Expression and regulation of ClC-5 chloride channels: effects of antisense and oxidants

T. X. Weng1, L. Mo1, H. L. Hellmich2, A. S. L. Yu3, T. Wood4, and N. K. Wills1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Xenopus beta -globin gene (a gift of Dr. Thomas Jentsch) were used for cRNA transcription and expression in Xenopus oocytes, following the methods of Mo et al. (22). For xClC-5 antisense transcription, plasmids containing xClC-5 cDNA were linearized with BamHI, extracted with phenol-chloroform, and then transcribed using commercially available kits (mMessage mMachine T7 kit; Ambion, Austin, TX).

For expression of ClC-5 in mammalian cells, a cDNA fragment from our previous pOcyte construct containing the xClC-5 cDNA and the Xenopus beta -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 MOmega . To form a gigaseal (10-50 GOmega ), the pipette was positioned onto the cell using a low-drift micromanipulator (5200; Burleigh). Data acquisition and analysis were achieved using a commercially available system (Axopatch 200B amplifier, DigiData 1200 analog-to-digital/digital-to-analog converter, and pCLAMP 6.03 software; Axon Instruments) controlled by an IBM PC (Gateway 2000, Pentium II). Current signals were low-pass filtered and digitized at a frequency at least twice the filter bandwidth. Pipette capacitance and series resistances were compensated using analog circuitry in the patch amplifier.

Data 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 (Delta i) divided by 20 mV (Delta V or gslope = Delta i/Delta V). Unless otherwise noted, results are presented as means + SE. Paired t-tests or nonparametric tests were employed to evaluate statistical significance as appropriate.

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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   A: immunoreaction of C1 polyclonal antibody (against ClC-5) with membranes from COS-7 cell membranes. Lane 1: untransfected COS-7 cells. Lane 2: cells transfected with cDNA for Xenopus ClC-5 (xClC-5) 2 days previously. Positive staining of an expected ~90-kDa band (arrow) was observed for xClC-5-transfected cells. B: fluorescence image from COS-7 cells transfected with xClC-5 cDNA 2 days previously and stained with C1 polyclonal antibody to ClC-5. No antibody staining was found when samples were preincubated with C1 blocking peptide (data not shown). C: confocal fluorescence image from A6 renal cell stained with antibody to ClC-5. As in COS-7 cells, no staining was observed in the presence of blocking peptide (data not shown).

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 -100 to +100 mV (in 20-mV increments) for GFP-transfected (upper left) and GFP + xClC-5-transfected (lower left) COS-7 cells. The holding potential was -20 mV and the postpotential was -60 mV. A large outwardly rectifying current was observed in xClC-5 cotransfected cells that was not found in controls (GFP transfection alone). The specific conductance for control cells (normalized to cell capacitance) averaged 32 ± 4 pS/pF (n = 4 at +100 mV) and remained stable for at least 10 min. No cells showed a conductance >38 pS/pF or current densities >1.2 pA/pF. In contrast, xClC-5-transfected cells showed larger conductances and time-independent currents, which averaged 358 ± 79 pS/pF and 18.1 ± 5.1 pA/pF, respectively, at +100 mV (n = 16). The mean steady-state I-V relationship is presented in Fig. 2 (right). We note that a variable, small inward current was also detected in some cells. This current usually occurred in cells that had been passaged many times before transfection.


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Fig. 2.   Whole cell current measurements in COS-7 cells under conditions of symmetrical Cl- concentrations. Left: recordings from representative control cell [transfected with green fluorescent protein (GFP) cDNA alone; top] and ClC-5-transfected cell (transfected with GFP and xClC-5 cDNAs; bottom) showing the current response to voltage steps ranging from -100 to +100 mV (in 20-mV increments). Right: mean steady-state current-voltage (I-V) relationships. , Cells cotransfected with xClC-5 and GFP cDNA (n = 16); open circle , mean results from control cells transfected with GFP cDNA alone (n = 4).

Relative Anion Permeability

ClC channels typically display a halide selectivity of Cl- > I- (11). Because of the extreme rectification of ClC-5, there is a lack of reversal potential shift. Consequently, in previous studies of ClC-5 expressed in Xenopus oocytes, we and others have compared the currents or slope conductances of the channel for various anions over a range of positive potentials (22, 27). In the present study, replacement of Cl- in the extracellular bathing solution by iodide, glutamate, or cyclamate decreased the currents (Fig. 3). The calculated slope conductances were significantly decreased after Cl- replacement and averaged 404 ± 47 pS/pF, 234 ± 36 pS/pF, 205 ± 39 pS/pF, and 197 ± 60 pS/pF (n = 4; paired measurements) for Cl-, iodide, glutamate, and cyclamate, respectively. Therefore, the relative anion conductance sequence was Cl- >I- > cyclamate >=  glutamate.


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Fig. 3.   Effects of anion replacement on transiently expressed xClC-5 currents in COS-7 cells. Outward currents were reduced when Cl- in the bathing solution was replaced by iodide, cyclamate, or glutamate. The relative anion conductivity sequence (gx/gCl) was 1:0.6 ± 0.2: 0.5 ± 0.2: 0.5 ± 0.2 for Cl-, iodide, cyclamate, and glutamate (n = 5).

Endogenous xClC-5 in Xenopus Oocytes and Effects
of Antisense

Before comparing the regulation of xClC-5 expressed in COS-7 cells and Xenopus oocytes, we first assessed endogenous ClC-5 expression in Xenopus oocytes. This functional expression system is widely used as an assay of ClC channels. However, in previous studies, we detected abundant mRNA for xClC-5 in Xenopus oocytes (16).

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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Fig. 4.   A: Western blot analysis of biotinylated surface membrane proteins (lane 1) or cytosolic proteins (lane 2) from oocytes injected with water. B: Western blot analysis of biotinylated plasma membranes for oocytes injected with xClC-5 cRNA (-) or with both xClC-5 cRNA and antisense xClC-5 cRNA (+). C: effects of xClC-5 antisense on ClC-5 currents in Xenopus oocytes. Control, water-injected oocytes (n = 8) and oocytes injected with antisense cRNA for xClC-5 (n = 13); Xenopus, cells injected with sense cRNA for xClC-5 (n = 13) or coinjected with sense and antisense cRNA for xClC-5 (n = 11); Human, injection of sense cRNA for human ClC-5 (hClC-5; n = 15) and coinjection with sense hClC-5 and antisense xClC-5 (n = 14).

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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Fig. 5.   Effects of acidic pH on xClC-5-induced currents in COS-7 cells (n = 4). A: comparison of slope conductances at +100 mV under pH 7.4, pH 5.3, and pH 8.7. B: mean steady-state I-V relationship at pH 7.4 (control, ), pH 8.7 (alkaline, open circle ), and pH 5.5 (acidic, black-down-triangle ).

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 (Delta i at +100 mV = 0.9 ± 0.2 pA/pF; n = 4), in agreement with previous studies (22, 26, 27).

                              
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Table 1.   Effects of pharmacological agents on xClC-5 currents expressed in mammalian cells and oocytes



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Fig. 6.   Effects of the cAMP-dependent protein kinase inhibitor, H-89, on expressed xClC-5 currents. A: effects on xClC-5 currents in oocytes. B: effects on xClC-5 currents in transfected COS-7 cells. Note that the xClC-5 currents at positive potentials were inhibited in both host cell types. Vm, voltage potential.

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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Fig. 7.   Effects of hydrogen peroxide (H2O2) on xClC-5 expressed in COS-7 Cells. A: whole cell conductances at +100 mV under control conditions (n = 7), after addition of H2O2 (100 µM, n = 7) to the extracellular solution, and after wash out (recovery, n = 4). B: mean steady-state I-V relationships for same experiments as shown in A: control, 100 µM H2O2, and recovery. C: representative traces showing xClC-5 currents from transfected COS-7 cells as a function of time.

Inhibitory effects of H2O2 were also found for ClC-5 channels expressed in Xenopus oocytes (see Fig. 8). Oocytes were injected with xClC-5 cRNA 48 h before each experiment. As before, xClC-5 currents showed strong outward rectification, and no effects of H2O2 were found at negative potentials. Unless otherwise noted, the following data represent current values measured at holding potentials of +100 mV. In preliminary experiments, exposure to H2O2 concentrations <1 mM had no significant effects on xClC-5 currents within 5 min. At higher H2O2 concentrations (10 mM), however, the average current was decreased by 14% ± 3% (n = 4, P < 0.05 paired t-test).


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Fig. 8.   Effects of H2O2 on xClC-5 expressed in Xenopus oocytes (n = 4). A: xClC-5 currents (at +100 mV) were measured at different times in the same oocytes. For the control group, oocytes bathed in normal Barth's solution were measured at time 0 (t = 0; solid bar) and 20 h later (t = 20; open bar). For the H2O2 group, xClC-5 currents were measured at time 0 (before H2O2 addition, solid bar) and after 20 h of incubation in H2O2 (1 mM; open bar). B: comparison of paired groups of oocytes injected with xClC-5 (left) or water (right). Mean values are shown for currents (at +100 mV) before (solid bars) and after 20 h of incubation in H2O2 (1 mM; open bars). The shaded bars show the currents 5 h after H2O2 was washed off.

In separate experiments, the effects of incubation in 1 mM H2O2 for 20 h were assessed in paired oocytes expressing xClC-5. As shown in Fig. 8A, xClC-5 currents were decreased by 57% ± 8% (n = 4, P < 0.01), compared with initial control values before exposure to H2O2. In contrast, xClC-5 currents for paired oocytes incubated in the absence H2O2 for 20 h increased 35% ± 6% (n = 4) compared with their initial control values, reflecting an expected time-dependent increase in the level of expression of xClC-5. No effects of H2O2 were found for water-injected oocytes when incubated in 1 mM H2O2 for 20 h (data not shown).

To control for time-related differences in the level of ClC-5 expression encountered in the experiments above, in an additional study, groups of oocytes were compared (Fig. 8B). One group was injected with the xClC-5 cRNA and not exposed to H2O2 (control), while a second group was similarly injected and incubated in the presence of 1 mM H2O2 for 20 h. The control group had a significantly higher average current (6.5 ± 0.7 µA, n = 16) compared with the group exposed to H2O2 (2.6 ± 0.3 µA, n = 18, P < 0.01). Within 5 h after wash off of H2O2, the xClC-5 current partially recovered to 4.3 ± 0.5 (n = 9). Similar results were found for oocytes expressing hClC-5 (data not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 protein alpha 2-macroglobin. Further work using ClC-5 tagged with fluorescent fusion proteins such as GFP to observe trafficking in living cells is needed resolve these discrepancies.

Implications 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 Cl- transport. Alternatively, it is conceivable that the conductance properties of ClC-5 could be altered by regulatory proteins that remain to be discovered.

As 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 Cl- channel, cystic fibrosis transmembrane conductance regulator (CFTR; data not shown). Therefore, the lack of regulation in oocytes apparently does not reflect a major deficit in intracellular signaling pathways in these cells.

In 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 Cl- channel activity in several types (14). In the present experiments, exposure to micromolar or millimolar concentrations of H2O2 inhibited ClC-5 currents. It is unclear whether this agent affected the ClC-5 channel protein directly or affected proteins that regulate ClC-5 channel activity. We note that ClC-5 expression has recently been found in human RPE cells (30) and human airway epithelial cells (12), two epithelia that are exposed to oxidative agents under various conditions. Airway epithelia are exposed to oxidative agents during inflammatory events (17), and RPE cells are also exposed to oxidants during phagocytosis of rod outer segments or by exposure to light (28). Recently, we demonstrated that the Cl- conductance of RPE cells is decreased after exposure to H2O2 (30). The present results raise the possibility that ClC-5 channels may be possible candidates for mediating these inhibitory effects of H2O2 on RPE cell conductances.

In 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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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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