvironment surrounding
photoreceptors and male germ cells, respectively. Localization of ClC-2
to the apical membrane of fetal lung epithelia suggests a role in
regulating airway surface fluid (7, 8). In addition, ClC-2 channel
function has been implicated in mediating intestinal fluid secretion,
as it has been localized close to the apical tight junctions of
intestinal epithelia (9, 10).
Detailed studies have revealed that ClC-2 channels are also localized
to intracellular vesicular compartments. Electron micrographs of
immunogold-labeled ClC-2 endogenously expressed in hippocampal neurons
show that it is localized not only to the plasma membrane of somata,
proximal dendrites, axon initial segments but also to small transport
vesicles in the cytosol (11). This expression pattern is similar to
that described previously for the In the present study, using an integrated approach involving
biochemical and cell imaging and electrophysiological techniques, we
show that ClC-2 interacts with the retrograde dynein motor complex
in vitro and in vivo. Further, this interaction
likely mediates trafficking of ClC-2 as the subcellular distribution and functional expression of this channel at the cell surface is
disrupted by inhibition of dynein function. To our knowledge, these are
the first results to demonstrate an in vivo interaction between an ion channel and the dynein motor complex.
Constructs and Antibodies--
The antisense ClC-2 construct was
made by cloning the rat ClC-2 open reading frame into pCDNA 3.1(
Several antibodies were screened for the detection of endogenous
dynein: two monoclonal antibodies against the dynein heavy chain (DHC)
and intermediate chain (DIC) subunits, respectively (Sigma), and one
against the intermediate chain (Chemicon). However, only the Chemicon
antibody exhibited positive immunoreactivity upon analysis of rat
hippocampal homogenate (anti-dynein IC; 1:1,000; Chemicon). Endogenous
ClC-2 was detected using polyclonal antibodies raised against either
the N or the C terminus of ClC-2 (directed against residues 31-74 and
869-907 of rat ClC-2, respectively). Where relevant, actin detection
with a monoclonal actin antibody (1:1,000; Sigma) was used as a control
for equal protein loading.
Cell Culture--
COS7 cells (obtained from the American Type
Culture Collection, Manassas, VA) were cultured at 37 °C in minimum
essential medium (Wisent Inc., Montreal, Canada) supplemented with 10%
fetal calf serum. Cells were generally grown to 30% confluency prior to transfection, as well as for patch clamp experiments. Transfections were performed on cells (seeded onto 35-mm coverslips 12 h prior to transfection) with SuperFect transfection reagent (Qiagen, Chatsworth, CA). Cells were incubated with transfection reagent for
3-6 h in 5% serum-supplemented medium, washed with phosphate-buffered saline (PBS; Wisent Inc.), and grown for 16-24 h.
For nocodazole experiments, cells were washed three times with PBS,
followed by a 30-min incubation with 35 µM nocodazole (methyl-(5-2-thienylcarbonyl)-1H-benzimidazol-2-yl)carbamate; Sigma) in serum-free medium, at either 4 or 37 °C. For experiments requiring drug recovery, cells were washed extensively with PBS following exposure to nocodazole and incubated with serum-supplemented medium for 120 min. In cycloheximide treatment experiments, cells were washed three times in PBS and then incubated with growth medium
supplemented with 100 µg/ml cycloheximide (Sigma) for 4 h to
arrest protein synthesis.
Immunofluorescence and Confocal Microscopy--
For
immunofluorescence experiments, cells were washed with PBS followed by
fixation with 4% paraformaldehyde in PBS for 20 min at room
temperature. Cells were permeabilized for 10 min with 0.1% Triton in
PBS. Coverslips were blocked in 5% goat serum (Vector Laboratories,
Burlingame, CA) in PBS for 2 h followed by a 1-h incubation in
primary antibody diluted to the desired concentration in blocking
buffer. Coverslips were washed in blocking buffer for 30 min before
incubation in secondary antibody for 1 h and washed again in PBS
before mounting with DAKO (DAKO, Copenhagen, Denmark) medium. For
double-labeling experiments, two primary antibodies developed in
different species were applied together, followed by simultaneous
detection using Cy3 and fluorescein isothiocyanate-coupled secondary
antibodies. Slides were analyzed with a Carl Zeiss confocal laser
microscope (LSM 510) under a ×63 objective. Optical sections were
generally 0.7-0.9 µm thick.
Image Analysis--
The extent of co-localization of ClC-2 with
EEA1 was quantified using a modification of methods described
previously (44, 45). Briefly, background-corrected EEA1-specific
immunofluorescence was subtracted from ClC-2-specific
immunofluorescence to yield a "difference" image (using
Scion Image; Scion Corporation). Vesicular structures within this
difference image represent ClC-2 and EEA1 co-localized within
endosomes. The extent of co-localization of ClC-2 with EEA1 was
determined as the percent intensity of the co-localized signal relative
to the total ClC-2-specific immunofluorescence.
ImageJ (NIH imaging) was employed for line scanning analysis to
quantitatively assess ClC-2 subcellular distribution in COS7 cells.
Lines were typically drawn from the periphery of the nucleus to the
cell edge through the intracellular staining pattern for ClC-2. This
line was then divided into four equal segments. The fluorescence
intensity for each segment was quantified and expressed as a fraction
of total fluorescence intensity.
Immunogold Electron Microscopy--
Paraformaldehyde- and
glutaraldehyde-fixed COS7 cells were infused with a solution of 10%
gelatin in PBS at 37 °C, allowed to gel at 4 °C, and infused with
2.3 M sucrose overnight. Ultrathin sections (100 nm) were
mounted on Formvar-coated nickel grids. Sections were labeled with
ClC-2 and/or dynein antibodies as described previously (9).
Controls included the omission of either of the primary antisera.
Specimens were examined and images acquired with a JEOL JEM
transmission electron microscope (JEOL USA, Peabody, MA) equipped with
a digital camera (AMT Corp., Danvers, MA).
Protein Affinity Columns and Immunoblotting--
1 ml Hi-Trap
columns (Amersham Biosciences) were injected with either 10 mg/ml
amylase (Sigma) or ClC-2, purified as described previously (14) in 0.2 M NaHCO3, 0.5 M NaCl, pH 8.3 (supplemented with 4% pentadecafluorooctanoic acid (Dakwood Products
Inc., West Columbia, SC) for the application of purified ClC-2).
After a 30-min incubation at room temperature, the column was washed
with Buffer A (0.5 M ethanolamine, 0.5 M NaCl,
pH 8.3), followed by Buffer B (0.1 M acetate, 0.5 M NaCl, pH 4.0). Mouse brain lysate was passed through the
column, equilibrated previously with 1% Triton in PBS, pH 8.0. Briefly, tissue was homogenized in 0.1% Triton X-100 in PBS with
protease inhibitors, 10 mg/ml aprotinin and leupeptin, 1 mM
benzamidine, 2 mM dithiothreitol, 10 µM E64, 0.1 mM sodium orthovanadate. Homogenate was spun at
100,000× g for 2 h to remove unsolubilized material
before application to the column. The column was then washed twice in
equilibration buffer, and bound protein was eluted with 4%
pentadecafluorooctanoic acid in 100 mM phosphate buffer, pH
4.0. 1-ml fractions were collected, and protein content was assayed by
measuring absorbance at 280 nm. Fractions containing the highest levels
of protein (18-22) were pooled and concentrated in an Amicon
Centriprep 50 concentrator (Millipore Corporation, Bedford, MA).
Following determination of protein concentration using a modified Lowry
protocol, eluant from all three columns was subjected to
SDS-polyacrylamide gel electrophoresis (4-12% Tris-glycine, NOVEX)
and silver staining.
For Western blotting analysis, antibodies were diluted to the required
working concentrations in blocking buffer (5% milk, 0.1% Tween 20 in
PBS). For sequential probing of the same samples, membranes were
stripped by washing in SDS-stripping buffer at 55-60 °C for 40 min.
Blots were first re-blocked and re-probed with secondary antibody to
confirm effective stripping, before incubation with the desired antibody.
MALDI-TOF Mass Spectrometry and Identification of
ClC-2-interacting Protein--
Matrix-assisted laser desorption
ionization time-of-flight (MALDI-TOF) mass spectrometry was performed
by the Mass Spectrometry Laboratory at the Molecular Medicine Research
Centre (University of Toronto). Briefly, the dominant protein band (100 kDa) eluted from the ClC-2 affinity column was excised from the
silver-stained gel, washed twice with acetonitrile followed by 0.1 M ammonium bicarbonate, and digested with 6.25 ng of
trypsin (Roche Molecular Biochemicals) in 4 mM
CaCl2, 50 mM ammonium bicarbonate at 37 °C
overnight. Tryptic peptides were extracted from the gel slice with two
washes of 100 µl each of 0.1 M ammonium bicarbonate. Pooled peptides were acidified by addition of acetic acid to 1% and
reverse phase extracted with a 1:1 suspension of C18 silica resin.
Resin was washed with two 100-µl aliquots of 2% acetonitrile, 1%
acetic acid, and peptides were eluted with 5 µl of 65% acetonitrile, 1% acetic acid. A MALDI-TOF peptide mass fingerprint was obtained with
an Applied Biosystems Voyager DE STR instrument using Co-immunoprecipitation--
Hippocampal slices (300-µm
thickness) were prepared from adult male rats (Sprague-Dawley, 150-200
g; Charles River) as described previously (13). A crude membrane
preparation was obtained from tissue homogenate (in PBS with protease
inhibitors by centrifugation at 100,000 × g for 1 h following a 10-min low speed spin at 2,000 × g to
pellet nuclei and unlysed cells. For co-immunoprecipitations, 500 µg
of protein from crude membrane preparations were solubilized in
modified RIPA buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.05% (w/v) SDS, 1%
Triton X-100 with protease inhibitors) at 37 °C for 10 min. Samples
were spun at 15,000 × g to sediment unsolubilized
protein aggregates and incubated with ClC-2 antibody (2 µg/ml) for
16 h at 4 °C. The antibody-protein complexes were adsorbed from
solution with protein A-Agarose beads (Roche Molecular Biochemicals).
Proteins were eluted from beads with SDS loading buffer containing 63 mM Tris-Cl, pH 6.8, 10% (v/v) glycerol, 2% (w/v) SDS, and
25 mM dithiothreitol. Samples were reduced with 10 mM Tris (2-carboxyethyl) phosphine and analyzed for the
presence of dynein and ClC-2.
Cell Surface Biotinylation--
COS7 fibroblasts grown to 70%
confluency on 15-cm dishes were incubated on ice for 30 min. Cells were
washed with biotinylation buffer (1 mM MgCl2, 1 mM CaCl2, pH 7.8) and then incubated with 1.0 mg/ml sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate (Pierce) for 30 min on ice. Biotinylation was quenched by rinsing cells
with 1% bovine serum albumin in PBS. Cells were then lysed with
modified RIPA buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% (w/v) SDS, 1% Triton
X-100 with protease inhibitors). Solubilized cell extracts were
collected by centrifugation at 15,000 × g for 10 min.
Supernatants were incubated with streptavidin beads (Pierce) for 1 h at 4 °C, and biotinylated proteins were eluted from beads with 63 mM Tris-Cl, pH 6.8, 10% (v/v) glycerol, 0.5 mM
EDTA, 2% (w/v) SDS, 2% Patch Clamp Studies of COS7 Cells--
COS7 membrane currents
were measured using conventional whole cell patch clamp technique as
described previously (10). Briefly, currents were measured using an
Axopatch 200A patch clamp amplifier (Axon Instruments, Foster City, CA)
and were filtered at 100 Hz with a six-pore Bessel Filter. Sampling
rate was 4 kHz for most data, and junction potentials were corrected.
Voltage clamp protocols were generated using pCLAMP software (version
7; Axon Instruments). Current-voltage relationships were determined in
a stepwise clamp protocol. From a holding potential of 30 mV, voltage
pulses of 3.0 s were applied from Statistics--
Patch clamp and immunofluorescence quantitation
data are presented as the means ± S.E. Most statistical analyses
were performed using the Student's unpaired t test.
Results obtained in patch clamp studies with drug-treated and
-transfected cells were analyzed using the unpaired Student's
t test. Differences between multiple treatment groups were
assessed by ANOVA, and differences between specific pairs were
subsequently analyzed using the Bonferroni method.
ClC-2 Interacts with Rat Brain Dynein in in Vitro Studies--
As
previous studies by Bali et al. (12) implicate a role for
vesicular trafficking in ClC-2 regulation, we sought to identify proteins that may interact with ClC-2 and serve to mediate or regulate
its translocation between the plasma membrane and intracellular vesicles. An affinity column bearing purified intact ClC-2 protein was
used to capture ClC-2-binding proteins. Recombinant rat ClC-2 protein,
engineered to possess a polyhistidine tail at its N terminus, was
expressed in Sf9 cells using the baculovirus expression system and purified by metal affinity using the methods described in our
previously published work (14). Purified ClC-2 was conjugated to a
Hi-Trap column, through which mouse brain homogenate was passed. Brain
tissue was used as starting material as ClC-2 is abundantly expressed
in this organ (Fig. 1A).
Control experiments were conducted simultaneously using an unconjugated
and an amylase-conjugated Hi-Trap column. Interacting proteins were
eluted using a pH gradient, and fractions containing the highest levels
of protein were pooled, concentrated, and analyzed by SDS-PAGE and
silver staining. Multiple silver-stained bands were detected
specifically in the eluant from the ClC-2 affinity column (Fig.
1B). We excised the prominent 100-kDa band for analysis by
mass spectrometry. Briefly, a MALDI-TOF peptide mass fingerprint of the
excised band was obtained, and monoisotopic protonated peptide masses
were used to search the NCBI sequence databases using PROFOUND. The
tentative protein identification as a fragment of DHC subunit was based
on the high number of matched peptide masses observed (25% sequence
coverage) and low mass error (<0.5 atomic mass unit).
The dynein motor complex is comprised of several different polypeptide
components; a functional complex is typically composed of two copies of
one of the heavy chain subunits, together with multiple copies of
intermediate, light intermediate, and light chains (15). Dissociated
heavy chain (isolated from purified bovine brain cytoplasmic dynein)
migrates with an apparent molecular mass slightly greater than 200 kDa
(15). As shown in Fig. 1B, silver stain analysis of the
ClC-2 column eluant detects a similarly sized protein. However,
commercially available antibodies were not effective in detecting the
putative DHC fragment in total brain lysate or in immunoblots of the
eluant from the ClC-2 affinity, possibly because of digestion of the
antibody epitope. Recent experiments conducted by King et
al. (15) report unstable DHC-containing subcomplexes generated
from potassium iodide-treated purified bovine dynein, which was
attributed to a loss in stability conferred by interactions with DIC
and dynein light chain subunits. In fact, SDS-PAGE analysis of the
DHC-containing complex revealed several distinct bands with molecular
masses of slightly greater than 100 kDa (15). It is therefore
possible that fragmentation of DHC may have occurred in our ClC-2
column because of a loss in stabilizing contacts with other dynein
subunits during gradient pH elution. We reasoned that other subunits of
the dynein motor complex may have been specifically eluted as multiple
silver-stained bands were seen in Fig. 1B. Indeed,
immunoblotting revealed the presence of DIC specifically in the ClC-2
column eluant (Fig. 1C). Furthermore, DIC migrates as a
75-80-kDa protein, and inspection of the silver-stained gel in Fig.
1B reveals the presence of a protein of this mass. These
in vitro findings suggest that ClC-2 protein can interact
(directly or indirectly) with the multi-subunit dynein motor complex.
ClC-2 Interacts with Dynein in in Vivo Studies--
To determine
whether the interaction between ClC-2 and dynein exists in
vivo, immunoprecipitation experiments were performed using rat
hippocampal membranes and polyclonal antibodies directed against the N
or the C terminus of ClC-2 or pre-immune IgG as a control. As with the
N-terminal antibody (Nt-ClC-2; see Fig. 1A), the
C-terminal-directed antibody (Ct-ClC-2) is specific as Western blotting
of membranes prepared from murine brain tissue reveals a single band
that migrates at 97 kDa that is absent in the ClC-2 knock-out sample
(generated by Melvin and co-workers (4); see Fig.
2C). Crude hippocampal
membranes were prepared, and immunoprecipitations were performed in the
presence of 1% Triton X-100 plus 0.05% SDS as described under
"Materials and Methods." Immunoprecipitates were subjected to
SDS-PAGE and probed using our ClC-2 antibody directed against the N
terminus. Similar levels of ClC-2 can be immunoprecipitated with both
anti-ClC-2 antibodies (Fig. 2A). As expected, the
nonspecific antibody (i.e. preimmune IgG) did not
immunoprecipitate ClC-2. Co-precipitation of dynein with ClC-2 was
assessed by probing the immunoprecipitates described above with
anti-dynein (IC) antibody. Dynein IC could be detected in
immunoprecipitates using our Ct-ClC-2 but not in immunoprecipitates
using nonspecific IgG (n = 3) or our Nt-ClC-2 (n = 1) (Fig. 2B). These findings suggest
that the ClC-2 interacts with the dynein motor protein complex in
vivo and implicate the N terminus of ClC-2 in mediating this
interaction.
ClC-2 Is Functionally Expressed at the Plasma Membrane of COS7
Cells--
The biochemical assays described in the preceding sections
suggest an interaction between ClC-2 and the dynein motor complex. In
the following studies, we assessed the functional significance of this
interaction in the COS7 fibroblasts as this cell line endogenously
expresses ClC-2 and can be readily transfected. Confocal micrographs of
ClC-2-specific immunofluorescence in COS7 cells revealed a predominant
perinuclear staining pattern with a fainter signal detected close to
the cell surface (Fig. 3A,
indicated by arrowheads). Electron micrographs of
immunogold-labeled ClC-2 support our claim that ClC-2 localizes to the
plasma membrane of COS7 cells (Fig. 3B). Further
confirmation of the presence of ClC-2 at the cell surface was achieved
using cell surface biotinylation. Intact COS7 fibroblasts were
incubated with sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate to selectively label cell surface-associated proteins. To ensure that biotin had not labeled intracellular protein,
we also assessed biotinylation of the abundant intracellular protein,
actin. As shown in Fig. 3D, Western blotting analysis of
biotinylated proteins isolated from COS7 extracts shows the presence of ClC-2 and the absence of actin. Hence, this biochemical assay reports cell surface incorporation of ClC-2 endogenously expressed in COS7 cells.
Whole cell patch clamp studies confirmed the functional expression of
ClC-2 at the cell surface. Currents typical of those conferred by ClC-2
expression, with activation at hyperpolarizing voltage steps of at
least The Retrograde Trafficking of ClC-2 May Involve the Endosomal
Pathway--
To determine whether ClC-2 is trafficked via endosomes in
COS7 cells, we examined the relative localization of ClC-2 with respect
to a marker of early endosomal vesicles; EEA1 (Fig.
4, A-C) using confocal
microscopy. We found that the pattern of ClC-2-specific staining in
COS7 cells at 37 °C showed no appreciable steady-state co-localization with EEA1 in COS7 cells, shown in Fig. 4A
(merged image). However, as ClC-2 channels may associate with this
compartment only transiently, we imposed a temperature block,
i.e. 20 °C for 2 h, a maneuver known to be
permissive to internalization but restrictive to trafficking out of
early endosomes. This treatment has been shown to result in the
formation of swollen early endosomes (17, 18). When COS7 cells were
analyzed immediately following the 20 °C incubation, ClC-2-specific
immunofluorescence retained a perinuclear staining pattern but also
acquired the appearance of swollen vesicular structures, which
partially overlapped with EEA1-specific immunofluorescence (Fig.
4B). Co-localization of ClC-2 in EEA1-positive compartments
was most clearly evident in cells at 20 °C, which had been treated
previously with cycloheximide to minimize protein expression in the
biosynthetic pathway (Fig. 4C).
Quantitative analysis of the percentage of overlapping ClC-2-specific
and EEA1-specific immunofluorescence relative to total ClC-2-specific
immunofluorescence was performed to provide a quantitative assessment
of protein co-localization under different experimental conditions. In
total, 71 cells exposed to the 20 °C treatment and 16 cells exposed
to 20 °C with cycloheximide were compared with 64 untreated cells.
Under control conditions, only 8.9 ± 0.9% of ClC-2 signal was
found to co-localize with EEA1. Temperature arrest of vesicular exit
from endosomes resulted in a statistically significant increase
(p < 0.0001) in ClC-2 co-localization with EEA1 signal
in the presence and in the absence of cycloheximide (22.0 ± 4.1%
and 39.8 ± 4.4% respectively; see Fig. 4D). These results suggest that ClC-2 undergoes trafficking via endosomes and is
retained in early endosomal compartments at 20 °C.
We would expect that the 20 °C trafficking block would decrease
ClC-2 expression on the cell surface, as this temperature is permissive
to internalization but inhibitory to trafficking out of early endosomes
via recycling endosomes (19). As is shown in Fig. 4E,
the level of ClC-2 expressed at the cell surface (assessed by
biotinylation band densitometry) decreased by almost 63% upon incubation at 20 °C (n = 3). Together with the
observation of increased co-localization of ClC-2 with EEA1 in
cycloheximide-treated cells, these results support the notion that
ClC-2 undergoes retrograde trafficking between the plasma membrane and endosomes.
ClC-2 Subcellular Distribution in COS7 Cells Is Dependent on
Microtubule Integrity and Motor Function of the Dynein Complex--
To
determine whether our biochemical findings suggesting an in
vivo interaction between ClC-2 and the dynein motor complex are
physiologically significant, we assessed the effect of disrupting microtubules and dynein motor function on ClC-2 localization. We first
compared the subcellular distribution of ClC-2 with that of dynein. As
shown in Fig. 5B, dynein
exhibited a perinuclear distribution similar to ClC-2 (Fig.
5A), with significant dynein-specific immunofluorescence
close to the cell surface. At a higher magnification, co-localization
of ClC-2 and dynein was observed in regions proximal to the plasma
membrane (Fig. 5, A and B, insets;
co-localization indicated by arrowheads). Co-localization of
these two proteins at the plasma membrane was evident in electron
micrographs of immunogold-labeled ClC-2 and dynein (Fig.
5C). ClC-2 and dynein were also found to co-localize on
continuous intracellular membranes (data not shown). Collectively,
these findings suggest a possible role for dynein in mediating
retrograde ClC-2-trafficking.
The microtubule-depolymerizing agent nocodazole has been reported to
inhibit dynein-based motility (20). If the dynein motor complex
participates in the regulation of ClC-2 localization we predict that
nocodazole treatment would perturb the normal subcellular distribution
of ClC-2. To test this prediction, COS7 cells were therefore subjected
to a 30-min incubation in serum-free growth medium supplemented with
either 35 µM nocodazole in dimethyl sulfoxide (Me2SO; final concentration 0.01%) (v/v) or 0.01%
Me2SO as control (Fig. 6,
B and A, respectively). Following immediate
fixation, cells were then processed for immunofluorescence analysis and labeled for ClC-2 and tubulin. As shown in Fig. 6B,
nocodazole appeared to cause a dispersion of ClC-2. This effect was
reversible as we found that ClC-2 localization was restored upon
removal of the drug from the culture medium (data not shown), also
ruling out the possibility of cytotoxic effects of the drug (21). When COS7 cells were subjected to nocodazole treatment at a reduced temperature (4 °C) at which microtubule depolymerization is still observed, but protein trafficking is halted, ClC-2 distribution was
found to be unaltered (data not shown).
We employed Image J software to quantify the relative effects of
nocodazole or Me2SO (vehicle) on ClC-2 distribution. Line scan analysis was conducted on 36 drug-treated cells, and 32 Me2SO-control cells (randomly chosen from three independent
trials). Mean values and their corresponding standard errors were
calculated and plotted as bar graph representations using Origin
software (Fig. 6C; black bars represent
nocodazole-exposed cells), and unpaired Student's t tests
were performed to determine whether any differences observed were
statistically significant. Typically 55.3 ± 1.6% (mean ± S.E.) of endogenous ClC-2 fluorescence signal was within the first quarter length of the cell, with 27.8 ± 1.3%, 8.7 ± 0.7%,
and 8.1 ± 0.6% in the second, third, and fourth quarter lengths,
respectively. Microtubule disruption was found to shift ClC-2
fluorescence signal away from the perinuclear region toward the
periphery of the cell. ClC-2 signal was distributed more evenly
throughout the cytosol, so that less than half the original signal was
in the first quarter length (26.0 ± 1.1%, p < 0.00001). A concomitant increase in fluorescence signal was seen in the
peripheral quadrant, (21.7 ± 0.9% versus 8.1 ± 0.6%, p < 0.00001), supporting our visual impression
that ClC-2 distribution is dependent on microtubule integrity.
Collectively, these findings suggest that ClC-2 localization is
regulated in part by a microtubule motor-dependent process.
As an alternative approach toward studying the role of dynein in ClC-2
trafficking, we investigated the effect of impaired dynein motor
activity on ClC-2 localization. This was achieved by the overexpression
of a dynactin subunit, dynamitin (22). Dynactin is believed to act as
an intermediary or adaptor complex that facilitates dynein binding to
its cargo (reviewed in Ref. 23). Overexpression of dynamitin results in
the disruption of the dynactin complex by separating the
p150glued subunit that interacts with dynein, from the Arp1
filament, which is thought to facilitate cargo/dynein protein
interactions (24). The overexpression of dynamitin has been reported to
disrupt dynein-dependent maintenance of membrane organelle
distribution (22) and microtubule organization (25). The
dynein-dynactin complex is also implicated in the movement of material
within the endocytic pathway, as dynactin disruption by dynamitin
overexpression was found to perturb endosomal trafficking (26). Similar
effects can be seen by overexpressing the coiled-coil 1 domain of
p150glued (CC1; amino acids 217-548), which binds dynein
intermediate chain in vitro (27, 28) and is thus thought to
be the dynein-binding domain of dynactin (25). To assess the effect of
impaired dynein function on ClC-2 localization, we first tested the
effectiveness of our system by labeling dynamitin-GFP-transfected COS7
cells with anti-EEA1 antibody. As expected on the basis of previous studies (22, 26), dynamitin-GFP-transfected cells were observed to have
an altered EEA1 (early endosome) staining pattern (Fig. 6D,
left panel). In contrast to its native compact juxtanuclear distribution, overexpression of dynamitin induced a redistribution of
EEA1 into a more dispersed pattern, extending throughout the cytosol
toward the periphery of the transfected cell (Fig. 6D, left panel; transfected cells indicated by GFP fluorescence,
right panel). Similarly, ClC-2 distribution was disrupted in
dynamitin overexpressing COS7 cells (Fig. 6E, left
panel). ClC-2 lost its perinuclear localization and exhibited a
more scattered distribution throughout the cytosol and toward the edge
of the cell. We also observed a significant increase in ClC-2
localization at the plasma membrane in certain transfected cells
(indicated by arrowheads; see Fig. 6E, left
panel). Similar changes in ClC-2 distribution were seen upon
transfection with the DSRedCC1 construct (data not shown), supporting
the hypothesis that the dynein motor complex mediates trafficking of
ClC-2 via the endosomal pathway.
Line scan analysis was performed to quantitatively assess the effect of
dynamitin-GFP expression on ClC-2-specific immunofluorescence. 51 transfected cells (identified by GFP signal) and 54 untransfected cells
were examined. The cells were randomly chosen from four independent
transfections, and control cells were taken from transfected coverslips
to minimize deviations in fluorescence signals attributed to
experimental variation. Mean values and their corresponding standard
errors were calculated and plotted as bar graph representations using
Origin software (Fig. 6F; black bars represent
transfected cells), and unpaired Student's t tests were
performed to determine whether any differences observed were
statistically significant. On average, 62.8 ± 1.5% (mean ± S.E.) of endogenous ClC-2 fluorescence signal was within the first
quarter length, with 24.7 ± 1.3%, 9.1 ± 0.7%, and
5.9 ± 0.2% in the second, third, and fourth quarter lengths,
respectively. As with the nocodazole treatment, overexpression of
dynamitin-GFP was found to shift this distribution away from the
perinuclear compartments, so that only 38.0 ± 0.7% signal was in
the first quarter length (p < 0.00001). A
statistically significant increase in fluorescence signal was seen in
the remaining three quarter lengths (28.8 ± 0.6%,
p < 0.05; 21.0 ± 0.7%, p < 0.00001; and 12.7 ± 0.5%, p < 0.00001),
suggesting that the overexpression of dynamitin resulted in a
redistribution of ClC-2 toward the periphery of the cell.
ClC-2 Functional Expression at the Cell Surface of COS7 Cells Is
Dependent on Motor Function of the Dynein Complex--
The functional
significance of disrupting ClC-2 localization by dynein inhibition was
assessed in patch clamp studies. We measured the amplitude of
hyperpolarization-activated currents in cells that were transfected
with dynamitin-GFP. Consistent with our line scan analysis, we observed
a significant increase in the amplitude of ClC-2-mediated current
density in dynamitin-GFP-transfected cells (Fig.
7A, bottom trace).
The current density measured at
To determine whether the observed increase in ClC-2 channel function
following disruption of dynein function is indicative of an increase in
the amount of ClC-2 protein at the cell surface, we assessed the effect
of EHNA on expression of ClC-2 in the plasma membrane by surface
biotinylation. We assessed the effect of this pharmacological inhibitor
of dynein function rather than the effect of dynamitin overexpression
to optimize the number of affected cells and enhance the gain of our
biochemical assay. Inhibition of dynein by the addition of EHNA was
found to increase the amount of biotinylated ClC-2 by ~4-fold (Fig.
8). These findings suggest that the
increase in hyperpolarization-activated currents caused by disruption
of dynein function reflects an increase in the amount of ClC-2 at the
cell surface.
In the present report, we provide biochemical evidence for an
in vitro and in vivo interaction between ClC-2
and dynein in brain tissue. First, we have shown that the dynein
complex can interact with purified intact ClC-2 protein in our in
vitro binding assay. This interaction likely also occurs in
vivo, as dynein and ClC-2 can be co-immunoprecipitated from rat
hippocampal slices. The functional consequences of this interaction
were elucidated in the COS7 cell line using confocal microscopy and
patch clamp electrophysiology. We demonstrate that dynein is important
in ClC-2 trafficking, as disruption of dynein function not only causes dispersion of ClC-2 subcellular localization toward the plasma membrane
but also increases ClC-2 expression and activity at the cell surface.
The molecular basis underlying the interaction between the dynein motor
complex and ClC-2 remains to the determined. Hypothetically, the dynein
motor complex may bind directly to ClC-2 as recent studies by Tai
et al. (31) showed that the membrane protein, rhodopsin, can
bind directly to the dynein light chain protein, Tctex. Interestingly,
we could successfully co-immunoprecipitate dynein with ClC-2 using our
C-terminal- but not our N-terminal-directed antibody. As both
antibodies are effective in immunoprecipitating similar amounts of
protein, this result possibly implicates the N terminus of ClC-2 in
mediating its interaction with the dynein complex. The N terminus of
ClC-2 has been implicated previously in the regulation of the channel
function of the protein (32). However, our preliminary in
vitro binding experiments (including GST-fusion pull-down and
enzyme-linked immunosorbent assay assays) failed to provide evidence
supporting a specific, direct interaction between purified dynein
(kindly provided by S. King and T. Schroer) and GST-fusion proteins
containing either the N terminus (residues 31-74) or C terminus
(residues 869-907) of ClC-2. On the other hand, it is well known that
dynactin interacts with and activates cytoplasmic dynein (33). Further,
it has been shown that dynactin may recruit dynein to cargo in membrane
vesicles directly (34) or indirectly via cytoskeletal proteins such as
spectrin (35-37). Hence, in our future work, we will test the
prediction that dynactin and spectrin may mediate the interaction
between ClC-2 and dynein.
A role for the dynein motor complex in endocytosis has been implicated
in previous studies. Burkhardt et al. (22) showed that
disruption of the dynactin-dynein complex in COS7 cells by overexpression of dynamitin led to dispersion of endosomes toward the
cell periphery. This observation was confirmed in the present work
(Fig. 6D) and supports the putative role for this complex in
retrograde endosomal trafficking (22, 26). Furthermore, cytoplasmic
dynein has been shown to participate in phosphatidylinositol 3-kinase-regulated GLUT4 internalization in adipocytes (38). However,
the present studies are the first to show that function of the dynein
motor complex contributes to retrograde endosomal trafficking of an ion
channel, thereby providing a novel molecular framework with which to
study the regulated trafficking of ClC-2 channels and possibly other
members of the ClC family of chloride channels. Two other members of
this family, ClC-3 and ClC-5, have been localized to endosomal
compartments (39-41). However, unlike ClC-2, which is thought to
mediate its primary function at the cell surface, both ClC-3 and ClC-5
are stably expressed in endomembranes where they are thought to
regulate the function of this organelle (39, 41). Hence, the molecular
components mediating localization and trafficking of ClC-3 and ClC-5
may be distinct from those mediating ClC-2 trafficking.
In light of our current findings, we suggest that ClC-2 channel
function at the cell surface can be regulated by modulation of vesicle
retrieval from or insertion into the plasma membrane by dynein.
In our future work, we plan to determine the role of dynein-mediated
vesicular trafficking in ClC-2 activation by experimental maneuvers
shown previously to regulate ClC-2 function at the cell surface. For
example, activation of protein kinase C, cyclin-dependent kinase p34cdc2/cyclin B, and phosphatidylinositol 3-kinase
have been shown to inhibit ClC-2 function at the cell surface (12, 32,
42, 43). Furthermore, Zheng et al. (43) showed that cyclin
B-dependent phosphorylation of rabbit ClC-2 led to its
enhanced ubiquitination and degradation. Finally, it will be important
to determine how post-translational modification of ClC-2 regulates its
interaction with molecular components within the ClC-2 trafficking
pathway, such as the dynein motor complex.
-aminobutyric acid, type A
receptor. However, complete understanding of the physiological
functions of ClC-2 requires elucidation of the molecular basis for its
regulation. Using cell imaging and biochemical and electrophysiological
techniques, we show that expression of ClC-2 at the cell surface may be
regulated via an interaction with the dynein motor complex. Mass
spectrometry and Western blot analysis of eluate from a ClC-2 affinity
matrix showed that heavy and intermediate chains of dynein bind ClC-2 in vitro. The dynein intermediate chain
co-immunoprecipitates with ClC-2 from hippocampal membranes suggesting
that they also interact in vivo. Disruption of dynein motor
function perturbs ClC-2 localization and increases the functional
expression of ClC-2 in the plasma membranes of COS7 cells. Thus, cell
surface expression of ClC-2 may be regulated by dynein motor activity. This work is the first to demonstrate an in vivo
interaction between an ion channel and the dynein motor complex.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-aminobutyric acid, type A
receptor and suggests that, as in the case of the
-aminobutyric
acid, type A receptor, the number of ClC-2 channels on the cell surface
may be regulated by vesicular transport. In fact, recent studies by
Bali et al. (12) showed that inhibitors of
phosphatidylinositol 3-kinase, a lipid kinase with a well established role in membrane trafficking, reduce the basal amplitude of
ClC-2-mediated chloride currents measured by patch clamp
electrophysiology on the cell surface (12). These findings suggest that
cell surface expression of ClC-2 may be regulated via membrane
trafficking. However, as yet, little is known regarding the molecular
components that regulate the cell surface expression of ClC-2.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
)
vector so that the reversed restriction sites on this vector would
reverse the orientation of the open reading frame to create the
antisense plasmid (10). The constructs for the expression of
GFP1-tagged dynamitin, and
DSRed-tagged CC1 were kindly provided by Dr. T. Schroer (The Johns
Hopkins University). For immunofluorescence analysis, endogenous ClC-2
was detected using a peptide-purified polyclonal antibody (8 µg/ml)
directed against the N terminus of ClC-2. Dynein complex was labeled
with a monoclonal antibody against the intermediate chain (IC) of
dynein (1:100; Chemicon, Pittsburgh, PA), early endosomal compartments
with monoclonal anti-EEA1 antibody (1:200; BD Biosciences), and
tubulin with monoclonal anti-
tubulin (1:2000; Sigma).
Fluorescein-conjugated donkey anti-mouse IgG (in 50% glycerol; 1:250;
Jackson ImmunoResearch Laboratories) and donkey anti-rabbit IgG
(1:1000; Molecular Probes, Eugene, OR) secondary antibodies were used.
For immunogold labeling, goat anti-rabbit IgG 10-nm gold complexes and
goat anti-mouse IgG 5-nm gold complexes (Amersham Biosciences)
were used.
-cyano hydroxycinnamic acid as a matrix.
-mercaptoethanol, and 50 mM
dithiothreitol. Eluants were then analyzed for the presence of
ClC-2.
140 to +40 mV in 20-mV
increments. The bath solution contained 140 mM
N-methyl-D-glutamine chloride, 2 mM
MgCl2, 2 mM CaCl2, 5 mM
HEPES, whereas the pipette solution contained 140 mM
N-methyl-D-glutamine chloride, 2 mM
MgCl2, 2 mM EGTA, and 5 mM HEPES.
Both pipette and bath solutions were adjusted to pH 7.4 and 260 milliosmoles.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
ClC-2 interacts with the dynein motor complex
in vitro. A, ClC-2 expression in
murine hippocampus. Western blotting reveals a dominant band at 97 kDa,
which is abolished upon pre-incubation of the ClC-2 antibody with
antigenic peptide (+). Lanes were probed for actin to assess
equal loading. B, eluted proteins from columns to which
purified ClC-2, amylase, or nothing was conjugated were subjected to
silver stain analysis. The major protein band (100 kDa) specific to the
ClC-2 eluant was excised and identified by MALDI-TOF mass spectrometry
to be a fragment of DHC. C, Western blotting analysis
confirms the presence of DIC (a single 75-80-kDa band, also seen in
whole brain homogenate; see Fig. 2B) in ClC-2 column eluant but not the
amylase-conjugated column eluant.
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Fig. 2.
Co-immunoprecipitation of dynein intermediate
chain with ClC-2. A, samples containing either 500 µg
(+) or 0 µg ( ) of detergent-solubilized hippocampal membrane
protein were incubated with anti-N-ClC-2, anti-C-ClC-2, or a
nonspecific IgG as control. Precipitated protein-antibody complexes (+)
or antibodies alone (
) were subjected to SDS-gel electrophoresis and
probed for ClC-2 using our anti-Nt-ClC-2 antibody. B,
protein/antibody complexes as described above were analyzed for the
presence of DIC. DIC can be pulled down using anti-C-ClC-2 but not
anti-N-ClC-2, implicating the N terminus of ClC-2 in mediating its
interaction with dynein. C, specificity of our polyclonal
C-terminal-directed ClC-2 antibody. Wild-type (+/+) and ClC-2 knock-out
(
/
) mice brain tissue membranes (4) were probed with anti-Ct-ClC-2.
Blots were also probed with anti-actin to assess equal protein
loading.
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Fig. 3.
ClC-2 is functionally expressed at the cell
surface of COS7 fibroblasts. A, immunofluorescence and
confocal microscopy reveal a prominent ClC-2 perinuclear staining
pattern, with detectable levels of protein at the cell surface. Unless
otherwise indicated, scale bar represents 10 µm. B,
immunogold electron microscopy confirms the surface localization of
ClC-2. Control panel (C) shows no signal in the absence of
primary antibody. Scale bar represents 100 nm. D,
COS7 cells were surface-biotinylated (+) at 4 °C. Control
experiments were conducted simultaneously where COS7 cells were
incubated with buffer alone, without added biotinylation reagent ( ).
Western blotting analysis of biotinylated proteins confirms the
presence of ClC-2. Intracellular actin could not be detected in the
eluant. E, whole cell currents obtained by voltage steps of
20-mV increments, applied from
140 to 40 mV in control, untransfected
(upper trace), and in antisense-transfected cells.
F, mean I-V curves of ClC-2-mediated currents in
untransfected and antisense transfected cells. The amplitude of ClC-2
currents is dramatically reduced when the cells are transfected with
antisense ClC-2 cDNA. Error bars indicate S.E.
60 mV and greater, and an inwardly rectifying current-voltage
relationship was detected (Fig. 3E). The observed current
density had a magnitude of
21.5 ± 1.3 pA/pF (mean ± S.E., n = 10) at a membrane potential of
140 mV.
To ensure that these measured currents were in fact mediated by ClC-2,
we employed an antisense strategy. We have previously used this
approach to confirm the functional expression of endogenous ClC-2 in
Caco-2 cells (10), as there are no known specific blockers of the
ClC-2-mediated chloride conductance (16). In this previous study, we
showed that antisense ClC-2 cDNA significantly suppressed ClC-2
protein expression and reduced hyperpolarization-activated currents.
This effect was specific, as an endogenous depolarization-activated chloride conductance was unaffected by antisense ClC-2 cDNA
transfection (10). As in our previous studies in Caco-2 cells, we found
that antisense ClC-2 cDNA transfection of COS7 cells led to
significant suppression of endogenous hyperpolarization-activated
chloride currents (Fig. 3, E and F). We observed
a dramatic reduction in the magnitude of hyperpolarization-activated
current densities (at
140 mV) in antisense-transfected cells (Fig.
3E;
7.2 ± 0.6 pA/pF, n = 10)
relative to control (
21.5 ± 1.3 pA/pF; see Fig. 3F,
n = 10; p < 0.0001). The abolition of
hyperpolarization-activated chloride currents upon ClC-2 antisense
(aClC-2) transfection demonstrates that ClC-2 channels are functionally
expressed in the cell surface of COS7 cells. Furthermore, as in the
previous Caco-2 cell studies (10), antisense ClC-2 cDNA had no
effect on endogenous depolarization-activated chloride currents (at +40
mV, 2.6 ± 0.1 pA/pF in control cells versus 2.0 ± 0.3 in antisense-transfected cells, n = 10;
p > 0.1) arguing that the effect was specific.
Therefore, ClC-2 channels are expressed in the plasma membrane of COS7
cells, and hence, this cell line is suitable for studies of vesicular
trafficking to and from the plasma membrane.
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Fig. 4.
A sub-population of intracellular ClC-2
localizes to early endosomes. A, minimal
co-localization of ClC-2 with EEA1 in COS7 cells at 37 °C increases
significantly upon incubation at 20 °C (B). C,
accumulation of steady state levels of stable ClC-2 in early endosomes
in cycloheximide-treated (CHX) cells. Scale bar
represents 10 µm. D, quantitative analysis of ClC-2/EEA1
co-localization. A statistically significant increase in ClC-2
co-localization with EEA1 (*, p < 0.0001) was observed
under both conditions. E, 20 °C temperature block causes
a significant decrease in ClC-2 biotinylated at the cell surface (*,
n = 3) reflecting endosomal sequestration.
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Fig. 5.
ClC-2 (A) and dynein (B)
co-localize at the cell surface of COS7 fibroblasts. Scale
bar represents 10 µm. C, confirmation of ClC-2 and
dynein co-localization at the plasma membrane using immunogold electron
microscopy. Ultrathin COS7 sections were sequentially labeled for ClC-2
and dynein. 10 nm gold label corresponds to ClC-2 (white
arrowhead), whereas 5 nm label identifies the dynein motor complex
(black). Scale bar represents 100 nm.
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Fig. 6.
ClC-2 localization in COS7 cells is
microtubule- and dynein-dependent.
A-C, effect of nocodazole on microtubule
integrity and ClC-2 distribution in COS7 cells. Control and
nocodazole-treated cells were labeled for ClC-2 (left) and
tubulin (right). In nocodazole-treated cells, ClC-2 loses
its perinuclear staining pattern and is redistributed throughout the
cytosol. C, line scanning analysis of ClC-2 signal in
Me2SO- (white) and nocodazole-treated
(black) cells. Error bars indicate S.E.
D, early endosomal localization is disrupted upon
dynamitin-GFP overexpression. Cells overexpressing dynamitin-GFP
exhibit a fragmented and more dispersed distribution of EEA1-positive
structures. E, transfected cells were also labeled for
ClC-2 (left). ClC-2 displays a more dispersed
distribution, extending throughout the cytosol and toward the edge of
the cell (see arrowheads). F, line scanning
analysis was conducted on 54 control (white) and 51 transfected cells (black). Differences between values
obtained for untransfected cells versus transfected cells
were statistically significant in all cases (p < 0.05).
140 mV increased 67%, from
21.5 ± 1.4 pA/pF (n = 10) to
35.9 ± 1.3 pA/pF (mean ± S.E., n = 14, p < 0.0001), suggesting that disruption of dynein function increases the
number of ClC-2 channels in the plasma membrane (Fig. 7C).
To ensure that the observed effect was specifically because of impaired
dynein function at the level of vesicular trafficking and not because
of impaired dynein biosynthesis, we assessed the effect of
erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA), an
inhibitor of the ATPase activity of the dynein complex (29, 30) (Fig.
7, B and D). Hyperpolarization-activated currents
were recorded prior to and following treatment with 50 µM
EHNA. ClC-2-mediated currents were typically recorded 10 min after
addition of EHNA (Fig. 7A, middle trace).
Multiple trials indicated a 2-fold increase in current density
(
43.2 ± 2.3 pA/pF, n = 10, p < 0.0001) at
140 mV (Fig. 7B). These data are summarized in
the bar graph shown in Fig. 7E. Identical experiments
performed on Caco2 intestinal epithelial monolayers revealed an even
greater increase in ClC-2 channel function at the plasma membrane upon dynein inhibition with EHNA (50 µM).
Hyperpolarization-activated chloride currents mediated by ClC-2
increased from
23.9 ± 2.5 pA/pF to
69.9 ± 12.9 pA/pF
(n = 7, p < 0.01; see Fig.
7D). Collectively, these findings are consistent with our
hypothesis that dynein may be important in the vesicular transport of
ClC-2.
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Fig. 7.
ClC-2-mediated chloride currents are
activated by disruption of dynein function in COS7 and Caco2
cells. A, whole cell recordings for
hyperpolarization-activated ClC-2 currents in control (untreated),
EHNA-treated, and dynamitin-GFP-transfected cells. Mean I-V curves of
ClC-2 currents in cells with normal dynein function (control,
Con) and with impaired dynein function either by EHNA
exposure (B) or dynamitin overexpression (C).
D, EHNA also increases ClC-2-mediated currents on the cell
surface of Caco2 cells. E, the effect of dynein inhibition
by both EHNA treatment and dynamitin-GFP overexpression significantly
increase ClC-2 currents at 140 mV (compared here with the effect of
antisense-ClC-2 transfected cells). Error bars indicate
S.E.
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Fig. 8.
EHNA inhibition of dynein results in an
increase in ClC-2 protein at the cell surface. A, COS7
cells were exposed to either vehicle alone or 250 µM EHNA
(0.025% Me2SO) for 20 min prior to cell surface
biotinylation. B, band densitometry analysis shows a 4-fold
increase (p < 0.0001) in the level of
surface-associated ClC-2 in EHNA-treated cells in duplicate
experiments. S.E. are indicated by error bars.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We are grateful to Professor T. Jentsch (Hamburg) for providing rClC-2 cDNA and Professor G. Cutting (The Johns Hopkins University) for providing hClC-2. We acknowledge Dr. Jeffrey Charuk (Mass Spectrometry Laboratory at the Molecular Medicine Research Centre, University of Toronto) for mass spectrometry analysis of the ClC-2 affinity column eluant. We are also grateful to Dr. Steven King (University of Connecticut) and Dr. Trina Schroer (The Johns Hopkins University) for providing us with purified bovine dynein and to Dr. Schroer for providing cDNA coding for dynamitin-GFP and dsRed-CC1. We also acknowledge Dr. Paul Park and Roberto Botelho for assistance with immunofluorescence image quantitation.
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FOOTNOTES |
---|
* This work was supported in part by a National Institutes of HealthGrant DK49096 (to C. E. B.) and by the Heart and Stroke Foundation of Canada.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 a studentship award from the Canadian Cystic Fibrosis Foundation.
** To whom correspondence should be addressed: Research Inst., Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Tel.: 416-813-5981; Fax: 416-813-5028; E-mail: bear@ sickkids.on.ca.
Published, JBC Papers in Press, February 23, 2003, DOI 10.1074/jbc.M209828200
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ABBREVIATIONS |
---|
The abbreviations used are: GFP, green fluorescent protein; IC, intermediate chain; DHC, dynein heavy chain; DIC, dynein IC; PBS, phosphate-buffered saline; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; EHNA, erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride; pA/pF, picoampere/picofarad.
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