Volume-sensitive Chloride Current Research Articles

Interleukin 1 beta-induced chloride currents are important in osteoarthritis onset: an in vitro study

Persistent hypotonic and inflammatory conditions in the joint cavity can lead to the loss of cartilage matrix and cell death, which are the important mechanisms of osteoarthritis (OA) onset. Previous studies have confirmed that the existence of a hypotonic environment is a red flag for inflammation, as hypotonic environment induces the opening of the chloride channel of the cell and promotes chloride ion efflux, which prompts the cell volume to increase. Chloride channels play an important role in the regulation of mineralization and chondrocyte death. Here, we reported that OA chondrocytes showed a significant increase of cell death rate and the imbalance of cartilage matrix catabolism. We found that the distribution of skeleton protein F-actin was disordered. In addition, the volume-sensitive chloride current of OA chondrocytes decreased significantly with the increase of the expression levels of inflammation-related proteins caspase-1, caspase-3, and NLRP3. Moreover, interleukin-1β (IL-1β) showed a potential to activate the chloride current of normal chondrocytes. These results indicate that IL-1β-induced chloride channel opening in chondrocytes may be closely related to the occurrence of OA. This chloride channel opening process may therefore be a potential target for the treatment of OA.

Acid- and Volume-Sensitive Chloride Currents in Human Chondrocytes.

Chondrocytes face extreme alterations of extracellular osmolarity and pH, which force them to appropriately regulate their cell volume (CV) and cellular pH. Perturbations of these mechanisms lead to chondrocyte death and ultimately to osteoarthritis (OA), the most common chronic joint diseases worldwide. OA hallmarks are altered cartilage hydration and severe fluid acidification. Impaired CV regulation and acidotoxicity contribute to disease progression and volume-sensitive anion channels are upregulated in OA. This study assessed the effect of hypotonicity and extracellular acidification on chondrocyte Cl– conductances and CV regulation. Cl– currents and membrane potentials were measured in human C28/I2 cells and primary human chondrocytes using the patch clamp technique. Intracellular pH was assessed by BCECF fluorescence, CV measurements were performed using the Coulter method, and cell viability/cell death by a resazurin assay. Hypotonic cell swelling caused activation of a volume-sensitive outwardly rectifying (VSOR) Cl– current followed by a regulatory volume decrease (RVD), which was attenuated by the Cl– channel blocker DCPIB. Extracellular, but not intracellular acidification to pH ≤ 5.0 elicited an acid-sensitive outwardly rectifying (ASOR) Cl– conductance. Activation of either current depolarized the cell membrane potential. Under simultaneous hypotonic and acidic stimulation, VSOR and ASOR currents transiently coactivated, giving rise to a mixed current phenotype. Over time the VSOR current gradually vanished and the residual conductance showed a pure ASOR current phenotype. Extracellular acidification caused an isotonic CV gain and a complete suppression of RVD under hypotonic conditions. The results suggest that deactivation of the VSOR current under acidic conditions impairs CV regulation in chondrocytes, which is likely to compromise chondrocyte viability.

Acid- and Volume-Sensitive Chloride Currents in Microglial Cells.

Many cell types express an acid-sensitive outwardly rectifying (ASOR) anion current of an unknown function. We characterized such a current in BV-2 microglial cells and then studied its interrelation with the volume-sensitive outwardly rectifying (VSOR) Cl− current and the effect of acidosis on cell volume regulation. We used patch clamp, the Coulter method, and the pH-sensitive dye BCECF to measure Cl− currents and cell membrane potentials, mean cell volume, and intracellular pH, respectively. The ASOR current activated at pH ≤ 5.0 and displayed an I− > Cl− > gluconate− permeability sequence. When compared to the VSOR current, it was similarly sensitive to DIDS, but less sensitive to DCPIB, and insensitive to tamoxifen. Under acidic conditions, the ASOR current was the dominating Cl− conductance, while the VSOR current was apparently inactivated. Acidification caused cell swelling under isotonic conditions and prevented the regulatory volume decrease under hypotonicity. We conclude that acidification, associated with activation of the ASOR- and inactivation of the VSOR current, massively impairs cell volume homeostasis. ASOR current activation could affect microglial function under acidotoxic conditions, since acidosis is a hallmark of pathophysiological events like inflammation, stroke or ischemia and migration and phagocytosis in microglial cells are closely related to cell volume regulation.

Fluid shear stress enhances the cell volume decrease of osteoblast cells by increasing the expression of the ClC-3 chloride channel.

ClC-3 is a volume-sensitive chloride channel that is responsible for cell volume adjustment and regulatory cell volume decrease (RVD). In order to evaluate the effects of fluid shear stress (FSS) stimulation on the osteoblast ClC-3 chloride channel, MC3T3-E1 cells were stimulated by FSS in the experimental group. Fluorescence quantitative polymerase chain reaction was used to detect changes in ClC-3 mRNA expression, the chloride ion fluorescent probe N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) was used to detect the chloride channel activity, and whole-cell patch clamping was used to monitor the changes in the volume-sensitive chloride current activated by a hypotonic environment following mechanical stimulation. The results show that the expression of the osteoblast chloride channel ClC-3 was significantly higher in the FSS group compared with the control group. MQAE fluorescence intensity was significantly reduced in the FSS group compared to the control group, suggesting that mechanical stimulation increased chloride channel activity and increased the efflux of intracellular chloride ions. Image analysis of osteoblast volume changes showed that osteoblast RVD was enhanced by mechanical stimulation. Whole-cell patch clamping showed that the osteoblast volume-sensitive chloride current was larger in the stimulated group compared to the control group, suggesting that elevated ClC-3 chloride channel expression results in an increased volume-sensitive chloride current. In conclusion, FSS stimulation enhances the RVD of osteoblast cell by increasing the expression of the ClC-3 and enhancing the chloride channel activity.

Intermediate-conductance Ca2+-activated potassium and volume-sensitive chloride channels in endothelial progenitor cells from rat bone marrow mononuclear cells

Bone marrow endothelial progenitor cells (BMEPCs) are believed to be a promising cell source for regenerative medicine; however, their electrophysiology properties have not been fully clarified, which is important to the clinical application of BMEPCs. The current study was designed to determine the transmembrane ion currents and mRNA expression levels of related ion channel subunits in rat BMEPCs. Bone marrow mononuclear cells were isolated by density gradient separation and cultured in EPC medium. The transmembrane ion currents were determined using whole-cell patch-voltage clamp technique, and the levels of mRNA and protein expressions of functional ionic channels were measured using RT-PCR and western immunoblot analysis. We observed two types of ionic currents inundifferentiated rat BMEPCs. One was Ca(2+) -activated potassium current (I(kca) ), which was seen in approx. 90% of cells when 1μm Ca(2+) was employed in pipette solution, and it was predominantly inhibited by intermediate-conductance I(kca) inhibitor clotrimazole. The other one was volume-sensitive chloride current (I(cl) ), which was detected in 85.7% of cells when BMEPCs were subjected to K(+) -free hypotonic extracellular solution, whose currents could be inhibited by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). The corresponding ion channel genes and proteins, KCNN4 for I(kca) and Clcn3 for I(cl) , were confirmed by RT-PCR and western immunoblot analysis of BMEPCs. Our results demonstrated for the first time that rat BMEPCs expressed intermediate-conductance Ca(2+) -activated potassium currents and volume-sensitive chloride currents, and corresponding genes and proteins of these two channels are KCNN4 and Clcn3 respectively.

Effects of ion channels on proliferation in cultured human cardiac fibroblasts

Our previous study demonstrated that multiple ion channels were heterogeneously expressed in human cardiac fibroblasts, including a large-conductance Ca2+-activated K+ current (BKCa), a volume-sensitive chloride current (ICl.vol), and voltage-gated sodium currents (INa). The present study was designed to examine the possible involvement of these ion channels in proliferation of cultured human cardiac fibroblasts using approaches of cell proliferation assay, whole-cell patch voltage-clamp, siRNA and Western blot analysis. It was found that the blockade of BKCa with paxilline (1–3μM) or ICl.vol with 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid disodium (DIDS, 100–200μM), but not INa with tetrodotoxin (0.1–10μM), remarkably suppressed proliferation in human cardiac fibroblasts. Knockdown of KCa1.1 or Clcn3 with specific siRNAs significantly reduced BKCa or ICl.vol current, mRNA and channel protein levels, and inhibited growth of human cardiac fibroblasts. Flow cytometry analysis showed accumulation of cardiac fibroblasts at G0/G1 phase and reduced cell number in S phase after inhibition of BKCa or ICl.vol with channel blockers or knock down of the corresponding channels with specific siRNAs; these effects were accompanied by a decreased expression of cyclin D1 and cyclin E. The present results demonstrate the novel information that BKCa and ICl.vol channels, but not INa channels, are involved in the regulation of proliferation in cultured human cardiac fibroblasts by promoting cell cycle progression via modulating cyclin D1 and cyclin E expression.

Control of volume-sensitive chloride channel inactivation by the coupled action of intracellular chloride and extracellular protons

The volume-sensitive chloride current (I(ClVol)) exhibit a time-dependent decay presumably due to channel inactivation. In this work, we studied the effects of chloride ions (Cl(-)) and H(+) ions on I(ClVol) decay recorded in HEK-293 and HL-60 cells using the whole-cell patch clamp technique. Under control conditions ([Cl(-)](e) = [Cl(-)](i) = 140 mM and pH(i) = pH(e) = 7.3), I(ClVol) in HEK cells shows a large decay at positive voltages but in HL-60 cells I(ClVol) remained constant independently of time. In HEK-293 cells, simultaneously raising the [Cl(-)](e) and [Cl(-)](i) from 25 to 140 mM (with pH(e) = pH(i) = 7.3) increased the fraction of inactivated channels (FIC). This effect was reproduced by elevating [Cl(-)](i) while keeping the [Cl(-)](e) constant. Furthermore, a decrease in pH(e) from 7.3 to 5.5 accelerated current decay and increased FIC when [Cl(-)] was 140 mM but not 25 mM. In HL-60 cells, a slight I(ClVol) decay was seen when the pH(e) was reduced from 7.3 to 5.5. Our data show that inactivation of I(ClVol) can be controlled by changing either the Cl(-) or H(+) concentration or both. Based on our results and previously published data, we have built a model that explains VRAC inactivation. In the model the H(+) binding site is located outside the electrical field near the extracellular entry whilst the Cl(-) binding site is intracellular. The model depicts inactivation as a pore constriction that happens by simultaneous binding of H(+) and Cl(-) ions to the channel followed by a voltage-dependent conformational change that ultimately causes inactivation.

Functional ion channels in mouse cardiac c-kit+ cells

Cardiac c-kit(+) cells are generally believed to be the major population of stem/progenitor cells in the heart and can be used as a cell source for cardiomyoplasty; however, the cellular electrophysiological properties are not understood in this type of cells. The present study was designed to investigate functional ion channels in undifferentiated mouse cardiac c-kit(+) cells using approaches of whole cell patch voltage clamp, RT-PCR, and cell proliferation assay. It was found that three types of ionic currents were present in mouse cardiac c-kit(+) cells, including a delayed rectifier K(+) current (IK(DR)) inhibited by 4-aminopyridine (4-AP), an inward rectifier K(+) current (I(Kir)) decreased by Ba(2+), and a volume-sensitive chloride current (I(Cl.vol)) inhibited by 5-nitro-1-(3-phenylpropylamino) benzoic acid (NPPB). RT-PCR revealed that the corresponding ion channel genes, Kv1.1, Kv1.2, and Kv1.6 (for IK(DR)), Kir.1.1, Kir2.1, and Kir2.2 (likely responsible for I(Kir)), and Clcn3 (for I(Cl.vol)), were significant in mouse cardiac c-kit(+) cells. The inhibition of I(Cl.vol) with NPPB and niflumic acid, but not IK(DR) with 4-AP and tetraethylammonium, reduced cell proliferation and accumulated the cell progression at G(0)/G(1) phase in mouse cardiac c-kit(+) cells. Our results demonstrate that three types of functional ion channel currents (i.e., IK(DR), I(Kir), and I(Cl.vol)) are present in mouse cardiac c-kit(+) cells, and I(Cl.vol) participates in regulating cell proliferation.

The volume‐sensitive chloride channel inhibitors prevent both contractile dysfunction and apoptosis induced by doxorubicin through PI3kinase, Akt and Erk 1/2

Contractile dysfunction and cardiomyopathies secondary to apoptotic cell death are limiting factors for treating cancer with doxorubicin. Inhibition of volume-sensitive chloride currents (I(Cl,vol)) has been reported to blunt doxorubicin-induced apoptosis in cardiomyocytes. To investigate cellular contractility during acute induction of apoptosis by doxorubicin and to determine whether I(Cl,vol) inhibitors are able to prevent the subsequent contractile dysfunction, electrically paced ventricular myocytes freshly isolated from adult rabbits were acutely exposed to doxorubicin in the presence and absence of I(Cl,vol) inhibitors IAA-94 or DIDS. Doxorubicin induced increases in both annexin V labelling and caspase-3 activity and decreases in cell volume. Alteration in cardiac contractility was observed after doxorubicin exposure. Both IAA-94 and DIDS abolished the doxorubicin-induced decreases in peak shortening and cell volume as well as the increases in caspase-3 activity and annexin V labelling. These protective effects of I(Cl,vol) inhibitors were abolished by previous inhibition of PI(3)kinase, Akt and Erk 1/2. Thus, I(Cl,vol) inhibitors prevent doxorubicin-induced apoptosis and subsequent contractile dysfunction through PI(3)kinase/Akt and Erk 1/2. Inhibition of I(Cl,vol) may represent a new pharmacological strategy for developing cytoprotective drugs against apoptotic cell death and contractile dysfunction.

Regulation of cardiac volume-sensitive chloride channel by focal adhesion kinase and Src kinase

The volume-sensitive chloride current (ICl,swell) is found in the mammalian myocardium and is activated by osmotic swelling. The goal of this study was to examine the importance of the tyrosine kinases focal adhesion kinase (FAK) and Src kinase in cardiac ICl,swell regulation. Neonatal rat ventricular myocytes were cultured on collagen membranes and infected with adenovirus expressing beta-galactosidase (AdLacZ), FAK, or FAK-related nonkinase. FAK-related nonkinase (FRNK) is an endogenous cardiac protein, which functions as an inhibitor of FAK. Whole cell patch-clamp recordings demonstrated that osmotic swelling was associated with the activation of an outward rectifying current in uninfected and AdLacZ-infected cells. Consistent with the properties of ICl,swell, this current displayed a reversal potential close to the equilibrium potential for Cl-; was inhibited by the Cl- channel blockers 4,4'-dinitrostilbene-2,2'-disulfonic acid, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, and tamoxifen; and was eliminated in hypertonic solution. In addition to activating ICl,swell, hypotonic swelling enhanced the tyrosine phosphorylation of multiple cardiac proteins including those in the range of 68-70 and 120-130 kDa. Pretreatment of the cells with the drug 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, an inhibitor of FAK and Src, diminished swelling-induced phosphorylation of these proteins but paradoxically increased ICl,swell. Furthermore, overexpression of FRNK but not FAK caused a twofold augmentation in I(Cl,swell) and increased the rate of current activation. Thus the tyrosine kinases FAK and Src contribute to the regulation of ICl,swell.

Ca2+- and Volume-sensitive Chloride Currents Are Differentially Regulated by Agonists and Store-operated Ca2+ Entry

Using patch-clamp and calcium imaging techniques, we characterized the effects of ATP and histamine on human keratinocytes. In the HaCaT cell line, both receptor agonists induced a transient elevation of [Ca2+]i in a Ca2+-free medium followed by a secondary [Ca2+]i rise upon Ca2+ readmission due to store-operated calcium entry (SOCE). In voltage-clamped cells, agonists activated two kinetically distinct currents, which showed differing voltage dependences and were identified as Ca2+-activated (ICl(Ca)) and volume-regulated (ICl, swell) chloride currents. NPPB and DIDS more efficiently inhibited ICl(Ca) and ICl, swell, respectively. Cell swelling caused by hypotonic solution invariably activated ICl, swell while regulatory volume decrease occurred in intact cells, as was found in flow cytometry experiments. The PLC inhibitor U-73122 blocked both agonist- and cell swelling–induced ICl, swell, while its inactive analogue U-73343 had no effect. ICl(Ca) could be activated by cytoplasmic calcium increase due to thapsigargin (TG)-induced SOCE as well as by buffering [Ca2+]i in the pipette solution at 500 nM. In contrast, ICl, swell could be directly activated by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a cell-permeable DAG analogue, but neither by InsP3 infusion nor by the cytoplasmic calcium increase. PKC also had no role in its regulation. Agonists, OAG, and cell swelling induced ICl, swell in a nonadditive manner, suggesting their convergence on a common pathway. ICl, swell and ICl(Ca) showed only a limited overlap (i.e., simultaneous activation), although various maneuvers were able to induce these currents sequentially in the same cell. TG-induced SOCE strongly potentiated ICl(Ca), but abolished ICl, swell, thereby providing a clue for this paradox. Thus, we have established for the first time using a keratinocyte model that ICl, swell can be physiologically activated under isotonic conditions by receptors coupled to the phosphoinositide pathway. These results also suggest a novel function for SOCE, which can operate as a “selection” switch between closely localized channels.

Volume-sensitive chloride currents in cardiomyocytes isolated from ClC-3 knockout mice

Volume-sensitive chloride currents in cardiomyocytes isolated from ClC-3 knockout mice

Differential effects of tyrosine kinase inhibitors on volume-sensitive chloride current in human atrial myocytes: evidence for dual regulation by Src and EGFR kinases.

To determine whether protein tyrosine kinase (PTK) modulates volume-sensitive chloride current (ICl.vol) in human atrial myocytes and to identify the PTKs involved, we studied the effects of broad-spectrum and selective PTK inhibitors and the protein tyrosine phosphatase (PTP) inhibitor orthovanadate (VO4 −3). ICl.vol evoked by hyposmotic bath solution (0.6-times isosmotic, 0.6T) was enhanced by genistein, a broad-spectrum PTK inhibitor, in a concentration-dependent manner (EC50 = 22.4 μM); 100 μM genistein stimulated ICl.vol by 122.4 ± 10.6%. The genistein-stimulated current was inhibited by DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid, 150 μM) and tamoxifen (20 μM), blockers of ICl.vol. Moreover, the current augmented by genistein was volume dependent; it was abolished by hyperosmotic shrinkage in 1.4T, and genistein did not activate Cl− current in 1T. In contrast to the stimulatory effects of genistein, 100 μM tyrphostin A23 (AG 18) and A25 (AG 82) inhibited ICl.vol by 38.2 ± 4.9% and 40.9 ± 3.4%, respectively. The inactive analogs, daidzein and tyrphostin A63 (AG 43), did not alter ICl.vol. In addition, the PTP inhibitor VO4 −3 (1 mM) reduced ICl.vol by 53.5 ± 4.5% (IC50 = 249.6 μM). Pretreatment with VO4 −3 antagonized genistein-induced augmentation and A23- or A25-induced suppression of ICl.vol. Furthermore, the selective Src-family PTK inhibitor PP2 (5 μM) stimulated ICl.vol, mimicking genistein, whereas the selective EGFR (ErbB-1) kinase inhibitor tyrphostin B56 (AG 556, 25 μM) reduced ICl.vol, mimicking A23 and A25. The effects of both PP2 and B56 also were substantially antagonized by pretreatment with VO4 −3. The results suggest that ICl.vol is regulated in part by the balance between PTK and PTP activity. Regulation is complex, however. Src and EGFR kinases, distinct soluble and receptor-mediated PTK families, have opposing effects on ICl.vol, and multiple target proteins are likely to be involved.

Modulation of volume-sensitive chloride current by noradrenaline in rabbit portal vein myocytes.

The effect of noradrenaline on the volume-sensitive chloride current (I(Cl(swell))) was studied with conventional whole-cell recording techniques in freshly dispersed isolated smooth muscle cells of the rabbit portal vein. In the absence of receptor antagonists, noradrenaline produced an increase in the amplitude of I(Cl(swell)) in some cells and a decrease in others. In the presence of the beta-adrenoceptor antagonist propranolol, noradrenaline increased I(Cl(swell)) and in the presence of the alpha(1)-adrenoceptor antagonist prazosin, noradrenaline reduced I(Cl(swell).) The phospholipase C (PLC) inhibitor U73122 reduced the amplitude of I(Cl(swell)) whereas the inactive analogue U73343 had no effect. The phorbol esters phorbol-12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate (PDBu) increased the amplitude of I(Cl(swell)) by approximately 60 and 100 %, respectively, in a voltage-independent fashion. Inhibitors of protein kinase C (PKC) chelerythrine and calphostin-C decreased the amplitude of I(Cl(swell)) in a concentration-dependent but voltage-independent manner. Bath application of 8-Br-cAMP decreased I(Cl(swell)) by about 60 % whereas the inhibitor of protein kinase A (PKA) KT5720 increased the amplitude of I(Cl(swell)) by approximately 80-90 %. In the presence of propranolol, chelerythrine prevented the increase of I(Cl(swell)) by noradrenaline; in the presence of prazosin, KT5720 blocked the inhibitory action of noradrenaline. The results show that in rabbit portal vein myocytes noradrenaline enhances I(Cl(swell)) by acting on alpha(1)-adrenoceptors and reduces I(Cl(swell)) by stimulating beta-adrenoceptors. The data suggest that the potentiating and inhibitory effects of noradrenaline are mediated, respectively, by PKC and PKA.

Block of volume-regulated anion channels by selective serotonin reuptake inhibitors.

We have used the whole-cell patch clamp technique to study the effects of the commonly used antidepressants sertraline, paroxetine, citalopram and fluvoxamine on the volume-regulated anion channel (VRAC) in endothelial cells. It was the purpose of the present experiments to investigate whether VRAC block is a general property of this group of selective serotonin reuptake inhibitors (SSRIs). At pH 7.4, all SSRIs induced a fast and reversible block of the volume-sensitive chloride current ( I(Cl,swell)), with an IC(50) value of 2.1+/-0.5 microM for sertraline, 2.7+/-0.2 microM for paroxetine, 12.3+/-1.4 microM for fluvoxamine and 27.7+/-2.8 microM for citalopram. The block was enhanced at more alkaline pH, indicating that it is mediated by the uncharged form. This study describes the effects of a variety of SSRIs on an anion channel. Our data reveal a potent block and suggest a hydrophobic interaction of high affinity between the uncharged SSRI and volume-regulated anion channels. We conclude that VRAC block is a general property of this pharmacological class of selective serotonin reuptake inhibitors.

Inhibition of volume-regulated anion channels in cultured endothelial cells by the anti-oestrogens clomiphene and nafoxidine.

1. We have used the whole-cell patch clamp technique to study the effect of the partial anti-oestrogens clomiphene and nafoxidine, the pure anti-oestrogens ICI 182,780 and RU 58,668 and the oestrogen ss-estradiol, on the volume-regulated anion channel (VRAC) in cultured pulmonary artery endothelial (CPAE) cells. 2. In contrast to the pure anti-oestrogens and ss-estradiol, clomiphene and nafoxidine potently inhibited the volume-sensitive chloride current, I(Cl,swell), activated by challenging CPAE cells with a 25% hypotonic solution. For clomiphene, the estimated IC(50) and Hill coefficient were 1.03+/-0.14 microM and 1.40+/-0.21 respectively. In the case of nafoxidine, these values were 1.61+/-0.29 microM and 1.24+/-0.19. 3. The inhibition induced by the pure enantiomers of clomiphene, zuclomiphene and enclomiphene, was not different from that of the racemic mixture, indicating that the interaction between clomiphene and VRAC is not stereoselective. 4. Clomiphene and nafoxidine inhibited proliferation of CPAE cells. Half-maximal inhibition was found at 1.98+/-0.17 and 1.66+/-0.21 microM respectively, concentrations similar to those for half-maximal block of VRAC. 5. In conclusion, the nonsteroidal partial anti-oestrogens nafoxidine and clomiphene are potent inhibitors of volume-regulated anion channels. The inhibition by clomiphene is not stereoselective and occurs at concentrations close to therapeutically relevant concentrations. Finally, both drugs inhibit the proliferation of endothelial cells.

Block by fluoxetine of volume-regulated anion channels.

1. We have used the whole-cell patch clamp technique to study the effect of fluoxetine, a commonly used antidepressant drug, on the volume-regulated anion channel (VRAC) in calf pulmonary artery endothelial (CPAE) cells. We also examined its effects on other Cl- channels, i.e. the Ca2(+)-activated Cl- current (I(Cl,Ca) and the cystic fibrosis transmembrane conductance regulator (CFTR) to assess the specificity of this compound for VRAC. 2. At pH 7.4 fluoxetine induced a fast and reversible block of the volume-sensitive chloride current (I(Cl,swell)), with a Ki value of 6.0+/-0.5 microM (n = 6-9). The blocking efficiency increased with increasing extracellular pH (Ki= 0.32+/-0.01 microM at pH 8.8, n = 3-9), indicating that the blockade is mediated by the uncharged form of fluoxetine. 3. Fluoxetine inhibited Ca2(+)-activated Cl(-) currents, I(Cl,Ca), activated by loading CPAE cells via the patch pipette with 1000 nM free Ca2+ (Ki= 10.7+/-1.6 microm at pH 7.4, n=3-5). The CFTR channel, transiently transfected in CPAE cells, was also inhibited with a Ki value of 26.9+/-9.4 microM at pH 7.4 (n = 3). 4. This study describes for the first time the effects of fluoxetine on anion channels. Our data reveal a potent block of VRAC at fluoxetine concentrations close to plasma concentrations. The results suggest a hydrophobic interaction with high affinity between uncharged fluoxetine and volume-activated chloride channels. Ca(2+)-activated Cl- currents and CFTR are also blocked by fluoxetine, revealing a novel characteristic of the drug as a chloride channel modulator.

Characterization of cell volume-sensitive chloride currents in freshly prepared and cultured pancreatic acinar cells from early postnatal rats.

1. In freshly prepared and cultured exocrine pancreatic acinar cells from 5- to 7-day-old rats a chloride-selective membrane conductance could be activated by intracellular application of GTPgammaS (40-100 microM), by application of positive pressure (5 cmH2O) to the pipette interior or by challenging the cells with a hyposmolar bath solution. Hyperosmolar bath solutions inhibited the cell volume-sensitive chloride currents. 2. The anion permeability sequence of the cell volume-sensitive chloride conductance was I- > Cl- approximately Br- > F- > methanesulphonate- > glutamate-. I- had a higher permeability but lower conductance than Cl-. The permeability ratio for Pglutamate/PCl was 0.12. 3. The cell volume-sensitive chloride conductance showed outward rectification. Membrane depolarization to high positive voltages (>= +60 mV) caused a time-dependent decay in outward currents. 4. DIDS (4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid) and SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid) reversibly inhibited the cell volume-sensitive chloride current in a voltage-dependent manner. NPPB (5-nitro-2-(3-phenylpropylamino)-benzoic acid), quinidine, quinine and tamoxifen caused voltage-independent current inhibition. 5. Combined fura-2 and whole-cell current measurements showed that activation of the cell volume-sensitive chloride current does not involve cytosolic Ca2+ signals. Furthermore, there is no evidence that Ca2+-activated chloride currents play a significant role in cultured pancreatic acinar cells from 5- to 7-day-old rats. 6. Polymerase chain reaction followed by DNA sequence analysis indicated the presence of mRNA homologous to the ClC-3 chloride channel in pancreatic tissue from 5-day-old rats.

Involvement of PKC-alpha in regulatory volume decrease responses and activation of volume-sensitive chloride channels in human cervical cancer HT-3 cells.

1. The present study was carried out to identify the specific protein kinase C (PKC) isoform involved in regulatory volume decrease (RVD) responses, and to investigate the signal transduction pathways underlying the activation of volume-sensitive chloride channels in human cervical cancer HT-3 cells. The role of Ca2+ in RVD and in the activation of chloride currents was also studied. 2. The time course of RVDs was prolonged by microinjection of PKC-alpha antibody but not by PKC-beta or PKC-gamma antibody, and also by exposure to Ca2+-free medium, in particular when combined with microinjection of EDTA. Immunofluorescence staining showed that hypotonic superfusion evoked the translocation of PKC-alpha to the cell membrane, whereas PKC-beta or PKC-gamma remained unaffected. The translocation of PKC-alpha was observed a few minutes after hypotonic stress, reaching peak intensity at 30 min, and returned to the cytoplasm 60 min after hypotonic exposure. Western blot analyses showed an increased PKC-alpha level in terms of intensity and phosphorylation in the cell membrane, while neither PKC-beta nor PKC-gamma was activated upon hyposmotic challenge. 3. Whole-cell patch-clamp studies demonstrated that neomycin and PKC blockers such as staurosporine and H7 inhibited volume-sensitive chloride currents. The inhibitory effect of neomycin on chloride currents can be reversed by the PKC activator phorbol 12-myristate, 13-acetate (PMA). Moreover, the PKC inhibitor and PKC-alpha antibody, but not PKC-beta or PKC-gamma antibody, significantly attenuated the chloride currents. The activation of volume-sensitive chloride currents were insensitive to the changes of intracellular Ca2+ but required the presence of extracellular Ca2+. 4. Our results suggest the involvement of PKC-alpha and extracellular Ca2+ in RVD responses and the activation of volume-sensitive chloride channels in HT-3 cells.

The list of potential volume-sensitive chloride currents continues to swell (and shrink).

The protein for the swelling-activated chloride conductance is one of the few ion channels left for which we do not have a universally acceptable clone. In this brief note, I hope to describe why chloride channels in general, and ICl.swell in particular, have been difficult to pin down. Dr. Strange