Volume-activated Chloride Currents Research Articles

Volume-activated chloride channels contribute to lipopolysaccharide plus nigericin–induced pyroptosis in bone marrow–derived macrophages

The representative morphological features of pyroptosis are excessive cell swelling and subsequent membrane rupture. However, the mechanism underlying the cell’s inherent inability to regulate volume during the progression of pyroptosis is poorly understood. In the current study, we found that both volume-activated chloride currents (Icl, vol) and the regulatory volume decrease (RVD) were markedly decreased in bone marrow–derived macrophages (BMDMs) undergoing pyroptosis induced by lipopolysaccharides (LPS) and nigericin. The inhibition of ICl, vol and RVD by the chloride channel blockers, tamoxifen or DCPIB, led to the emergence of pyroptosis-like phenotypes such as activated-caspase-1, pyroptotic-body-like bubbles, and a fried-egg–like appearance. Moreover, the expression of the volume-activated chloride channel (VRAC) constituent protein Leucine-Rich Repeat-Containing 8A (LRRC8A) was significantly down-regulated in pyroptotic BMDMs treated with LPS and nigericin. The silencing of LRRC8A expression by small interfering RNA (si)-LRRC8A transfection not only reduced ICl, vol and RVD, but also caused BMDMs to show pyroptosis-like manifestations such as activated-caspase-1, membrane bubbles, and have a fried-egg–like appearance. These results reveal a new mechanism for the loss of volume regulation in the process of pyroptotic cell swelling and strongly suggest that a functional deficiency of VRAC/LRRC8A plays a key role in this disorder.

Different properties between spontaneous and volume-activated chloride currents in human nasopharyngeal carcinoma and its normal counterpart cells.

Although the spontaneous chloride currents (SCC) have been well studied in the normal cells, its properties and roles in neoplasms cells are still unknown. Here, we found that the SCC was manifested in the poorly differentiated human nasopharyngeal carcinoma CNE-2Z cells, with some differences such as lower occurrence and bigger current density than those of the volume-activated chloride currents (VACC). NPPB, a chloride channel blocker, inhibited the SCC much stronger than the VACC. Down-regulation of chloride channel -3 (ClC-3), a volume and mechanically dependent ion channel, could significantly decrease the VACC, but not in SCC. The occurrence, latency, and mean density of the SCC were much lower in the normal nasopharyngeal NP69-SV40T cells than those in CNE-2Z cells. Our results demonstrated that the spontaneous electrical reactivity of neoplasm cells is higher than that of normal cells, which probably relates to their high physiological activity of neoplasm cells. SIGNIFICANCE OF THE STUDY: Spontaneous chloride currents (SCC) are well known in excitable tissues and regulate a variety of physiological and pathophysiological processes. During our researching on the volume-activated chloride currents (VACC) in human nasopharyngeal carcinoma CNE-2Z cells, SCC could be also observed with different properties from VACC. Meanwhile, the occurrence, latency, and mean density of the SCC were much higher in CNE-2Z cells than those in normal nasopharyngeal NP69-SV40T cells. Our results revealed the expression and characteristics of SCC in carcinoma cells and provided a preliminary experimental basis for further exploring the function of SCC in tumour cell biology.

Down-Regulation of ClC-3 Expression Reduces Epidermal Stem Cell Migration by Inhibiting Volume-Activated Chloride Currents.

ClC-3, a member of the ClC chloride (Cl(-)) channel family, has recently been proposed as the primary Cl(-) channel involved in cell volume regulation. Changes in cell volume influence excitability, contraction, migration, pathogen-host interactions, cell proliferation, and cell death processes. In this study, expression and function of ClC-3 channels were investigated during epidermal stem cell (ESC) migration. We observed differential expression of CLC-3 regulates migration of ESCs. Further, whole-cell patch-clamp recordings and image analysis demonstrated ClC-3 expression affected volume-activated Cl(-) current (I Cl,Vol) within ESCs. Live cell imaging systems, designed to observe cellular responses to overexpression and suppression of ClC-3 in real time, indicated ClC-3 may regulate ESC migratory dynamics. We employed IMARIS software to analyze the velocity and distance of ESC migration in vitro to demonstrate the function of ClC-3 channel in ESCs. As our data suggest volume-activated Cl(-) channels play a vital role in migration of ESCs, which contribute to skin repair by migrating from neighboring unwounded epidermis infundibulum, hair follicle or sebaceous glands, ClC-3 may represent a new and valuable target for stem cell therapies.

The ClC-3 chloride channel associated with microtubules is a target of paclitaxel in its induced-apoptosis

Recent evidences show that cationic fluxes play a pivotal role in cell apoptosis. In this study, the roles of Cl− channels in paclitaxel-induced apoptosis were investigated in nasopharyngeal carcinoma CNE-2Z cells. Chloride current and apoptosis were induced by paclitaxel and inhibited by chloride channel blockers. Paclitaxel-activated current possessed similar properties to volume-activated chloride current. After ClC-3 was knocked-down by ClC-3-siRNA, hypotonicity-activated and paclitaxel-induced chloride currents were obviously decreased, indicating that the chloride channel involved in paclitaxel-induced apoptosis may be ClC-3. In early apoptotic cells, ClC-3 was up-regulated significantly; over-expressed ClC-3 was accumulated in cell membrane to form intercrossed filaments, which were co-localized with α-tubulins; changes of ultrastructures and decrease of flexibility in cell membrane were detected by atomic force microscopy. These suggest that ClC-3 is a critical target of paclitaxel and the involvement of ClC-3 in apoptosis may be associated with its accumulation with membrane microtubules and its over activation.

The ClC-3 chloride channel protein is a downstream target of cyclin D1 in nasopharyngeal carcinoma cells

It has been demonstrated previously by us that cyclin D1 and ClC-3 play important roles in regulation of the cell cycle in nasopharyngeal carcinoma cells. The action of cyclin D1 on the functional activities and expression of chloride channels were investigated in nasopharyngeal carcinoma CNE-2Z cells in this study. The results indicated that enhanced cyclin D1 expression increased the activation of volume-activated chloride currents and promoted the expression of ClC-3 chloride channel proteins. The fluorescence resonance energy transfer (FRET) experiments demonstrated that the distance between cyclin D1 and ClC-3 was less than 10nm, and there existed interaction between the two proteins. ClC-3 was partially colocalized with cyclin D1 and CDK4/6. Dialyzing CDK4 antibodies into cells via recording pipettes activated a chloride current, but dialysis of CDK6 antibodies inhibited basal and volume-activated Cl− currents. The CDK4/6 inhibitor fascaplysin chloride hydrate (highly selective for CDK4/cyclin D1 with IC50=0.35μM and less selective for CDK6/D1 with IC50=3.4μM) activated a chloride current in low concentration, but did not show significantly facilitative effects on the current in high concentration. In conclusion, our data suggest that the ClC-3 chloride channel is an important target of cyclin D1. Cyclin D1 may regulate the functional activities of the chloride channel via CDK4 and CDK6, and/or the expression of the chloride channel. Cyclin D1–CDK4 complexes may phosphorylate chloride channels resulting in inhibition or inactivation of the channels, and cyclin D1–CDK6 complexes may facilitate the activation of chloride channels.

Volume-Activated Chloride Currents in Fetal Human Nasopharyngeal Epithelial Cells

Volume-activated chloride channels have been studied by us extensively in human nasopharyngeal carcinoma cells. However, the chloride channels in the counterpart of the carcinoma cells have not been investigated. In this study, volume-activated chloride currents (I(cl,vol)) were characterized in normal fetal human nasopharyngeal epithelial cells using the whole-cell patch-clamp technique. Under isotonic conditions, nasopharyngeal epithelial cells displayed only a weak background current. Exposure to 47% hypotonic solution activated a volume-sensitive current. The reversal potential of the current was close to the calculated equilibrium potential for Cl(-). The peak values of the hypotonicity-activated current at +80 mV ranged from 0.82 to 2.71 nA in 23 cells. Further analysis indicated that the density of the hypotonicity-activated current in most cells (18/23) was smaller than 60 pA/pF. Only five cells presented a current larger than 60 pA/pF. The hypotonicity-activated current was independent of the exogenous ATP. Chloride channel inhibitors ATP, tamoxifen and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), inhibited the current dramatically. The anion permeability of the hypotonicity-activated chloride channels was I(-) > Br(-) > Cl(-) > gluconate. Unexpectedly, in isotonic conditions, ATP (10 mM) activated an inward-rectified current, which had not been observed in the nasopharyngeal carcinoma cells. These results suggest that, under hypotonic challenges, fetal human nasopharyngeal epithelial cells can produce I(cl,vol), which might be involved in cell volume regulation.

Differential expression and roles of volume-activated chloride channels in control of growth of normal and cancerous nasopharyngeal epithelial cells

We have previously shown that chloride channel activities were cell cycle-dependent and were involved in cell proliferation in nasopharyngeal carcinoma cells. In this study, the expression and roles of volume-activated chloride channels in cell growth were investigated in the poorly-differentiated human nasopharyngeal carcinoma cell (CNE-2Z) and its counterpart, the normal human nasopharyngeal epithelial cell (NP69-SV40T). Consistent with growth ability, the background chloride currents recorded under isotonic condition, the volume-activated chloride currents induced by 47% hypotonic challenges and the hyponinicity-induced regulatory volume decrease (RVD) were much larger in CNE-2Z cells than in NP69-SV40T cells, suggesting the up-regulation of expression of volume-activated chloride channels in cancerous cells. This was proved by the up-regulation of ClC-3 proteins, a candidate of volume-activated chloride channels, in the cancerous cells. Functional inhibition of chloride channel activities by the chloride channel blockers, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and tamoxifen, and knock-down of ClC-3 expression by specific ClC-3 siRNA attenuated the background currents, suppressed the activation of volume-activated chloride currents, decreased the hyponinicity-induced RVD and inhibited cell growth in the cancerous and normal cells. However, the sensitivities of the cancerous cells were much higher than that of the normal cells. Our data suggest that volume-activated chloride channels play a more important role in control cell proliferation in the cancerous cells than in the normal cells; the growth of cancerous cells is more dependent on the activities of volume-activated chloride channels than that of the normal cells. ClC-3 protein may be considered as a potential tumor marker and therapeutic target for human nasopharyngeal carcinoma

Volume-Sensitive Chloride Channels are Involved in Maintenance of Basal Cell Volume in Human Acute Lymphoblastic Leukemia Cells

Chloride channels are expressed ubiquitously in different cells. However, the activation and roles of volume-activated chloride channels under normal isotonic conditions are not clarified, especially in lymphatic cells. In this study, the activation of basal and volume-activated chloride currents and their roles in maintenance of basal cell volume under isotonic conditions were investigated in human acute lymphoblastic leukemia Molt4 cells. The patch-clamp technique and time-lapse image analysis were employed to record whole-cell currents and cell volume changes. Under isotonic conditions, a basal chloride current was recorded. The current was weakly outward-rectified and volume-sensitive and was not inactivated obviously in the observation period. A 47% hypertonic bath solution and the chloride channel blockers NPPB and tamoxifen suppressed the current. Exposure of cells to 47% hypotonic bath solution activated further the basal current. The hypotonicity-activated current possessed properties similar to those of the basal current and was inhibited by NPPB, tamoxifen, ATP and hypertonic bath solution. Furthermore, extracellular hypotonic challenges swelled the cells and induced a regulatory volume decrease (RVD). Extracellular applications of NPPB, tamoxifen and ATP swelled the cells under isotonic conditions and inhibited the RVD induced by hypotonic cell swelling. The results suggest that some volume-activated chloride channels are activated under isotonic conditions, resulting in the appearance of the basal chloride current, which plays an important role in the maintenance of basal cell volume in lymphoblastic leukemia cells. Chloride channels can be activated further to induce a regulatory volume recovery when cells are swollen.

Lack of association between stretch‐activated and volume‐activated Cl− currents in hepatocellular carcinoma cells

Stretch-activated chloride currents (I(Cl,SA) ) have been considered to be a component of volume-activated chloride currents (I(Cl,vol) ) for some time. This is due to a similarity in biophysical and pharmacological properties that involve a membrane curvature-induced mechanism and rearrangement of the cytoskeleton induced by cell swelling or membrane stretch. In the present study, we demonstrated that current density, along with the time taken from the activation of currents to the peak, were significantly different between the two currents, in highly metastatic human hepatocellular carcinoma cells. In addition, the activation of I(Cl,vol) or I(Cl,SA), induced maximally by hypotonic solutions or membrane stretch, respectively, did not affect the following activation of the other one. Moreover, neither inhibition of I(Cl,vol) by sh-ClC-3 transfection, nor functional blocking of I(Cl,vol) by intracellular dialysis of anti-ClC-3 antibody had an effect on the activation and properties of I(Cl,SA). Collectively, our results suggest that I(Cl,SA) is different from I(Cl,vol) in activation mechanism and/or in molecular entity responsible for formation of the currents. ClC-3 is involved in the activation of I(Cl,vol), but not of I(Cl,SA).

Characterization of Volume-Activated Chloride Currents in Regulatory Volume Decrease of Human Cholangiocyte

Volume-activated chloride channel (VACC) plays vital roles in many physiological functions. In bile duct epithelium, VACC actively participates in biliary secretion and cell volume regulation, and it mediates regulatory volume decrease (RVD). Recently, we have shown that mouse cholangiocytes have an intact RVD via VACC and K(+) conductance. However, such cell volume regulation was not studied in the normal human cholangiocyte. Volume measurement by Coulter counter and whole-cell patch clamp technique were used to characterize the RVD and VACC in human cholangiocyte cell line (HBDC). When exposed to hypotonic solution, HBDC exhibited an intact RVD, which was inhibited by 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), NPPB (5-nitro-2'- (3-phenylpropylamino)-benzoate), DIDS (4,4'-diisothiocyanatostilbene-2-disulfonic acid), and tamoxifen, but was not affected by the removal of extracellular calcium. During RVD, HBDC exhibited large, outwardly rectifying currents and time-dependent inactivation at positive potential. The amplitude of the outward current was approximately 3 times of that of the inward current, and this volume-activated current returned to the baseline when switched to isotonic solution. The amplitude and reversal potential of the volume-activated current was dependent on Cl(-) concentration, and the VACC was significantly inhibited by replacing chloride with gluconate, glutamate, sucrose, and acetate in the hypotonic solution. In addition, classical VACC inhibitors, such as NPPB or tamoxifen, inhibited the VACC. These inhibitory effects were reversible with washing out the inhibitors from the bath solution. The present study is the first to characterize and show that HBDC has an intact RVD, mediated by VACC, which has similar electrophysiological characteristics as that in mouse cholangiocytes.

Role of Calcium in Volume-Activated Chloride Currents in a Mouse Cholangiocyte Cell Line

Volume-activated Cl(-) channels (VACCs) play vital roles in many cells including cholangiocytes. Previously, we characterized the VACCs in mouse cholangiocytes. Since calcium plays an important role in VACC regulation in many cells, we have studied the effect of calcium modulation on the regulatory volume decrease (RVD) and VACC currents in mouse bile duct cells (MBDCs). Cell volume measurements were assessed by a Coulter counter with cell sizer, and conventional whole-cell patch-clamp techniques were used to study the role of calcium on RVD and VACC currents. Cell volume study indicated that MBDCs exhibited RVD, which was inhibited by 5-nitro-2'-(3-phenylpropylamino)-benzoate (NPPB), 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra-acetoxymethyl ester (BAPTA-AM) but not by removal of extracellular calcium. During hypotonic challenge, MBDCs exhibited an outwardly rectified current, which was significantly inhibited by administration of classical chloride channel inhibitors such as NPPB and tamoxifen. Chelation of the intracellular calcium with BAPTA-AM or removal of extracellular calcium and calcium channel blocker had no significant effect on VACC currents during hypotonic challenge. In addition to VACC, MBDC had a calcium-activated chloride channel, which was inhibited by NPPB. The present study is the first to systemically study the role of calcium on the VACC and RVD in mouse cholangiocytes and demonstrates that a certain level of intracellular calcium is necessary for RVD but the activation of VACC during RVD does not require calcium. These findings suggest that calcium does not have a direct regulatory role on VACC but has a permissive role on RVD in cholangiocytes.

Roles of volume-activated Cl- currents and regulatory volume decrease in the cell cycle and proliferation in nasopharyngeal carcinoma cells.

Previously it has been shown, that the volume-activated plasma membrane chloride channel is associated with regulatory volume decrease (RVD) of cells and may play an important role in control of cell proliferation. We have demonstrated that both expression of the channel and RVD capacity are actively regulated in the cell cycle. In this study, we aimed to further study the role of the volume-activated chloride current and RVD in cell cycle progression and overall in cell proliferation. Whole-cell currents, RVD, cell cycle distribution, cell proliferation and cell viability were measured or detected with the patch-clamp technique, the cell image analysis technique, flow cytometry, the MTT assay and the trypan blue assay respectively, in nasopharyngeal carcinoma cells (CNE-2Z cells). The Cl- channel blockers, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and tamoxifen, inhibit the volume-activated chloride current, RVD and proliferation of CNE-2Z cells in a dose-dependent manner. Analysis of relationships between the current, RVD and cell proliferation showed that both the current and RVD were positively correlated with cell proliferation. NPPB (100 microM) and tamoxifen (20 microM) did not significantly induce cell death, but inhibited cell proliferation, implying that the blockers may inhibit cell proliferation by affecting cell cycle progression. This was verified by the observation that tamoxifen (20 microM) and NPPB (100 microM) inhibited cell cycle progress and arrested cells at the G0/G1 phase boundary. Activity of the volume-activated chloride channel is one of the important factors that regulate the passage of cells through the G1 restriction point and that the Cl- current associated with RVD plays an important role in cell proliferation.

Volume-activated chloride currents in interstitial cells of Cajal

Interstitial cells of Cajal (ICC) undergo marked morphological changes on contraction of the musculature, making it essential to understand properties of mechanosensitive ion channels. The whole cell patch-clamp technique was used to identify and to characterize volume-activated Cl- currents in ICC cultured through the explant technique. Hypotonic solutions (approximately 210 mosM) activated an outwardly rectifying current, which reversed near the equilibrium potential for Cl-. Time-dependent inactivation occurred only at pulse potentials of +80 mV, with a time constant of 478 +/- 182 ms. The degree of outward rectification was calculated using a rectification index, the ratio between the slope conductances of +65 and -55 mV, which was 13.9 +/- 1.5 at 76 mM initial extracellular Cl- concentration. The sequence of relative anion permeability of the outwardly rectifying Cl- channel was I- > Cl- > aspartate-. The chloride channel blockers, DIDS and 5-nitro-2-(3-phenlypropl-amino)benzoic acid, caused a voltage-dependent block of the outwardly rectifying Cl- current, inhibition occurring primarily at depolarized potentials. On exposure to hypotonic solution, the slope conductance significantly increased at the resting membrane potential (-70 mV) from 1.2 +/- 0.2 to 2.0 +/- 0.4 nS and at the slow-wave plateau potential (-35 mV) from 2.1 +/- 0.3 to 5.0 +/- 1.0 nS. The current was constitutively active in ICC and contributed to the resting membrane potential and excitability at the slow-wave plateau. In conclusion, swelling or volume change will depolarize ICC through activation of outwardly rectifying chloride channels, thereby increasing cell excitability.

Electrophysiological characterization of volume-activated chloride currents in mouse cholangiocyte cell line

Recent electrophysiological and radioisotope efflux studies have demonstrated various Cl(-) channels in cholangiocytes including volume-activated Cl(-) channels (VACC). Because VACCs play prominent roles in many vital cellular functions and physiology in cholangiocytes, we have examined their electrophysiological characteristics in mouse cholangiocytes to provide an important framework for studying in the future. The present study is to characterize VACCs expressed in the mouse bile duct cell (MBDC) line, conditionally immortalized by SV40 virus. Conventional whole cell patch-clamp techniques were used to study the electrophysiological characteristics of VACC in MBDC. When the MBDCs were exposed to hypotonic solution, they exhibited an outwardly rectified current, which was significantly inhibited by replacing chloride in the bath solution with gluconate or glutamate and by administration of classic chloride channel inhibitors 5-nitro-2-(3-phenylpropylamino)-benzoate, glybenclamide, DIDS, and tamoxifen. These inhibitory effects were reversible with washing them out from the bath solution. Moreover, the ion selectivity of the volume-activated channel to different anions indicates that it is more permeable to SCN(-) > I(-) >/= Cl(-) > F(-) >/= acetate >/= glutamate >/= gluconate. These electrophysiological characteristics demonstrate that the volume-activated current observed is a VACC. In addition, the VACC in MBDC has electrophysiological characteristics similar to those of the VACC in human cholangiocarcinoma cell line. The present study is the first to characterize the VACC in mouse cholangiocyte and will provide an important framework for further studies to examine and understand the role of the VACC in biliary secretion and ion-transport physiology.

Cell cycle-dependent expression of volume-activated chloride currents in nasopharyngeal carcinoma cells.

Patch-clamping and cell image analysis techniques were used to study the expression of the volume-activated Cl(-) current, I(Cl(vol)), and regulatory volume decrease (RVD) capacity in the cell cycle in nasopharyngeal carcinoma cells (CNE-2Z). Hypotonic challenge caused CNE-2Z cells to swell and activated a Cl(-) current with a linear conductance, negligible time-dependent inactivation, and a reversal potential close to the Cl(-) equilibrium potential. The sequence of anion permeability was I(-) > Br(-) > Cl(-) > gluconate. The Cl(-) channel blockers tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), and ATP inhibited I(Cl(vol)). Synchronous cultures of cells were obtained by the mitotic shake-off technique and by a double chemical-block (thymidine and hydroxyurea) technique. The expression of I(Cl(vol)) was cell cycle dependent, being high in G(1) phase, downregulated in S phase, but increasing again in M phase. Hypotonic solution activated RVD, which was cell cycle dependent and inhibited by the Cl(-) channel blockers NPPB, tamoxifen, and ATP. The expression of I(Cl(vol)) was closely correlated with the RVD capacity in the cell cycle, suggesting a functional relationship. Inhibition of I(Cl(vol)) by NPPB (100 microM) arrested cells in G(0)/G(1). The data also suggest that expression of I(Cl(vol)) and RVD capacity are actively modulated during the cell cycle. The volume-activated Cl(-) current associated with RVD may therefore play an important role during the cell cycle progress.

Ransom CB, O'Neal JT, Sontheimer H. Volume-activated chloride currents contribute to the resting conductance invasive migration of human glioma cells.

Ransom CB, O'Neal JT, Sontheimer H. Volume-activated chloride currents contribute to the resting conductance invasive migration of human glioma cells.

Volume-Activated Chloride Currents Contribute to the Resting Conductance and Invasive Migration of Human Glioma Cells

We used an in vitro model for glioma cell invasion (transwell migration assay) and patch-clamp techniques to investigate the role of volume-activated Cl(-) currents (I(Cl,Vol)) in glioma cell invasion. Hypotonic solutions ( approximately 230 mOsm) activated outwardly rectifying currents that reversed near the equilibrium potential for Cl(-) ions (E(Cl)). These currents (I(Cl,Vol)) were sensitive to several known Cl(-) channel inhibitors, including DIDS, tamoxifen, and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB). The IC(50) for NPPB inhibition of I(Cl,Vol) was 21 microm. Under isotonic conditions, NPPB (165 microm) blocked inward currents (at -40 mV) and increased input resistance in both standard whole-cell recordings and amphotericin perforated-patch recordings. Reducing [Cl(-)](o) under isotonic conditions positively shifted the reversal potential of whole-cell currents. These findings suggest a significant resting Cl(-) conductance in glioma cells. Under isotonic and hypotonic conditions, Cl(-) channels displayed voltage- and time-dependent inactivation and had an I(-) > Cl(-) permeability. To assess the potential role of these channels in cell migration, we studied the chemotactic migration of glioma cells toward laminin or vitronectin in a Boyden chamber containing transwell filters with 8 microm pores. Inhibition of I(Cl,Vol) with NPPB reduced chemotactic migration in a dose-dependent fashion with an IC(50) of 27 microm. Time-lapse video microscopy during patch-clamp recordings revealed visible changes in cell shape and/or movement that accompanied spontaneous activation of I(Cl,Vol), suggesting that I(Cl,Vol) is activated during cell movement, consistent with the effects of NPPB in migration assays. We propose that I(Cl,Vol) contributes to cell shape and volume changes required for glioma cell migration through brain tissue.

Volume-Activated Chloride Currents in HeLa Cells are Blocked by Tamoxifen But Not by a Membrane Impermeant Quaternary Analogue

Background/Aims: Tamoxifen has been shown to inhibit volume activated chloride currents in many cell types. Tamoxifen has also been reported to inhibit a number of cation channels as well as cytosolic proteins such as calmodulin. The mechanism of channel block by tamoxifen is not known but three hypotheses can be proposed: i) a direct effect following binding to the channel protein from the aqueous environment or ii) a direct effect on the channel protein after partitioning into the lipid membrane or iii) an indirect mechanism via binding to intracellular regulatory proteins after diffusion across the lipid membrane. The aim of these experiments was to distinguish between these hypothoses using membrane permeant and impermeant antioestrogens. Methods: Volume activated chloride currents were recorded from single HeLa cells using whole cell patch clamp technique. The ability of tamoxifen and its membrane impermeant quaternary derivative ethyl bromide tamoxifen (EBT) to inhibit these currents was examined. Results: Extracellular tamoxifen at 3µM inhibited volume activated chloride currents in HeLa cells whereas EBT had no effect up to 10µM when applied either to the extracellular bathing solution or the intracellular solution via the patch pipette. Conclusion: Eliminating the ability of tamoxifen to cross the plasma membrane abolishes its channel blocking activity against volume activated chloride channels in HeLa cells.

The role of ClC-3 in volume-activated chloride currents and volume regulation in bovine epithelial cells demonstrated by antisense inhibition.

1. A chloride current with mild outward rectification was induced in the native bovine non-pigmented ciliary epithelial (NPCE) cells by a 23 % hypotonic solution. The current showed no or little inactivation at depolarized steps. 2. ATP blocked 88 and 61 % of the outward and inward components of the volume-activated chloride current (ICl,vol) with an IC50 of 5.3 and 9.6 mM, respectively. 3. The volume-activated chloride current was decreased and the activation of the current was delayed by inhibiting endogenous ClC-3 expression using a ClC-3 antisense oligonucleotide. The inhibition of the current as a function of antisense concentration was asymptotic with a maximum about 60 %. The remaining current was probably not derived from ClC-3 and was inhibited by ATP. 4. ClC-3 expression in the bovine NPCE cells was verified by immunofluorescence studies. ClC-3 immunofluorescence was distributed throughout the cells but with the predominant location within the nucleus. The expression of ClC-3 protein was diminished by the ClC-3 antisense oligonucleotide with the greatest diminution occurring in the nuclear region. 5. The size of the volume-activated chloride current was positively correlated with the ClC-3 immunofluorescence level. 6. Regulatory volume decrease of the NPCE cells was reduced by ClC-3 antisense oligonucleotide. 7. We conclude that endogenous ClC-3 is associated with the volume-activated chloride current and is involved in cell volume regulation, but that it can only contribute towards a proportion of the current in NPCE cells. 8. The nuclear predominance of ClC-3 immunofluorescence in NPCE cells, the absence of basal activity of chloride current and the marked pharmacological differences between IClC-3 and ICl,vol argue against ClC-3 being the only, or even the main, volume-activated chloride channel in NPCE cells.

Functional and molecular characterization of a volume-sensitive chloride current in rat brain endothelial cells.

1. Volume-activated chloride currents in cultured rat brain endothelial cells were investigated on a functional level using the whole-cell voltage-clamp technique and on a molecular level using the reverse transcriptase-polymerase chain reaction (RT-PCR). 2. Exposure to a hypotonic solution caused the activation of a large, outward rectifying current, which exhibited a slight time-dependent decrease at strong depolarizing potentials. The anion permeability of the induced current was I- (1.7) > Br- (1.2) > Cl- (1.0) > F- (0. 7) > gluconate (0.18). 3. The chloride channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB, 100 microM) rapidly and reversibly inhibited both inward and outward currents. The chloride transport blocker 4,4'-diisothiocyanatostilbene-2, 2'-disulphonic acid (DIDS, 100 microM) also blocked the hypotonicity-induced current in a reversible manner. In this case, the outward current was more effectively suppressed than the inward current. The volume-activated current was also inhibited by the antioestrogen tamoxifen (10 microM). 4. The current was dependent on intracellular ATP and independent of intracellular Ca2+. 5. Activation of protein kinase C by phorbol 12,13-dibutyrate (PDBu, 100 nM) inhibited the increase in current normally observed following hypotonic challenge. 6. Extracellular ATP (10 mM) inhibited the current with a more pronounced effect on the outward than the inward current. 7. Verapamil (100 microM) decreased both the inward and the outward hypotonicity-activated chloride current. 8. RT-PCR analysis was used to determine possible molecular candidates for the volume-sensitive current. Expression of the ClC-2, ClC-3 and ClC-5 chloride channels, as well as pICln, could be shown at the mRNA level. 9. We conclude that rat brain endothelial cells express chloride channels which are activated by osmotic swelling. The biophysical and pharmacological properties of the current show strong similarities to those of ClC-3 channel currents as described in other cell types.