Departments of 1 Physiology and Biophysics, 2 Internal Medicine, and 4 Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555; and 3 Renal Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02155
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ABSTRACT |
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Genetic mutations of the
Cl channel ClC-5 cause Dent's disease in humans. We
recently cloned an amphibian ortholog of Xenopus ClC-5
(xClC-5) from the A6 cell line. We now compare the properties and regulation of ClC-5 currents expressed in mammalian (COS-7) cells
and Xenopus oocytes. Whole cell currents in COS-7 cells transfected with xClC-5 cDNA had strong outward rectification, Cl
> I
anion sensitivity, and were
inhibited at low pH, similar to previous results in oocytes. In
oocytes, antisense xClC-5 cRNA injection had no effect on endogenous
membrane currents or the heterologous expression of human ClC-5.
Activators of cAMP and protein kinase C inhibitors had no
significant effects on ClC-5 currents expressed in either COS-7 cells
or oocytes, whereas H-89, a cAMP-dependent protein kinase (PKA)
inhibitor, and hydrogen peroxide decreased the currents. We conclude
that the basic properties of ClC-5 currents were independent of the
host cell type used for expression. In addition, ClC-5 channels may be
modulated by PKA and reactive oxygen species.
Dent's disease; Xenopus oocytes; mammalian COS-7 cells; patch clamp; hydrogen peroxide
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INTRODUCTION |
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CLC CHANNELS are a
rapidly growing and large family of voltage-gated Cl
channels that are found in a wide variety of organisms including bacteria, yeast, plants, fish, amphibians, and mammals (11, 21,
23). To date, at least nine different human ClC genes have been
isolated. Loss of function mutations of one member of this family,
ClC-5, have been linked to the human hereditary renal disorder, Dent's
disease (18).
Recently, we isolated a homologue of the Xenopus ClC-5 channel (xClC-5) from the amphibian cultured renal cell line A6 (16, 22). When expressed in Xenopus oocytes (22, 26), xClC-5 currents showed strong outward rectification and were inhibited by acidic extracellular pH. These conductance properties were essentially identical to those of human ClC-5 (hClC-5) or rat ClC-5 (rClC-5) expressed in Xenopus oocytes (18, 27).
Although the predicted amino acid sequences for ClC-5 channels contain sites for putative phosphorylation by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), cAMP activators had no significant effects on rClC-5 currents expressed in oocytes (27). It is presently unclear whether this apparent lack of regulation by PKA and PKC is related to the host cell environment or other factors. For example, it is unclear whether endogenous intracellular messenger levels in oocytes are sufficient to support ClC-5 activation. In addition, oocytes lack some anchoring proteins necessary for modulation of channel activity by protein kinases (6). Similarly, it is conceivable that interactions with other endogenous proteins could affect ClC-5 channel activity.
Another issue that could affect ClC-5 channel properties is the
possibility of heteromultimeric channel formation. For example, expression in oocytes of heteromultimeric channels composed of ClC-1
and ClC-2 monomers resulted in Cl channels with novel
properties (19). Because we and others have detected ClC-5
mRNA and protein (xClC-5) in Xenopus oocytes (4,
16), it is important to know whether endogenous ClC-5 proteins
in oocytes affect the properties of ClC-5 currents expressed in this
system. Likewise, it is important to determine whether ClC-5 channel
properties and regulation are similar in mammalian expression systems.
To date, there have been only a few reports of ClC-5 channel functional
expression in mammalian somatic cells. In whole cell patch studies of
Chinese hamster ovary cells stably transfected with rClC-5, Sakamoto et
al. (25) observed a slightly outwardly rectifying current
that could be blocked by DIDS and had an anion selectivity sequence of
I > Cl
. In contrast, Friedrich et al.
(5) reported that transiently transfected HEK-293 cells
had a strong outwardly rectifying ClC-5-induced current that was more
permeable to Cl
than I
. Moreover, there
have been no reports of regulation of these outwardly rectifying ClC-5
channels expressed in mammalian cells. For this reason, we sought to
evaluate the effects of cAMP-related agents on these currents.
Recently, oxidative agents, including hydrogen peroxide
(H2O2), have been found to be potentially
important inhibitors of Cl channel activity in several
cell types, including cardiac myocytes (1), gastric
mucosal cells (24), and cultured human retinal pigment
epithelial (RPE) cells (30). It is presently unknown whether ClC channels are affected by oxidants. For this reason, we also
investigated the effects of H2O2 on ClC-5
currents expressed in mammalian cells and oocytes.
The aims of the present study were threefold. First, we compared the conductance properties of ClC-5 currents expressed in two systems, mammalian cultured cells and Xenopus oocytes. Second, the effects of endogenous ClC-5 protein expression in Xenopus oocytes were assessed using antisense methods. Third, several putative regulators of ClC-5 channels, including PKA and PKC activators and inhibitors, pH, and H2O2 on ClC-5 conductance properties were evaluated.
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METHODS |
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Cell Culture Methods
COS-7 cells were grown in plastic flasks at 37°C in an atmosphere containing 5% CO2 in Dulbecco's modified Eagle's medium (Fischer, Houston, TX) supplemented with 10% fetal bovine serum. For transfection, the cells were plated at a subconfluent density onto sterile glass coverslips coated with polylysine (50 ng/ml in water; Sigma-Aldrich, St. Louis, MO) and incubated at 37°C overnight. A6 cells were grown using similar methods for amphibian cells as described by Wills and Millinoff (29).cDNA Constructs
An expression vector (pOcyte) containing Xenopus ClC-5 (xClC-5) isolated from a cDNA library from Xenopus kidney cells (22) or hClC-5 cDNA flanked by the untranslated regions (UTR) of the XenopusFor expression of ClC-5 in mammalian cells, a cDNA fragment from our
previous pOcyte construct containing the xClC-5 cDNA and the
Xenopus -globin 5'-UTR was subcloned into a modified version of the pcDNA3.1 vector in which a cytomegalovirus (CMV) intermediate early promoter was inserted upstream of the multiple cloning site (CMV xClC-5).
Oocyte Preparation, Injection, and Electrical Recordings
Oocytes were prepared and injected as described by Mo et al. (22). Briefly, oocytes were subjected to a collagenase digestion protocol and injected 3-4 h later with 0.1-0.2 ng/nl cRNA solution. After incubation at 17°C for 2-3 days, induced membrane currents were measured using a two-electrode voltage clamp.Transfection
COS-7 cells on glass coverslips were subjected to transfection (FuGENE 6; Boehringer Mannheim, Indianapolis, IN) with a 10:1 mixture of cDNAs for xClC-5 (CMV xClC-5) and green fluorescent protein (GFP; pEGFR-N1 vector, Clontech). Cells were incubated for 2-3 days before current measurements. Control cells were transfected with pEGFR-N1 vector alone or sham transfected with pEGFR-N1 vector and CMV vector without xClC-5.Patch-Clamp Recordings of Whole Cell Currents
Ionic current recordings were made using whole cell patch-clamp techniques (8). The cells were mounted on the stage of an inverted microscope (Zeiss IM) and bathed in an extracellular solution consisting of (in mM) 130 tetramethylammonium (TMA)-Cl, 2 NaH2PO4, 2 calcium cyclamate, 1 MgSO4, 5 glucose, and 10 HEPES, pH 7.4 (300 mosmol/kgH2O). Patch pipettes were made from borosilicate glass (cat. no. 1B150F-3; World Precision Instruments) and were filled with intracellular solution [(in mM): 130 TMA-Cl, 0.2 calcium cyclamate, 3 MgSO4, 2 EGTA, 10 HEPES, and 3 Na-ATP, pH 7.4 (270 mosmol/kgH2O)]. The patch electrode was connected to an Ag-AgCl wire that was referred to a 3 M KCl-agar bridge in the bath solution. Pipette resistances were ~3-6 MData Analysis and Statistics
Steady-state currents were used to calculate the current-voltage (I-V) relationships. The outward slope conductance was calculated as the difference in current between +80 and +100 mV (Protein Isolation and Western Blot
The protocol for Western blot analysis was modified from Chillaron et al. (2). Untransfected COS-7 cells or COS-7 cells 3 days after transfection with xClC-5 cDNA were washed in Dulbecco's PBS (Sigma-Aldrich) and homogenized in fractionating medium (FM; 25 mM Tris · HCl and 100 mM mannitol, pH 7.2) with 0.5 mM phenylmethylsulfonyl fluoride (PMSF). Cells were then lysed by sonication on ice for 15 s and centrifuged for 15 min at 10,000 rpm to remove insoluble components. Cell membranes were pelleted at 100,000 g and resuspended in FM with 0.5 mM PMSF. For Western blot, 25 µg of each protein sample was boiled for 5 min in sample buffer (Nupage LDS; Novex, San Diego, CA), loaded in each lane of a precast gel (Nupage Bis Tris; Novex), electrophoresed, transferred to nitrocellulose membrane (Novex), and blotted with a polyclonal antibody to ClC-5 (C1 antibody raised against residues 570-677 of rClC-5) (20). For peptide-blocked controls, membranes were incubated overnight with a mixture containing a 5:1 ratio of C1 antigenic peptide to C1 antibody. Reacting proteins were detected using the enhanced chemiluminescence reagent system (Amersham Pharmacia Biotech, Buckinghamshire, UK) and photographic film (CL-Xposure; Pierce, Rockford, IL).Surface Biotinylation of Oocyte Proteins
Plasma membranes from Xenopus oocytes were labeled using NHs-reactive biotin ester (Sulfo-NHS-LC-Biotin; Pierce). Groups of five oocytes were incubated on ice for 30 min in 0.5 ml of wash solution containing 0.25 mg of biotin ester. Cells were then washed three times with chilled wash solution and homogenized in lysis buffer containing protease inhibitor cocktail (Sigma-Aldrich; 1:1,000 dilution) and 100 µM PMSF. After centrifugation, the supernatant was precipitated with streptavidin-coated beads, and the proteins were run on a precasted gel and subjected to Western blot analysis as described above.Immunocytochemistry
To allow visualization of ClC-5 proteins, A6 cells, COS-7 cells, and COS-7 cells that were transfected 2 days previously with xClC-5 cDNA were subjected to immunocytochemical analysis. Cells were fixed and permeabilized by immersion in chilled methanol for 8 min, washed with sterile filtered PBS, and incubated overnight at 4°C with the C1 primary antibody for ClC-5. After a wash in PBS and incubation at room temperature for 1 h with secondary antibody (Alexa 488), the specimens were washed again in PBS and mounted in Fluorosave (Calbiochem). In parallel experiments, the specificity of antibody staining was evaluated using blocking protocols. In these experiments, the specimen was exposed to a mixture of C1 antibody and a four- to eightfold excess of antigenic peptide and incubated overnight at 4°C as described above. Cells were viewed using a Nikon eclipse E800 epifluorescent microscope equipped with a digital camera and interfaced to a laboratory computer (Micron Electronics) or a confocal microscope system (Zeiss, Axomat).Drugs and Solutions
Forskolin, cAMP, PMSF, mammalian protease inhibitor cocktail, H2O2, and DIDS were from Sigma-Aldrich. 3-Isobutyl-1-methylxanthine (IBMX) and H-89 were from Biomol (Plymouth Meeting, PA). All drugs were dissolved in DMSO except cAMP, which was dissolved in distilled water, and PMSF, which was dissolved in isopropanol. Restriction enzymes were from New England Biolabs (Beverly, MA).Unless otherwise noted, PBS solutions consisted of (in mM) 81 Na2HPO4 and 19 NaH2PO4, pH 7.4. For biotinylation experiments, the solution washing buffer was composed of (in mM) 138 NaCl, 2.7 KCl, 1.5 KH2PO4, and 8 Na2HPO4. The lysis solution was 150 mM NaCl, 1% Triton X-100, and 20 mM Tris · HCl (pH 7.6).
Incubation solution for the oocyte experiments was modified Barth's solution [in mM: 88 NaCl, 1.0 KCl, 2.4 NaHCO3, 5 Tris · HCl, 0.82 MgSO4, 0.33 Ca(NO3)2, and 0.41 CaCl2, pH 7.5]. For current recording experiments, oocytes were bathed in ND96 buffer containing (in mM) 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES titrated with NaOH to pH 7.5.
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RESULTS |
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In the following sections, we first present the properties of xClC-5 transiently expressed in cultured mammalian cells. Next, we assess the effects of endogenous ClC-5 proteins on membrane currents in Xenopus oocytes and the heterologous expression of a mammalian ClC-5 channel in this system. Last, we compare the regulation of ClC-5 conductances expressed in mammalian cells or Xenopus oocytes.
xClC-5 Expression In Mammalian Cells
Endogenous mRNA for ClC-5 was not detected in Northern blot analysis of untransfected COS-7 cells (data not shown). Figure 1A shows the results of Western blot analysis of membrane proteins for untransfected COS-7 cells or cells that were transfected with cDNA for xClC-5 and incubated for 2 days. Untransfected control cells (lane 1) showed no detectable protein staining to polyclonal antibody to ClC-5, consistent with the earlier negative results for xClC-5 mRNA in Northern blot analysis. However, xClC-5 transfected cells (lane 2) showed a strong band of staining near the expected size for xClC-5 (89 kDa).
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Immunocytochemical methods of the transfected COS-7 cells also confirmed protein expression. Figure 1B shows a typical fluorescence image of COS-7 cells transfected with xCLC-5 cDNA 2 days previously as described above. Cells were stained with the C1 antibody to ClC-5 and subjected to confocal microscopy. Although positive staining to the ClC-5 antibody could be seen near the cell plasma membrane in most cells, most staining was found in intracellular compartments and in the perinuclear region. Untransfected cells or transfected cells incubated with antigenic peptide for ClC-5 did not show staining (data not shown).
To determine the pattern of ClC-5 staining in renal cells, A6 cells were stained with the ClC-5 antibody using identical methods. As shown in Fig. 1C, as in the case of COS-7 cells, staining was located near the nucleus, throughout the cell cytoplasm, with bright staining in the region of the cell membrane and filamentous membrane processes. Cells incubated in the presence of blocking peptide showed no detectable staining (data not shown). Consequently, ClC-5 appeared to be located intracellularly and near the cell membrane when expressed endogenously in A6 renal cells and after heterologous overexpression in COS-7 cells. For this reason, we next characterized the properties of xClC-5 currents overexpressed in COS-7 cells.
Conductance Properties
Currents induced by xClC-5 expression in COS-7 cells were recorded using the whole cell voltage-clamp technique (8). Figure 2 presents representative experiments showing the current response to voltage steps from
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Relative Anion Permeability
ClC channels typically display a halide selectivity of Cl
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Endogenous xClC-5 in Xenopus Oocytes and Effects
of Antisense
As a first step in assessing endogenous ClC-5 expression, Western blot
analyses of oocyte membranes were performed using the C1 antibody for
ClC-5 as before. Figure 4A
shows results for biotinylated surface membrane proteins (lane
1) and the cytosolic fraction (lane 2) for
water-injected oocytes. No staining was found in biotinylated surface
membrane proteins, whereas multiple bands, including a 90-kDa band near
the predicted size for xClC-5 (89 kDa), were found in the cytosolic
fraction. The staining of all bands in the cytosolic fraction could be
blocked with antigenic peptide for the C1 antibody (data not shown). We
next determined the effects of xClC-5 cRNA injections on protein
expression in the biotin-labeled surface proteins. As shown in Fig.
4B, left lane, a band of ~90 kDa was now
evident. In contrast, injection of equal amounts of xClC-5 cRNA and
antisense cRNA for xClC-5 reduced intensity of the 90-kDa band.
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To determine the effects of xClC-5 antisense on ClC-5 currents, three groups of paired oocytes (from at least 3 frogs) were injected as follows: group 1 was injected with antisense for xClC-5, group 2 was injected with both sense and antisense cRNA for xClC-5, and group 3 was injected with sense cRNA for hClC-5 and antisense for xClC-5. Paired controls for each group were injected with water (group 1), sense xClC-5 cRNA (group 2), or sense hClC-5 cRNA (group 3). After 3 days of injection, oocyte currents and protein expression at the plasma membrane were investigated using a two-electrode voltage clamp and Western blot analysis of biotinylated proteins, respectively.
The mean results for ClC-5-induced currents (normalized to the values at +100 mV) measured in oocytes are summarized in Fig. 4C for all three groups. Injection of antisense xClC-5 cRNA alone had no effect, i.e., the currents were similar to that of water-injected controls (Fig. 4C, control). In contrast, coinjection of antisense xClC-5 with sense xClC-5 essentially abolished the xClC-5-induced currents (Fig. 4C, Xenopus). This effect was specific for xClC-5 since no effects of antisense xClC-5 coinjection were found for cells expressing hClC-5 (Fig. 4C, human). Similarly, Western blot analysis indicated that the expression of hClC-5 was not affected by xClC-5 antisense coinjection (data not shown).
The results demonstrate that antisense xClC-5 is capable of blocking xClC-5 expression. Together with the findings for water-injected oocytes, the results suggest that endogenous expression of xClC-5 has negligible effects on endogenous currents or the expression of hClC-5 in oocytes. This lack of effect on hClC-5 conductance properties also indicates that the action of antisense xClC-5 cRNA is specific to the Xenopus isoform, as expected, due to sequence differences in the NH2-terminal region of human and Xenopus ClC-5.
Regulation of ClC-5 Properties
Modulation of the membrane conductance by bath pH.
Acidic bathing solutions decrease ClC-5 currents expressed in oocytes
(5, 22, 26). As summarized in Fig.
5 in COS-7 cells, reduction of the
extracellular bathing solution pH from 7.4 to 5.3 rapidly reduced the
outward currents and conductance by 35 ± 6% and 49 ± 5%,
respectively, at +100 mV (n = 4, P < 0.05), similar to our previous findings for xClC-5 expressed in
oocytes.
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PKA and PKC regulation of
xClC-5.
As discussed above, the predicted amino acid sequence for ClC-5
contains several sites for potential phosphorylation by PKA and PKC.
However, previous studies of mammalian ClC-5 expressed in
Xenopus oocytes found no effects of agents that elevate PKA activity such as cAMP, forskolin, or IBMX (27). Table
1 summarizes the effects of these agents
on xClC-5 currents expressed in oocytes and COS-7 cells. In
general, addition of 10 µM forskolin or a cAMP cocktail (containing
250 µM cAMP, 25 µM forskolin, and 100 µM IBMX) had no effect on
xClC-5 currents expressed in these two systems. Similarly, application
of the PKC inhibitor calphostin C (1 µM) also had no significant
effects on the currents. However, H-89 (10 µM), a PKA inhibitor,
decreased xClC-5 currents by 41% ± 5% (n = 4, P < 0.01) within 5 min for xClC-5-transfected COS-7 cells and by 31% ± 4% (n = 5, P < 0.01) for xClC-5-injected oocytes within 1 h (see also Fig.
6, A and B). The
effects of the Cl channel blocker DIDS (1 mM) were also
assessed. No significant effects were found (
i at +100
mV = 0.9 ± 0.2 pA/pF; n = 4), in agreement
with previous studies (22, 26, 27).
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Effects of oxidative agents.
To test the possible effects of the oxidative agent
H2O2 on ClC-5 channel activity, whole cell
currents in COS-7 cells expressing xClC-5 channels were measured
before, during, and after addition of 100 µM
H2O2 to the extracellular bathing solution.
H2O2 addition reduced the xClC-5 currents by
44% ± 8% (Fig. 7; n = 7, P < 0.01) within 5 min of exposure to
H2O2. These inhibitory effects were largely
reversible, as currents recovered 84% ± 7% within 5 min after wash
off of H2O2.
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DISCUSSION |
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The results of these experiments indicate that endogenous ClC-5 proteins in Xenopus laevis oocytes are largely located in intracellular compartments and do not significantly contribute to the membrane conductances in these cells. In contrast, evidence for both intracellular and membrane localization was obtained for ClC-5 proteins endogenously expressed by cultured renal epithelial A6 cells and heterologously expressed in COS-7 cells. Furthermore, transient expression of xClC-5 in COS-7 cells induced membrane currents with properties and regulation that were similar to those observed in oocytes (Refs. 22, 26, 27, and present results). These findings suggest that at least some basic features of ClC-5 currents and their regulation are independent of the host cell type used for expression. A novel inhibition of ClC-5 currents by H2O2 was also observed, suggesting the possibility that ClC channels are modulated by oxidative agents.
Endogenous ClC-5 Proteins: Lack of Effect on Membrane Currents
The results of antisense experiments indicate that endogenous ClC-5 proteins do not significantly affect the endogenous membrane conductance properties of oocytes. Although Lorenz et al. (19) previously reported that at least some ClC channels expressed in oocytes form heteromultimeric channels, several lines of evidence suggest that such heteromultimer formation is unlikely to occur for ClC-5 channels. First, coinjection of xClC-5 antisense cRNA had no effect on the expression of hClC-5 channels in Xenopus oocytes. Second, Steinmeyer et al. (27) found no effects of coexpression of rClC-5 with other ClC channels. Moreover, similar properties were found for mammalian and amphibian ClC-5 channels expressed in Xenopus oocytes or mammalian cells, including the I-V relationship and anion sensitivity (Ref. 5 and present results). Lastly, the present results show similar regulation of ClC-5 channels expressed in amphibian or mammalian cells, including inhibition by acidic pH, H-89, and H2O2.Immunodetection of Amphibian ClC-5
In the present study, we used the C1 antibody against ClC-5 for Western blot analysis of COS-7 and oocyte membrane proteins. In both cases, a protein band near the predicted size (~90 kDa) for xClC-5 was detected in cells transfected with xClC-5 cDNA or injected with xClC-5 mRNA, respectively, but not for untransfected or water-injected (control) cells. Previous studies of ClC-5 have produced discrepancies regarding the size of ClC-5 proteins from different species. The present results are in agreement with studies of hClC-5 or rClC-5 expressed in Xenopus oocytes (7). We note that Dowland et al. (4), using the same C1 antibody to ClC-5, recently reported immunostaining of a smaller 65-kDa protein band for porcine ClC-5 (predicted size: 83 kDa) expressed in cultured LLC-PK1 cells. Apparently, this difference is unrelated to the use of the C1 antibody.Channel Subcellular Localization
Western blot analysis of biotinylated surface oocyte membranes and immunofluorescence staining of xClC-5 transiently expressed in COS-7 cells indicated that ClC-5 was predominantly located in intracellular compartments, although some staining was also apparent in surface membranes for A6 cells and transfected COS-7 cells. Similar findings of a predominantly intracellular localization of ClC-5 proteins have been previously reported by Günther et al. (7) and Dowland et al. (4). In the latter study, Dowland et al. reported ClC-5 colocalization to an endosomal population that entraps FITC-dextran but not albumin or ricin. They concluded that ClC-5 was localized to early endosomes of the apical membrane fluid-phase endocytotic pathway. In contrast, Devuyst et al. (3) reported colocalization of ClC-5 with FITC-albumin but not FITC-dextran for cultured opossum kidney cells. They concluded that ClC-5 is associated with receptor-mediated endocystosis rather than a fluid-phase endocytotic pathway. Similarly, Günther et al. (7) observed that ClC-5 colocalized with early endosomes that internalized the proteinImplications of ClC-5 Current Properties and Regulation
Membrane currents in COS-7 cells that were transiently expressing xClC-5 showed strong outward rectification, similar to previous studies of xClC-5 and other ClC-5 homologues expressed in oocytes (18, 22, 26, 27). Recently, Friedrich et al. (5) also reported currents with strong outward rectification for HEK-293 cells transiently transfected with human ClC-5; however, anion conductance properties and regulation of channel activity were not reported in these experiments. The I-V properties of the ClC-5 channel are puzzling because cells do not usually encounter positive membrane potentials. However, at least some epithelial cells from so-called "tight" epithelia can demonstrate positive apical membrane potentials under conditions of high Na+ transport (15). Although the rectification properties of ClC-5 channels are poised to facilitate apical membrane NaCl entry into renal A6 epithelial cells, it is presently unknown whether these channels are trafficked to the apical membrane or whether they play a significant role in transepithelial Na+ or ClAs noted in RESULTS, in some COS-7 cells expressing xClC-5, a small linear inward current component was observed. These currents began to appear in cells that had been passaged many times over a period of several months. Linear components in the currents were infrequently found in cells from fresh stocks from the original passage number. Although we did not assess the cells for mutations, it is conceivable that this difference could result from some as yet unidentified genetic change or other possible alteration in the cells over time. Therefore, the use of cells with relatively low passage numbers may be an important variable in obtaining reproducible expression of currents.
Regulation
In agreement with previous studies of ClC-5 expressed in oocytes (18, 26, 27), ClC-5 currents were not significantly altered by cAMP or other PKA activators or PKC inhibitors of ClC-5 channel activity. This was the case both for ClC-5 currents expressed in Xenopus oocytes and for those expressed in COS-7 mammalian cells. Although PKA activators and PKC inhibitors were ineffective in altering ClC-5 activity in the present studies, these agents were clearly effective in investigations in paired oocytes expressing an unrelated ClIn contrast, H-89, a member of the H series of chemically synthesized inhibitors of cAMP-activated PKA (9, 10), inhibited ClC-5 currents in both expression systems. Kajita et al. (13) reported that H-89 inhibited ClC-2 channel activity in choroid plexus epithelial cells. These channels were stimulated by vasoactive inhibitory peptide via elevations in intracellular cAMP. One possible explanation for the lack of cAMP effects on ClC-5 activity in the present study is that the intracellular cAMP levels in both host cell types are sufficient to maximally activate the ClC-5 currents. However, the activation of CFTR by cAMP in paired cells would seem to argue against elevated endogenous cAMP levels. The inhibition of ClC-5 currents by the PKA inhibitor H-89, not by the PKC inhibitor calphostin C, is also consistent with activation of ClC-5 currents through a cAMP pathway but could also indicate a direct inhibitory effect of H-89. Further work assessing phosphorylation or site-directed mutagenesis of putative PKA binding sites in ClC-5 channels is needed to resolve this issue.
In agreement with previous reports from oocytes and HEK-293 cells (5, 22, 26), exposure to extracellular acidic bathing solutions led to an inhibition of ClC-5 currents expressed in COS-7 cells. In previous studies of ClC-5 expressed in oocytes (22, 26), the effects of extracellular pH were assessed over a range of values from 4.5 to 9.5. These studies reported no significant changes in ClC-5 currents for pH values above ~7.5 and maximal changes between pH 5 and pH 6. The inhibitory effect of acidic pH is opposite to the stimulation found for other ClC channels, such as ClC-1. Friedrich et al. (5) have proposed that ClC-5 channels located in endosomes might facilitate acidification of the endosomal lumen. However, the inhibition of these currents by acidic external pH appears to be inconsistent with this hypothesis. To resolve this issue, further work is needed to determine the mechanism of pH effects on ClC-5 channels in situ.
Oxidative Agents
Oxidative agents are known to regulate ClIn summary, the properties of amphibian ClC-5 currents, including outward rectification, anion dependence, inhibition by acidic extracellular pH, the PKA inhibitor H-89, and H2O2, were similar when this protein was expressed in cultured mammalian cells or Xenopus oocytes. Thus the observed properties of ClC-5 channels were apparently independent of the heterologous expression system employed. Further work is needed to elucidate the molecular mechanisms of ClC-5 regulation.
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ACKNOWLEDGEMENTS |
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We are indebted to Dr. Brian Button and Ana Pajor for advice and assistance with the Western blot analysis and to Drs. Harvey Fishman, Eric Detrait, and Soonmoon Yoo for equipment and assistance with the confocal images.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-53352 (to N. K. Wills).
Address for reprint requests and other correspondence: N. K. Wills, Dept. of Physiology and Biophysics, Univ. of Texas Medical Branch, Galveston, TX 77555 (E-mail: nkwills{at}utmb.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 26 October 2000; accepted in final form 11 January 2001.
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