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The Journal of Neuroscience, July 2, 2003, 23(13):5572-5582
Previous Article | Next Article
Expression of Voltage-Gated Chloride Channels in Human Glioma Cells
M. L. Olsen, S. Schade, S. A. Lyons, M. D. Amaral, andH. Sontheimer Department of Neurobiology and Civitan International Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294
Abstract
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Abstract
Introduction
Voltage-gated chloride channels have recently been implicated as being important for cell proliferation and invasive cell migration of primary brain tumors cells. In the present study we provide several lines of evidence that glioma Cl– currents are primarily mediated by ClC-2 and ClC-3, two genes that belong to the ClC superfamily. Transcripts for ClC-2 thru ClC-7 were detected in a human glioma cell line by PCR, whereas only ClC-2, ClC-3, and ClC-5 protein could be identified by Western blot. Prominent ClC-2, -3, and -5 channel expression was also detected in acute patient biopsies from low- and high-grade malignant gliomas. Immunogold electron microscopic studies as well as digital confocal imaging localized a portion of these ClC channels to the plasma membrane. Whole-cell patch-clamp recordings show the presence of two pharmacologically and biophysically distinct Cl– currents that could be specifically reduced by 48 hr exposure of cells to channel-specific antisense oligonucleotides. ClC-3 antisense selectively and significantly reduced the expression of outwardly rectifying current with pronounced voltage-dependent inactivation. Such currents were sensitive to DIDS (200–500 µM) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (165 µM). ClC-2 antisense significantly reduced expression of inwardly rectifying currents, which were potentiated by hyperpolarizing prepulses and inhibited by Cd2+ (200–500 µm). Currents that were mediated by ClC-5 could not be demonstrated. We suggest that ClC-2 and ClC-3 channels are specifically upregulated in glioma membranes and endow glioma cells with an enhanced ability to transport Cl–. This may in turn facilitate rapid changes in cell size and shape as cells divide or invade through tortuous extracellular brain spaces.
Key words: ClC channel; brain tumor; patch clamp; antisense knockdown; cell migration; cell proliferation
Introduction
Most primary brain tumors are derived from glial cells and are collectively referred to as gliomas. This heterogeneous group of tumors includes astrocytomas, glioblastomas, and oligodendrogliomas among others. Their precise lineage relationship to glial cells and the mechanisms underlying their malignant transformation are poorly understood (Linskey, 1997). In addition to their uncontrolled proliferation, glioma cells show an unusual propensity to disperse from the tumor site and invade the healthy brain tissue (Merzak et al., 1994
In many aspects, migrating glioma cells mirror the migration of progenitor cells during embryonic brain development (Levison et al., 1993
Several studies have reported on the expression of Cl– channels in glioma cells, some requiring volume changes for activation (Jackson and Strange, 1993
In the present study we set out to examine the expression and functional activity of endogenous voltage-gated Cl– channels in glioma cells. We demonstrate the expression of ClC-2, ClC-3, and ClC-5 at the mRNA and protein levels. Additionally, whole-cell patch-clamp recordings show two distinct Cl– currents that can be attributed to ClC-2 and ClC-3, respectively, using antisense knock-down strategies.
Materials and Methods
Cell culture. All experiments were performed on the glioma cell lines D54-MG [glioblastoma multiforme (GBM), World Health Organization (WHO) grade IV], a gift from Dr. D. Bigner (Duke University), U251-MG (GBM; a gift from Dr. Y. Gillespie (University of Alabama at Birmingham). U-138 (GBM), U118 (GBM), U87 (GBM), and STTG-1 (anaplastic astrocytoma, WHO grade III) were obtained from the American Type Tissue Collection (Rockville, MD). Cells were cultured in either DMEM/F12 (Invitrogen, Grand Island, NY) supplemented with 7% fetal calf serum (FCS) (Hyclone, Logan, UT) or DMEM supplemented with 10% FCS. No difference was observed between cells cultured in either media.Electrophysiology. Whole-cell voltage-clamp recordings were obtained via standard methods (Hamill et al., 1981
. Recordings were made on the stage of an inverted Nikon Diaphot microscope equipped with Hoffman Modulation Contrast Optics. Current recordings were obtained with an Axopatch 200A amplifier (Axon Instruments, Foster City, CA). Current signals were low-pass filtered at 2 kHz and were digitized on-line at 10–20 kHz, using a Digidata 1200 digitizing board (Axon Instruments) interfaced with an IBM-compatible computer (Dell XPS R400). Data acquisition and storage were conducted with the use of pClamp 8.2 (Axon Instruments). Cell capacitances and series resistances were measured directly from the amplifier, with the upper limit for series resistance being 10 M
Solutions. Unless stated otherwise KCl pipette solution was used with 2 mM TEA in the extracellular bathing solution to block outward K + currents. The standard KCl pipette solution contained (in mM): 145 KCl, 1 MgCl2, 10 EGTA, 10 HEPES sodium salt, pH adjusted to 7.3 with Tris-base. CaCl2 (0.2 mM) was added to the pipette solution just before recording, resulting in a free-calcium concentration of 1.9 nM. Cells were perfused continuously at room temperature with a saline solution containing (in mM): 125 NaCl, 5.0 KCl, MgSO4, 1.0 CaCl2, 1.6 Na2HPO4, 0.4 Na2H2PO4, 10.5 glucose, 32.5 HEPES acid, and 2 TEA, pH adjusted to 7.4 with NaOH. The osmolarity of this solution was
300 mOsm. Drugs were added directly to these solutions, and unless stated otherwise all drugs were purchased from Sigma (St. Louis, MO). When CdCl2 was added to the bath solution, phosphates and sulfates were omitted to prevent precipitation of CdPO4 and CdSO4. Similar results were observed when KCl pipette solution and 2 mM TEA in the bathing solution were replaced with a CsCl pipette solution (KCl in pipette replaced with 145 mM CsCl). For ion replacement studies, Cl – ions in the bathing solution were replaced with an equal amount of the substituting ion.
PCR. Total RNA was extracted from D54-MG cells using Trizol (Invitrogen) using the manufacturer's protocol, treated with DNase (Promega) using the manufacturer's protocol, alcohol extracted with phenol/chloroform/isoamyl, precipitated, and resuspended in 1 mM sodium citrate, pH 6.4 (Ambion). Starting with 2 µg total D54 RNA as template, cDNA was synthesized using 500 ng random hexamers at 70°C for 10 min before placing the reaction on ice. Tris-HCl (14.7 mM), pH 8.3, 22 mM KCl, 0.9 mM MgCl2, 12 mM dithiothreitol, 0.6 mM each dNTP and 12 U Superscript Reverse Transcriptase II (Invitrogen) were added in a final volume of 17 µl, and the reaction was incubated at 25°C for 15 min, 42°C for 120 min, and 92°C for 2 min. For (–) reverse transcriptase (RT) reactions, water was substituted for the Superscript RT II. After the RT reaction was complete, the cDNA was precipitated using 0.1 vol of 5 M ammonium acetate and 2.5 vol of 100% EtOH at –20°C for at least 2 hr. Precipitated reactions were pelleted by centrifugation and resuspended in 10 µl of water. Half of the cDNA was used as template for each PCR reaction. DNA was amplified by adding 100 ng each gene specific primers and Platinum PCR Supermix (Invitrogen: 22 mM Tris-HCl, pH 8.4, 55 mM KCl, 1.65 mM MgCl2, 220 µM each dNTP and 22 U/ml TaqDNA polymerase with Platinum Taq antibody) for a final reaction volume of 50 µl. PCR cycling conditions were as follows: an initial denaturation step of 94°C for 5 min, 94°C for 1 min, annealing at 57°C for 1 min for ClC-1, and elongation at 72°C for 1 min. A final elongation step of 10 min at 72°C occurred on the last cycle. All PCR reactions were cycled 30 times except for ClC-4, which required 35 cycles. For ClC-2 and ClC-4, the annealing temperature used was 55°C; for ClC-3, -5, -6, and -7 the temperature was 50°C. The PCR primers for ClC-1, ClC-2, ClC-4, and ClC-7 were created with molecular biology software (Vector NT and Gene-Tool); ClC-3 and ClC-5 primers have been published previously (Enz et al., 1999
Western blot analysis. Cells were lysed using RIPA buffer [50 mM TrisCl, pH 7.5, 150 mM NaCl, 1% Nondet P-40 (NP-40), 0.5% sodium deoxycholate, 1% SDS] for 30 min supplemented with protease inhibitor mixture obtained from Sigma. Homogenates were centrifuged for 5 min at 12,000 x g at 4°C. Protein quantification was performed on the supernatant using a DC protein assay kit from Bio-Rad (Hercules, CA). Protein was boiled for 5 min in Laemmli-SDS sample buffer containing 600 mM
-mercaptoethanol. Equal amounts of protein were loaded into each lane of a 7.5 or 4–20% gradient precast acrylamide SDS-PAGE gel (Bio-Rad). Proteins were separated at 120 V constant. Gels were transferred onto polyvinylidene difluoride paper (Millipore, Bedford, MA) at 200 mA constant for 2 hr at room temperature, and membranes were blocked in blocking buffer (5% nonfat dried milk, 2% bovine serum albumin, and 2% normal goat serum in TBS plus 0.1% Tween 20). Blots were incubated in primary antibody according to manufacturer's instructions. The membranes were then rinsed three times for 10 min and then incubated with HRP-conjugated secondary antibodies for 90 min. Blots were once again washed three times for 10 min and developed with enhanced chemiluminesence (Amersham Biosciences, Arlington Heights, IL) on Hyperfilm (Amersham Biosciences). For negative controls blots were stripped and reprobed with the appropriate control peptide incubated with antibody according to manufacturer's instructions. Recent controversy has focused on the specificity of voltage-gated chloride channel antibodies. For that reason we chose to use two sets of antibodies to confirm Western blot and immunocytochemistry results. One set of ClC-2, ClC-3, and ClC-5 polyclonal antibodies was obtained from Alpha Diagnostics (San Antonio, TX). Alternative ClC-2 and ClC-3 antibodies were obtained from Alomone Labs (Jerusalem, Israel), and ClC-5 was a generous gift from Thomas Jentsch (University of Hamburg, Hamburg, Germany). Actin and secondary HRP-conjugated antibodies were purchased from Sigma.
Immunocytochemistry. Cells plated on coverslips (12 mm round; Macalaster Bicknell, New Haven, CT) were washed two times with PBS and fixed with 4% paraformaldehyde for 15 min. Cells were then washed two more times with PBS and then permeabilized in PBS, 0.3% Triton X-100, and 3% goat serum [permeabilization buffer (PB)] for 30 min. Primary antibody was diluted in PB and added according to manufacturer's suggestion overnight at 4°C. The cells were washed three times in PBS before adding an FITC-conjugated goat anti-rabbit secondary antibody (Molecular Probes, Eugene, OR) diluted at 1:500 in PB for 1 hr at room temperature. Cells were then washed two times with PBS, washed once with DAPI (10 – 4 mg/ml; Sigma), and diluted in PBS for 5 min. DAPI was rinsed off with PBS, and then cells were mounted onto clean coverslips with Gel/Mount (Biomedia Corporation, Forest City, CA). Fluorescent images (400 and 1000x) were acquired on a Leica DMRB fluorescent microscope (Leica, Heerbrugg, Germany). Digital confocal images (400 nM sections) were acquired with a Zeiss Axiovert 200M (München, Germany).
Immunogold electron microscopy. D54-MG cells were fixed in 4% paraformaldehyde for 30 min and in 0.25% glutaraldehyde for 30 min at room temperature and then permeabilized with 0.1% Triton X-100 in PIPES for 45 sec at room temperature. After rinsing and blocking, the cells were incubated with anti-ClC antibodies (Alomone) ClC-2, ClC-3, and Jentsch ClC-5 (1:100) for 4 hr at 4°C, washed, and incubated with 6 nm gold-labeled goat anti-rabbit IgG (1:10; Electron Microscopy Sciences) overnight at 4°C. Cells were rinsed, incubated with 1% OsO4 for 60 min at room temperature, dehydrated, and embedded in SPURR's resin (Electron Microscopy Sciences). Ultrathin sections (<90 nm) obtained on a Reichert Ultracut S (Leica, Heerbrugg, Germany) were contrasted with uranyl acetate and lead citrate and then examined on a JOEL 100 CX electron microscope (Joel, Peabody, MA).
Immunohistochemistry. Human glioma tissues with pathology reports were obtained from three separate sources: The Cooperative Human Network (Eastern and Southern Divisions), the Brain Tumor Tissue Bank (London, UK, and Ontario, Canada), and the University of Alabama at Birmingham Brain Bank (Birmingham, AL). Frozen tissue samples were cryosectioned into 6–8 µm. The basic procedure for fixing and staining fresh-frozen tissue slices has been described previously (Lyons et al., 2002
Antisense and nonsense oligonucleotide knockdown. Phosphorothiate-modified, 5' end fluorescein-tagged antisense oligonucleotide primers were purchased from Invitrogen Custom Primers (Rockville, MD). The antisense oligonucleotide primer sequences used were as follows: ClC2: 5'-CGCCGCGGCCGCCAT-3'; ClC3: 5'-TCCATTTGTCATTGT-3'. ClC-3 antisense will eliminate both the short and long form of ClC-3 (Shimada et al., 2000
Statistical analysis. Current–responses to varied voltage steps and ramps were analyzed and measured in Clampfit (Axon Instruments); the resulting raw data were graphed and plotted in Origin 6.0 (MicroCal, Northampton, MA). Unless stated otherwise, all values are reported ± SE, with n being the number of cells sampled. Two-tailed t tests were performed to evaluate statistical significance, and p values are given in Results (Origin). The constant field potential equation PX/PCl = [Cl]o * e –(
Erev(zF/RT)/[X]o (Hille, 1992
Results
Glioma cells express two biophysically and pharmacologically distinct Cl– currents
To examine the expression of Cl– channels in glioma cells, we first examined whole-cell currents elicited from cultured D54-MG glioma cells by patch clamp. These recordings were obtained under iso-osmotic conditions, as were all subsequent recordings. To avoid activation of swelling activated currents, we maintained the pipette osmolarity 10% below that of bath solution. To ensure that we were indeed recording Cl– currents, we initially replaced intracellular K+ with Cs+. However, prolonged recordings with CsCl-containing pipette solutions lead to extensive membrane blebbing, a phenomenon that we have not observed in other cells using identical solutions. In turn, blebbing often resulted in the spontaneous activation or enhancement of outward and inward currents. These currents were reminiscent of swelling-activated Cl– currents described previously in these cells (Ransom et al., 2001
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Figure 1. Voltage-dependent outward Cl– currents in human glioma cells. A, Representative examples of whole-cell Cl– currents that were evoked with voltage steps from –60 to +100 mV from a holding potential of –40 mV (in the presence of 2 mM TEA to block outward potassium currents). Traces demonstrate currents before and after gluconate, and subtraction of the two traces yielded the gluconate-sensitive current. B, Whole-cell currents using the same voltage step protocol before and after DIDS (500 µM). C, I–V plot of peak gluconate-sensitive current before and after DIDS (500 µM). D, Whole-cell Cl– currents before and after NPPB (165 µM). E, I–V plot of peak gluconate-sensitive currents before and after application of NPPB (165 µM). B, D, E, CsCl pipette solution.
When we altered the stimulus protocol and applied hyperpolarizing voltage steps ranging from –80 to –140 mV, we observed small inward currents (Fig. 2). It has been demonstrated that inward Cl– currents can be enhanced if voltage steps are preceded by a 30 sec prepulse to –120 mV (Bond et al., 1998
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Figure 2. Voltage-dependent inward Cl– currents in human glioma cells. A, Inward currents were evoked with voltage steps from –140 to +20 mV from a holding potential of –40 mV. Cells were hyperpolarized to –120 mV for a minimum of 20 sec to increase activation of inward current. Representative traces of inward Cl– current before (top) and after Cd2+ (200 µM) (middle) and the subtracted Cd2+ sensitive current (bottom) are shown. B, I–V plot of Cd2+ (200 µM)-sensitive Cl– currents evoked from the same voltage step protocol. Currents returned after washout of Cd2+, and removal of the 5 mM [K]o or addition of 200 µM Ba2+ had no effect (data not shown).
Although specific Cl– channel blockers are few, differential sensitivity of ClC channels to DIDS, 9-AC, NPPB, niflumic acid, tamoxifen, and Cd2+ has been useful for the pharmacological characterization of ClC Cl– channels. For example, inward Cl– currents mediated by ClC-2 are typically sensitive to Cd2+ or Zn2+ (Clark et al., 1998
Another defining feature of Cl– channels is their permeability to a number of halide and non-halide anions. Indeed, the relative permeability to I–, Br–, and F– has been used as a distinguishing feature of ClC channels and ICLswell. We therefore examined whether replacement of extracellular Cl– with other anions could sustain these Cl– currents. Indeed, both I– and Br– produced slightly larger outward currents while reducing the inward current; additionally, outward currents were reduced in gluconate (Fig. 3). Relative permeability of ions through channels are typically derived from shifts in tail current reversal potentials; however, ClC channel gating is coupled to the permeating anion, disallowing this approach (Pusch et al., 1995
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Figure 3. Effects of halide ion replacement on D54 glioma cells. Representative recording of Cl– current from a linear voltage ramp protocol (–160 to +160 mV, holding at –40 mV) is shown. Extracellular Cl– (thick black line) in the bath solution was replaced with 130 mM NaI (gray line), NaBr (black dashed line), or Na-gluconate (light gray line). Inset magnifies the region around the reversal potential.
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Table 1. Chloride ion replacement shifts the reversal potential in D54 glioma cells
Glioma cells and acute patient biopsies show expression of ClC-2, -3, and -5
We were ultimately interested in determining whether the above described currents could be mediated by known Cl– channels of the ClC family. In an initial effort to examine this question, we used RT-PCR using specific primers for ClC-1 through ClC-7 (Table 2) and mRNA from D54-MG glioma cells. RT-PCR performed with the specific primers yielded fragments of the predicted molecular weight (Table 2) for ClC-2, -3, -4, -5, -6, and -7, but lacked transcripts for ClC-1 (Fig. 4). The latter has been shown to be specific for muscle (Jentsch et al., 1995
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Table 2. Primer sets used to RT-PCR ClC channels from D54 glioma cells
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Figure 4. RT-PCR of ClC-1 through ClC-7 in D54 glioma cells. Lane 1 is a 100 bp marker (Invitrogen). Lanes denoted with + are RT-PCR reactions with primers for the designated ClC channel. Lanes denoted with – are identical reactions with water substituted for reverse transcriptase. Using D54-MG total RNA as a template, only the muscle-specific ClC-1 primers did not yield a product. ClC-2 through ClC-7 mRNA was present in D54-MG cells as judged by amplification of the expected size PCR products using gene-specific primers.
To examine channel expression at the protein level, we performed Western blots on glioma cell lysates (D54, U251, U158, U87, and STTG1) using antibodies to ClC-2, -3, -4, and -5 (Fig. 5). We did not observe any immunoreactivity with ClC-4 (data not shown); however, we consistently saw bands corresponding to ClC-2, -3, and -5. Because there remains significant controversy concerning the specificity of these antibodies (Stobrawa et al., 2001
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Figure 5. Western blot analysis demonstrating expression of ClC-2, ClC-3, and ClC-5 in the human glioma cell lines U251, D54, U138, U118, STTG1, and U87. A, Top, Alpha Diagnostics ClC-2 antibody recognizes a doublet at
To illustrate the distribution of ClC-2, ClC-3, and ClC-5 in human glioma cells, we used immunocytochemistry with FITC-conjugated secondary antibodies. Distinct localization was observed for each, with differential staining of the cytoplasm versus cell surface (Fig. 6). Figure 6, A and B, demonstrates 400x and 1000x images, respectively. The images in Figure 6C are digital confocal images. All images shown were probed with Alpha Diagnostics antibodies; however, similar results were obtained with the alternate set of antibodies. Interestingly, all three ClC channels (ClC-2, -3, and -5) appear to associate prominently with lamellipodia at the leading edges of the cells, and overall they appear in clusters on the membrane. In addition, both Alpha Diagnostics and Jentsch ClC-5 antibody recognized large vesicular type structures in the cell cytoplasm.
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Figure 6. Immunoreactivity for ClC-2, ClC-3, and ClC-5 demonstrates intracellular and plasma membrane labeling of D54 glioma cells (Alpha Diagnostics). Left panels are 400 x images; middle panels are 1000 x images. Right panels demonstrate 400 nM sections of digital confocal images. Similar results were observed with Alomone ClC-2 and ClC-3 and Jentsch ClC-5 antibodies.
To further confirm the surface expression of these channels, we obtained immunogold electron microscopy (EM) images from D54 glioma cells in which ClC-2, -3, and -5 were each conjugated to 6 nm gold particles. As is demonstrated in Figure 7A–C (white arrows), immunoreactivity for each channel was found in surface clusters.
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Figure 7. Immunogold EM with 6 nm gold particles localizes ClC-2, ClC-3, and ClC-5 to the plasma membrane of glioma cells. A–C show localization of a portion of ClC-2, ClC-3, and ClC-5 with the plasma membrane of human glioma cells (Alomone ClC-2 and ClC-3 and Jentsch ClC-5).
Because the above studies were performed on cultured cells, we sought to confirm these findings by examining ClC expression in acute biopsies from patients with glioblastoma multiforme and pilocytic astrocytoma. Several such biopsies were examined, and a representative example of each tumor type is illustrated in Figure 8. Paraffin sections of these biopsies were stained with ClC antibodies, followed by secondary antibodies detected with DAB (a brown reaction product). These studies show prominent expression of ClC-2, -3, and -5 and by and large confirmed our findings in cultured cells. Taken together, our biochemical and immmunohistochemical studies suggest that ClC-2, -3, and -5 are expressed in glioma cells in vivo and that, at least in isolated glioma cells, a significant percentage of these channels is localized in the plasma membrane.
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Figure 8. Human biopsy samples stain positive for ClC-2, ClC-3, and ClC-5. Consecutive cryostat sections of frozen patient samples of a glioblastoma tumor (A) and a pilocytic astrocytoma tumor (B) were immunohistochemically stained with antibodies to ClC-2 (a1, b1), ClC-3 (a2, b2), and ClC-5 (a3, b3) and detected with a DAB reaction (a brown reaction product). The slices were counterstained with methyl green to detect cell nuclei. The control stainings (a4, b4) were performed under identical conditions omitting only the primary antibodies.
Antisense studies suggest that glioma Cl– currents are mediated in part by ClC-2 and ClC-3
We next sought to determine whether any of the currents observed in glioma cells (Figs. 1, 2, 3) could be attributed to defined ClC channels. Because of the current profiles, we hypothesized that the inactivating, outwardly rectifying currents were attributable to ClC-3, whereas the activating, inwardly rectifying currents were attributable to ClC-2. The lack of specific Cl– channel blockers led us to use antisense knockdown techniques to investigate our hypothesis. We used specific antisense primers for ClC-2 and ClC-3 (sequences given in Materials and Methods). D54-MG cells were recorded 48 hr after transfection with fluorescently tagged antisense and nonsense oligonucleotides. Current densities of successfully transfected cells (identified by their fluorescence) were analyzed. Representative traces for ClC-3 antisense knockdown demonstrated a significant reduction in whole-cell currents (Fig. 9A). Mean current densities of nonsense- and antisense-treated cells exhibited significant reductions in whole-cell currents at potentials that typically show the greatest activation (50% at the peak current, p < 0.01) (Fig. 9B). The specificity of the ClC-3 antisense oligonucleotides is demonstrated in Figure 9C. When an equal amount of protein is loaded (as evidenced by actin loading control), ClC-3 protein was dramatically reduced when D54 cells were treated with the ClC-3 antisense oligonucleotides.
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Figure 9. ClC-3 antisense oligonucleotides inhibit whole-cell outward Cl– currents in D54 glioma cells. A, Whole-cell outward currents from a representative control (nonsense- or ClC-2 antisense-treated cell) and ClC-3 antisense-treated cell before and after Na-gluconate bath solution and the subtracted gluconate-sensitive current. Currents elicited by voltage step protocol are shown in the inset. B, Antisense treatment significantly reduced whole-cell outward Cl– currents at peak voltages: +80, 40%; +100 mV, 48%; +120 mV, 50%. Interestingly, the current that remained appeared to be the leak current that was not sensitive to gluconate (n=number of cells examined). C, Western blot analysis demonstrates the specificity of the ClC-3 antisense oligonucleotides (lane 1, nonsense-treated cells; lane 2, ClC-2 antisense-treated cells; lane 3, ClC-3 antisense-treated cells; lane 4, ClC-3 antisense-treated cells with a threefold higher concentration of DNA). Of note, for electrophysiology experiments ClC-3 was used at a three- to fourfold higher concentration than ClC-2 (see Materials and Methods).
Cells treated with antisense to ClC-2 demonstrated a marked reduction in inward current (Fig. 10A). Once again, peak current densities at hyperpolarized potentials were significantly reduced by 60% (p < 0.03) (Fig. 10B). Interestingly, treatment with ClC-2 antisense oligonucleotides also increased the input resistance of these cells (1239 ± 270 M
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Figure 10. ClC-2 antisense oligonucleotides inhibit inward Cl– currents and increase input resistance in D54 glioma cells. A, Whole-cell outward Cl– currents from a representative nonsense- and ClC-2 antisense-treated cell before and after application of Cd2+ (200 µM)and the Cd2+ -sensitive subtracted currents. Currents were elicited by the voltage-step protocol shown in the inset. B, Antisense treatment significantly reduced inward Cl– currents at peak voltages: –140 mV before hyperpolarization, 60%; 140 mV after hyperpolarization, 58%; after hyperpolarization at –120 mV, 55%; after hyperpolarization at –100 mV, 60%. C, Treatment of D54 glioma cells with ClC-2 antisense oligonucleotides significantly increased the input resistance of these cells relative to nonsense or ClC-3 antisense-treated cells (p < 0.01 relative to nonsense) (n = number of cells examined). D, Once again Western blotting demonstrated the specificity of the ClC-2 antisense oligonucleotides. Only cells treated with ClC-2 antisense oligonucleotides show a decrease in ClC-2 protein (lane 1, nonsense-treated cells; lane 2, ClC-2 antisense-treated cells; lane 3, ClC-3 antisense-treated cells).
The reduction in whole-cell currents of cells treated with antisense suggested that both ClC-2 and ClC-3 contribute to distinctly different Cl– currents in D54 glioma cells.
Of note, antisense knockdown specifically interrupts the synthesis of new protein but has no affect on existing protein. Hence the effective depletion of functional channels in the membrane depends primarily on the turnover of these proteins. We currently do not know the turnover time for ClC channels. However, for voltage-gated Na+ channels, a half-life of 26 hr in neuroblastoma cells (Waechter et al., 1983
Discussion
In the present study we demonstrate the presence of a subset of ClC genes and their proteins in cultured glioma cells. Specifically, we provide evidence for expression of ClC-2, -3, and -5 protein in glioma cell membranes, often associated with lamellipodia. Importantly, the same complement of channels was observed in acute biopsies from patients who had these tumors removed surgically. We overcame the absence of specific pharmacological drugs for ClC channels subtypes through the use of an antisense knockdown strategy. These studies suggest that Cd2+-sensitive inward Cl– currents can be reduced with ClC-2 antisense, whereas outwardly rectifying, DIDS-sensitive currents are selectively reduced after ClC-3 knockdown.Cl– channels have been referred to as "the problem children of ion channels" (Clapham, 2001
Biophysical and pharmacological properties of ClC currents in glioma cells
The signature features reported for recombinant ClC-2 currents are (1) inward rectification, (2) time-dependent activation, (3) potentiation by negative holding potentials, and (4) sensitivity to Cd2+ and Zn2+ (Clark et al., 1998Currents thought to be mediated by ClC-3 have been described as outwardly rectifying and DIDS and NPPB sensitive, and they often show pronounced voltage-dependent inactivation (Duan et al., 1997
Although we were able to detect ClC-5 at both the mRNA and protein levels, we lacked conclusive biophysical evidence for functional channels in glioma cells. As reported previously, ClC-5 gives rise to outwardly rectifying currents that are unaffected by all known Cl– channel inhibitors (Mo et al., 1999
We routinely used ion replacements to verify that the recorded currents were indeed carried by Cl–. We always observed a potentiation of outward currents by Br– and I– as was reported for recombinant ClC-3 (Duan et al., 1997
Localization of ClC channels
ClC-2 appears to be a ubiquitous Cl– channel that has been identified previously on the plasma membrane of many cell types. In the nervous system, ClC-2 channels are found on the end feet of astrocytes and on the cell body, axons, and dendrites of hippocampal neurons, where they have been implicated in chloride homeostasis and Cl– movements associated with GABAergic synaptic transmission (Sik et al., 2000Functional implications
Chloride channels have been implicated in a multitude of cellular functions that include osmoregulation, salt secretion, cell migration, and cell proliferation (for review, see Jentsch et al., 2002For other aspects of biology, Cl– channel function has been less well studied, yet a few studies have implicated Cl– channels in cell shape changes that may occur in conjunction with cell division or cell migration. For example, it has been demonstrated that cell division is associated with a transient increase in cell volume (Premack and Gardner, 1991
Other evidence suggests that Cl– channels may also serve important functions in the context of cell migration. In rat astrocytes, changes in cell morphology are sufficient to induce Cl– currents (Lascola and Kraig, 1996
Footnotes
Received Dec. 12, 2002; revised Apr. 23, 2003; accepted Apr. 23, 2003.This work was supported by National Institutes of Health Grant RO1-NS36692 and Human Development Grant P30HD-38985. We thank Dr. Thomas Jentsch for providing the ClC-5 channel-specific antibodies. We also thank Jessy Deshane, Patricia Ritch, Tara Spears, and Ed Phillips for technical assistance.
Correspondence should be addressed to Dr. Harald Sontheimer, 1719 Sixth Avenue South, Civitan International Research Center 545, Birmingham, AL 35294. E-mail: sontheimer{at}uab.edu.
S. Schade's present address: Transmolecular Inc., 3800 Colonnade Parkway, Suite 240, Birmingham, AL 35243.
Copyright © 2003 Society for Neuroscience 0270-6474/03/235572-11$15.00/0
References
Amberger VR, Avellana-Adalid V, Hensel T, Baron-Van Evercooren A, Schwab ME (1997) Oligodendrocyte-type 2 astrocyte progenitors use a metalloendoprotease to spread and migrate on CNS myelin. Eur J Neurosci 9: 151–162.[Web of Science][Medline]
Arreola J, Begenisich T, Nehrke K, Nguyen HV, Park K, Richardson L, Yang B, Schutte BC, Lamb FS, Melvin JE (2002) Secretion and cell volume regulation by salivary acinar cells from mice lacking expression of the Clcn3 Cl(–) channel gene. J Physiol (Lond) 545: 1–16.
[Free Full Text] Bakhramov A, Fenech C, Bolton TB (1995) Chloride current activated by hypotonicity in cultured human astrocytoma cells. Exp Physiol 80: 373–389.[Abstract]
Bond TD, Ambikapathy S, Mohammad S, Valverde MA (1998) Osmosensitive Cl– currents and their relevance to regulatory volume decrease in human intestinal t84 cells: outwardly vs. inwardly rectifying currents. J Physiol (Lond) 511: 45–54.
[Abstract/Free Full Text] Bordey A, Sontheimer H (1998) Electrophysiological properties of human astrocytic tumor cells in situ: enigma of spiking glial cells. J Neurophysiol 79: 2782–2793.
[Abstract/Free Full Text] Brandt S, Jentsch TJ (1995) Clc-6 and Clc-7 are two novel broadly expressed members of the Clc chloride channel family. FEBS Lett 377: 15–20.[Web of Science][Medline]
Clapham D (2001) How to lose your hippocampus by working on chloride channels. Neuron 29: 1–3.[Web of Science][Medline]
Clark S, Jordt SE, Jentsch TJ, Mathie A (1998) Characterization of the hyperpolarization-activated chloride current in dissociated rat sympathetic neurons. J Physiol (Lond) 506: 665–678.
[Abstract/Free Full Text] Coca-Prados M, Sanchez-Torres J, Peterson-Yantorno K, Civan MM (1996) Association of Clc-3 channel with Cl– transport by human nonpigmented ciliary epithelial cells. J Membr Biol 150: 197–208.[Web of Science][Medline]
Crépel V, Panenka W, Kelly MEM, MacVicar BA (1998) Mitogen-activated protein and tyrosine kinases in the activation of astrocyte volume-activated chloride current. J Neurosci 18: 1196–1206.
[Abstract/Free Full Text] Duan D, Winter C, Cowley S, Hume JR, Horowitz B (1997) Molecular identification of a volume-regulated chloride channel. Nature 390: 417–421.[Medline]
Duan D, Cowley S, Horowitz B, Hume JR (1999) A serine residue in Clc-3 links phosphorylation-dephosphorylation to chloride channel regulation by cell volume. J Gen Physiol 113: 57–70.
[Abstract/Free Full Text] Eggermont J, Buyse G, Voets T, Tytgat J, De Smedt H, Droogmans G, Nilius B (1997) Alternative splicing of ClC-6 (a member of the CIC chloride-channel family) transcripts generates three truncated isoforms one of which, ClC-6c, is kidney-specific. Biochem J 325: 269–276.
Ehrengruber MU, Deranleau DA, Coates TD (1996) Shape oscillations of human neutrophil leukocytes: characterization and relationship to cell motility. J Exp Biol 199: 741–747.[Abstract]
Enz R, Ross BJ, Cutting GR (1999) Expression of the voltage-gated chloride channel ClC-2 in rod bipolar cells of the rat retina. J Neurosci 19: 9841–9847.
[Abstract/Free Full Text] Fahlke C (2001) Ion permeation and selectivity in ClC-type chloride channels. Am J Physiol Renal Physiol 280: F748–F757.
[Abstract/Free Full Text] Friedrich T, Breiderhoff T, Jentsch TJ (1999) Mutational analysis demonstrates that ClC-4 and ClC-5 directly mediate plasma membrane currents. J Biol Chem 274: 896–902.
[Abstract/Free Full Text] Garber SS, Cahalan MD (1997) Volume-regulated anion channels and the control of a simple cell behavior. Cell Physiol Biochem 7: 229–241.
Gunther W, Luchow A, Cluzeaud F, Vandewalle A, Jentsch TJ (1998) Clc-5, the chloride channel mutated in Dent's disease, colocalizes with the proton pump in endocytotically active kidney cells. Proc Natl Acad Sci USA 95: 8075–8080.
[Abstract/Free Full Text] Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques for high resolution current recording from cells and cell free membrane patches. Pflügers Arch 391: 85–100.[Web of Science][Medline]
Hermoso M, Satterwhite CM, Andrade YN, Hidalgo J, Wilson SM, Horowitz B, Hume JR (2002) ClC-3 is a fundamental molecular component of volume-sensitive outwardly rectifying Cl– channels and volume regulation in HeLa cells and Xenopus laevis oocytes. J Biol Chem 277: 40066–40074.
[Abstract/Free Full Text] Hille B (1992) Ionic channels in excitable membranes. Sunderland, MA: Sinauer.
Huang P, Liu J, Di A, Robinson NC, Musch MW, Kaetzel MA, Nelson DJ (2001) Regulation of human ClC-3 channels by multifunctional Ca2+/calmodulin-dependent protein kinase. J Biol Chem 276: 20093–20100.
[Abstract/Free Full Text] Jackson PS, Strange K (1993) Volume-sensitive anion channels mediate swelling-activated inositol and taurine efflux. Am J Physiol 265: 489–500.
Jackson PS, Strange K (1995) Characterization of the voltage-dependent properties of a volume-sensitive anion conductance. J Gen Physiol 105: 661–676.
[Abstract/Free Full Text] Jentsch TJ, Günther W, Pusch M, Schwappach B (1995) Properties of voltage-gated chloride channels of the ClC gene family. J Physiol (Lond) [Suppl] 482: 19S–25S.
Jentsch TJ, Stein V, Weinreich F, Zdebik AA (2002) Molecular structure and physiological function of chloride channels. Physiol Rev 82: 503–568.
[Abstract/Free Full Text] Kaba SE, Kyritsis AP (1997) Recognition and management of gliomas. Drugs 53: 235–244.[Medline]
Kawasaki M, Uchida S, Monkawa T, Miyawaki A, Mikoshiba K, Marumo F, Sasaki S (1994) Cloning and expression of a protein kinase C-regulated chloride channel abundantly expressed in rat brain neuronal cells. Neuron 12: 597–604.[Web of Science][Medline]
Komuro H, Rakic P (1998) Orchestration of neuronal migration by activity of ion channels, neurotransmitter receptors, and intracellular Ca 2+ fluctuations. J Neurobiol 37: 110–130.[Web of Science][Medline]
Lascola CD, Kraig RP (1996) Whole-cell chloride currents in rat astrocytes accompany changes in cell morphology. J Neurosci 16: 2532–2545.
[Abstract/Free Full Text] Lascola CD, Nelson DJ, Kraig RP (1998) Cytoskeletal actin gates a Cl – channel in neocortical astrocytes. J Neurosci 18: 1679–1692.
[Abstract/Free Full Text] Levison SW, Chuang C, Abramson BJ, Goldman JE (1993) The migrational patterns and developmental fates of glial precursors in the rat subventricular zone are temporally regulated. Development 119: 611–622.
[Abstract/Free Full Text] Linskey ME (1997) Glial ontogeny and glial neoplasia: the search for closure. J Neurooncol 34: 5–22.[Medline]
Lyons SA, O'Neal J, Sontheimer H (2002) Chlorotoxin, a scorpion-derived peptide, specifically binds to gliomas and tumors of neuroectodermal origin. Glia 39: 162–173.[Web of Science][Medline]
Merzak A, Pilkington GJ (1997) Molecular and cellular pathology of intrinsic brain tumours. Cancer Metastasis Rev 16: 155–177.[Web of Science][Medline]
Merzak A, McCrea S, Koocheckpour S, Pilkington GJ (1994) Control of human glioma cell growth, migration and invasion in vitro by transforming growth factor
Mo L, Hellmich HL, Fong P, Wood T, Embesi J, Wills NK (1999) Comparison of amphibian and human ClC-5: similarity of functional properties and inhibition by external pH. J Membr Biol 168: 253–264.[Web of Science][Medline]
Nehrke K, Arreola J, Nguyen HV, Pilato J, Richardson L, Okunade G, Baggs R, Shull GE, Melvin JE (2002) Loss of hyperpolarization-activated Cl(–) current in salivary acinar cells from Clcn2 knockout mice. J Biol Chem 277: 23604–23611.
[Abstract/Free Full Text] Nilius B, Prenen J, Voets T, Eggermont J, Droogmans G (1998) Activation of volume-regulated chloride currents by reduction of intracellular ionic strength in bovine endothelial cells. J Physiol (Lond) 506: 353–361.
[Abstract/Free Full Text] Noble M, Mayer-Pröschel M (1997) Growth factors, glia and gliomas. J Neurooncol 35: 193–209.[Medline]
Pappas CA, Ritchie JM (1998) Effect of specific ion channel blockers on cultured Schwann cell proliferation. Glia 22: 113–120.[Web of Science][Medline]
Pastan I, Chaudhary V, Fitzgerald DJ (1992) Recombinant toxins as novel therapeutic agents. Annu Rev Biochem 61: 331–354.[Web of Science][Medline]
Phipps DJ, Branch DR, Schlichter LC (1996) Chloride-channel block inhibits T lymphocyte activation and signaling. Cell Signal 8: 141–149.[Web of Science][Medline]
Premack BA, Gardner P (1991) Role of ion channels in lymphocytes. J Clin Immunol 11: 225–238.[Web of Science][Medline]
Pusch M, Ludewig U, Rehfeldt A, Jentsch TJ (1995) Gating of the voltage-dependent chloride channel CIC-O by the permeant anion. Nature 373: 527–531.[Medline]
Ransom CB, Sontheimer H (2001) BK channels in human glioma cells. J Neurophysiol 85: 790–803.
[Abstract/Free Full Text] Ransom CB, O'Neal JT, Sontheimer H (2001) Volume-activated chloride currents contribute to the resting conductance and invasive migration of human glioma cells. J Neurosci 21: 7674–7683.
[Abstract/Free Full Text] Roman RM, Smith RL, Feranchak AP, Clayton GH, Doctor RB, Fitz JG (2001) ClC-2 chloride channels contribute to HTC cell volume homeostasis. Am J Physiol Gastrointest Liver Physiol 280: G344–G353.
[Abstract/Free Full Text] Rouzaire-Dubois B, Bostel S, Dubois JM (1999) Evidence for several mechanisms of volume regulation in neuroblastoma x glioma hybrid NG108–15 cells. Neuroscience 88: 307–317.[Web of Science][Medline]
Rouzaire-Dubois B, Milandri JB, Bostel S, Dubois JM (2000) Control of cell proliferation by cell volume alterations in rat C6 glioma cells. Pflügers Arch 440: 881–888.[Web of Science][Medline]
Rutledge E, Bianchi L, Christensen M, Boehmer C, Morrison R, Broslat A, Beld AM, George AL, Greenstein D, Strange K (2001) CLH-3, a ClC-2 anion channel ortholog activated during meiotic maturation in C. elegans oocytes. Curr Biol 11: 161–170.[Web of Science][Medline]
Rutledge E, Denton J, Strange K (2002) Cell cycle- and swelling-induced activation of a Caenorhabditis elegans ClC channel is mediated by CeGLC-7alpha/beta phosphatases. J Cell Biol 158: 435–444.
[Abstract/Free Full Text] Schlichter LC, Sakellaropoulos G, Ballyk B, Pennefather PS, Phipps DJ (1996) Properties of K + and Cl – channels and their involvement in proliferation of rat microglial cells. Glia 17: 225–236.[Web of Science][Medline]
Schmidt JW, Catterall WH (1986) Biosynthesis and processing of the a-subunit of the voltage-sensitive sodium channel in rat brain neurons. Cell 46: 437–445.[Web of Science][Medline]
Schmieder S, Lindenthal S, Ehrenfeld J (2002) Cloning and characterisation of amphibian ClC-3 and ClC-5 chloride channels. Biochim Biophys Acta 1566: 55–66.[Medline]
Schneider SW, Pagel P, Rotsch C, Danker T, Oberleithner H, Radmacher M, Schwab A (2000) Volume dynamics in migrating epithelial cells measured with atomic force microscopy. Pflügers Arch 439: 297–303.[Web of Science][Medline]
Shen MR, Droogmans G, Eggermont J, Voets T, Ellory JC, Nilius B (2000) Differential expression of volume-regulated anion channels during cell cycle progression of human cervical cancer cells. J Physiol (Lond) 529: 385–394.
[Abstract/Free Full Text] Shimada K, Li X, Xu G, Nowak DE, Showalter LA, Weinman SA (2000) Expression and canalicular localization of two isoforms of the Clc-3 chloride channel from rat hepatocytes. Am J Physiol Gastrointest Liver Physiol 279: G268–276.
[Abstract/Free Full Text] Sik A, Smith RL, Freund TF (2000) Distribution of chloride channel-2-immunoreactive neuronal and astrocytic processes in the hippocampus. Neuroscience 101: 51–65.[Web of Science][Medline]
Simpson PB, Armstrong RC (1999) Intracellular signals and cytoskeletal elements involved in oligodendrocyte progenitor migration. Glia 26: 22–35.[Web of Science][Medline]
Soroceanu L, Manning TJ Jr, Sontheimer H (1999) Modulation of glioma cell migration and invasion using Cl – and K + ion channel blockers. J Neurosci 19: 5942–5954.
[Abstract/Free Full Text] Steinmeyer K, Schwappach B, Bens M, Vandewalle A, Jentsch TJ (1995) Cloning and functional expression of rat Clc-5, a chloride channel related to kidney disease. J Biol Chem 270: 31172–31177.
[Abstract/Free Full Text] Stobrawa SM, Breiderhoff T, Takamori S, Engel D, Schweizer M, Zdebik AA, Bosl MR, Ruether K, Jahn H, Draguhn A, Jahn R, Jentsch TJ (2001) Disruption of ClC-3, a chloride channel expressed on synaptic vesicles, leads to a loss of the hippocampus. Neuron 29: 185–196.[Web of Science][Medline]
Ullrich N, Sontheimer H (1996) Biophysical and pharmacological characterization of chloride currents in human astrocytoma cells. Am J Physiol 270: C1511–C1521.
Ullrich N, Bordey A, Gillespie GY, Sontheimer H (1998) Expression of voltage-activated chloride currents in acute slices of human gliomas. Neuroscience 83: 1161–1173.[Web of Science][Medline]
Voets T, Szucs G, Droogmans G, Nilius B (1995) Blockers of volume-activated Cl– currents inhibit endothelial cell proliferation. Pflügers Arch 431: 132–134.[Web of Science][Medline]
von Weikersthal SF, Barrand MA, Hladky SB (1999) Functional and molecular characterization of a volume-sensitive chloride current in rat brain endothelial cells. J Physiol (Lond) 516: 75–84.
[Abstract/Free Full Text] Voura EB, Sandig M, Kalnins VI, Siu C (1998) Cell shape changes and cytoskeleton reorganization during transendothelial migration of human melanoma cells. Cell Tissue Res 293: 375–387.[Web of Science][Medline]
Waechter CJ, Schmidt JW, Catterall WA (1983) Glycosylation is required for maintenance of functional sodium channels in neuroblastoma cells. J Biol Chem 258: 5117–5123.
[Abstract/Free Full Text] Walz W (2002) Chloride/anion channels in glial cell membranes. Glia 40: 1–10.[Web of Science][Medline]
Wang GL, Wang XR, Lin MJ, He H, Lan XJ, Guan YY (2002) Deficiency in ClC-3 chloride channels prevents rat aortic smooth muscle cell proliferation. Circ Res 91: E28–E32.
Wang L, Chen L, Jacob TJ (2000) The role of Clc-3 in volume-activated chloride currents and volume regulation in bovine epithelial cells demonstrated by antisense inhibition. J Physiol (Lond) 524: 63–75.
[Abstract/Free Full Text] Wilson GF, Chiu SY (1993) Mitogenic factors regulate ion channels in Schwann cells cultured from newborn rat sciatic nerve. J Physiol (Lond) 470: 501–520.
[Abstract/Free Full Text] Wondergem R, Gong W, Monen SH, Dooley SN, Gonce JL, Conner TD, Houser M, Ecay TW, Ferslew KE (2001) Blocking swelling-activated chloride current inhibits mouse liver cell proliferation. J Physiol (Lond) 532: 3–72.
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