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PubMed1994

Drugs transported by P-glycoprotein inhibit a 40 pS outwardly rectifying chloride channel.

P-glycoprotein functions as an ATP-dependent pump for a diverse spectrum of compounds. Recently, it has been shown that P-glycoprotein may be bi-functional and act as a chloride channel as well as a pump. The single channel properties of this conductance are unknown, however, as macroscopic, whole cell currents are inhibited by substrates for P-glycoprotein transport, the single channels underlying this response should also be blocked by these compounds. We found that colchicine, vinblastine, daunomycin and verapamil (50 microM) caused block of a 40 pS outwardly-rectifying chloride channel in cells expressing P-glycoprotein. The inhibitory effect of these compounds appeared specific for the 40 pS chloride channel as a large, 300 pS chloride channel found in the same cells was unaffected by addition of drug. These results suggest that the 40 pS chloride channel may be associated with P-glycoprotein expression.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1996

Volume-activated chloride channels in neuroblastoma cells are blocked by the antiestrogen toremifene.

The presence of volume-activated chloride channels has been examined in neuroblastoma C1300 cells using the whole-cell configuration of the patch-clamp technique. Chloride channels could not be detected under isotonic conditions. However, hypotonic challenge induced slowly developed inward and outward anionic currents that exhibited outward rectification and inactivation at the most depolarizing potentials, features that were similar to the currents described in other cell preparations where volume-activated Cl- channels have been associated with the expression of P-glycoprotein. This hypotonicity-activated Cl- currents could be reversibly blocked by extracellular exposure to toremifene, a novel synthetic antioestrogen. The fact that toremifene and its analog tamoxifen, have been shown to block P-glycoprotein-associated chloride channels and to reverse P-glycoprotein associated multidrug resistance in a number of cell lines suggest that P-glycoprotein could be involved in the generation of hypotonic-induced chloride conductance in neuroblastoma cells.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1999

Localised fibre cell swelling characteristic of diabetic cataract can be induced in normal rat lens using the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid.

Chloride channels are known to be involved in the regulated volume decrease that occurs when the rat lens is exposed to hypotonic challenge. We now report that chloride channel blockage makes the rat lens gain water under isotonic conditions, suggesting that chloride and water fluxes may also play an important role under resting conditions. Histological comparison of hypotonically and isotonically swollen rat lenses revealed a significant difference: in the former, fibre cells were swollen from the periphery inwards, while in the latter, swollen fibre cells were confined to a discrete cortical zone which was located 150-200 micron from the lens surface with cells on either side of this zone appearing unaffected. This localised fibre cell swelling is remarkable because of its similarity to the situation in the diabetic rat lens.

Animals↗

PubMed2004

Inhibition of CLC-2 chloride channel expression interrupts expansion of fetal lung cysts.

Normal lung morphogenesis is dependent on chloride-driven fluid transport. The molecular identity of essential fetal lung chloride channel(s) has not been elucidated. CLC-2 is a chloride channel, which is expressed on the apical surface of the developing respiratory epithelium. CLC-2-like pH-dependent chloride secretion exists in fetal airway cells. We used a 14-day fetal rat lung submersion culture model to examine the role of CLC-2 in lung development. In this model, the excised fetal lung continues to grow, secrete fluid, and become progressively cystic in morphology (26). We inhibited CLC-2 expression in these explants, using antisense oligonucleotides, and found that lung cyst morphology was disrupted. In addition, transepithelial voltage (V(t)) of lung explants transfected with antisense CLC-2 was inhibited with V(t) = -1.5 +/- 0.2 mV (means + SE) compared with -3.7 +/- 0.3 mV (means + SE) for mock-transfected controls and -3.3 +/- 0.3 mV (means + SE) for nonsense oligodeoxynucleotide-transfected controls. This suggests that CLC-2 is important for fetal lung fluid production and that it may play a role in normal lung morphogenesis.

Animals↗

PubMed1996

Chloride channel blockers decrease intracellular pH in cultured renal epithelial LLC-PK1 cells.

The effects of chloride channel blockers upon intracellular pH (pHi) were examined in renal epithelial monolayers of LLC-PK1 cells. A significant intracellular acidification was found with addition of 100 microM 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), niflumic acid, flufenamate and diphenylamine-2-carboxylate (DPC) but not with 4,4'-diisothiocyanatostilbene-2-2'disulphonic acid (DIDS). The effects of these agents upon pHi was dose-dependent with apparent K0.5 values of: 16.7 +/- 0.3 microM, 34.2 +/- 0.9 microM and 740 +/- 13 microM for niflumic acid, flufenamate and DPC respectively. The results indicate that at concentrations commonly used to block channel activity these chloride channel blockers have profound effects upon pHi.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2003

Expression of voltage-gated chloride channels in human glioma cells.

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 microm) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (165 microm). ClC-2 antisense significantly reduced expression of inwardly rectifying currents, which were potentiated by hyperpolarizing prepulses and inhibited by Cd2+ (200-500 microm). 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.

Antibodies↗

PubMed1997

Actions of pyrethroid insecticides on voltage-gated chloride channels in neuroblastoma cells.

Pyrethroid insecticides have the potential to act at several sites in excitable tissues in addition to their primary effect on sodium channels. We have assessed one such site, the voltage gated chloride channel. Following our previous demonstration of actions of deltamethrin on the voltage-gated chloride channel in rats and in neuroblastoma cells, we examined the dose-response relationship for deltamethrin by patch clamp analysis of voltage-gated channels in partially differentiated NIE115 cells. Open channel probability (P0) was assessed in channels stepped from 0 to +20 mV for 10 seconds. Deltamethrin significantly decreased P0 by 0.237+/-0.070 at 10(-10) M and, although construction of a dose-response relationship was rendered difficult by the very low water solubility of the pyrethroids, a decrease of 0.968+/-0.140 was reached at the highest concentration tested, 10(-4) M. A second Type II pyrethroid cypermethrin also decreased P0, by 0.430+/-0.09 at 5 x 10(-6) M, but the Type I pyrethroid cismethrin produced a decrease of only 0.188+/-0.08 at 2 x 10(-5) M. These effects were seen in 340 pS conductance, calcium-independent channels. A sample of 9 calcium-dependent channels with a conductance of 225 pS failed to show any response to deltamethrin. We conclude that actions on calcium-independent voltage-gated chloride channels are likely to contribute significantly to type II pyrethroid toxicity.

Animals↗

PubMed1999

Chloride channels in renal disease.

Recent studies of hereditary renal tubular disorders have facilitated the identification and roles of chloride channels and cotransporters in the regulation of the most abundant anion, Cl-, in the ECF. Thus, mutations that result in a loss of function of the voltage-gated chloride channel, CLC-5, are associated with Dent's disease, which is characterized by low-molecular weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. Mutations of another voltage-gated chloride channel, CLC-Kb, are associated with a form of Bartter's syndrome, whereas other forms of Bartter's syndrome are caused by mutations in the bumetanide-sensitive sodium-potassium-chloride cotransporter (NKCC2) and the potassium channel, ROMK. Finally, mutations of the thiazide-sensitive sodium-chloride cotransporter (NCCT) are associated with Gitelman's syndrome. These studies have helped to elucidate some of the renal tubular mechanisms regulating mineral homeostasis and the role of chloride channels.

Amino Acid Sequence↗

PubMed1994

Nonsense and missense mutations in the muscular chloride channel gene Clc-1 of myotonic mice.

In mature vertebrate muscle, the chloride channel Clc-1 is necessary for the stabilization of the resting potential. Its functional defect leads to the disease myotonia. The ADR mouse (phenotype ADR, genotype adr/adr) is an animal model for human myotonias. The adr gene is a member of a family of non-complementing recessive autosomal mutations ("alleles" of adr) that cause myotonia in the mouse. The standard allele adr has arisen by the insertion of a retroposon into the chloride channel gene Clc-1 (Steinmeyer, K., Klocke, R., Ortland, C., Gronemeier, M., Jockusch, H., Gründer, S., and Jentsch, T. J. (1991) Nature 354, 304-308). In order to study the nature of two other alleles, adrmto and adrK, we have analyzed overlapping Clc-1 cDNA amplification products by the hydroxylamine and osmium tetroxide modification technique and direct sequencing. A comparison between ADR*MTO and C57BL/6 wild type showed six base pair substitutions, one of which resulted in a stop codon in position 47, whereas the five others are either silent or lead to amino acid substitutions in non-conserved regions of the Clc-1 sequence and were already present in the wild type inbred SWR/J strain from which adrmto was derived. The detection of the stop codon in the adrmto allele is further indication of the identity of the Clc-1 chloride channel with the adr myotonia gene in the mouse, because a chain termination close to the N terminus would necessarily destroy gene function. For the ethylnitrosourea-induced mutation adrK, an Ile-->Thr exchange in codon 553 was identified. As this affects a conserved residue within a highly conserved region of the Clc-1 gene, a functional significance of this residue is suggested.

Amino Acid Sequence↗

PubMed1992

Separation of drug transport and chloride channel functions of the human multidrug resistance P-glycoprotein.

The human multidrug resistance P-glycoprotein is an active transporter that pumps cytotoxic drugs out of cells. Expression of P-glycoprotein is also associated with a volume-activated chloride channel. Here we address the relationship between these two functions. Drug transport requires ATP hydrolysis while, in contrast, ATP binding is sufficient to enable activation of the chloride channel. The chloride channel and drug transport activities of P-glycoprotein appear to reflect two distinct functional states of the protein that can be interconverted by changes in tonicity. Transportable drugs prevent channel activation but have no effect on channel activity once it has been preactivated by hypotonicity. The transport and channel functions of P-glycoprotein have been separated by directed mutations in the nucleotide-binding domains of the protein. These data provide further evidence that P-glycoprotein is bifunctional with both transport and channel activities. Implications for the design of chemotherapeutic drugs and for the function of the related cystic fibrosis gene product, CFTR, are discussed.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1990

Benzodiazepine agonist and inverse agonist actions on GABAA receptor-operated chloride channels. II. Chronic effects of ethanol.

Mice were made tolerant to and dependent on ethanol by administration of a liquid diet. Gamma-aminobutyric acid (GABA) receptor-dependent uptake of 36Cl- by mouse cortical microsacs was used to study the actions of benzodiazepine (BZ) agonists and inverse agonists. Chronic exposure to ethanol attenuated the ability of a BZ agonist, flunitrazepam, to augment muscimol-stimulated uptake of 36Cl- and enhanced the actions of BZ inverse agonists, Ro15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,4]-benzodiazepine - 3-carboxylate) and DMCM (methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate), to inhibit GABAA receptor-operated chloride channels. Augmentation of chloride flux by pentobarbital was not reduced by chronic ethanol exposure. Attenuation of flunitrazepam efficacy was transient and returned to control levels within 6 to 24 hr after withdrawal from ethanol, but increased sensitivity to Ro15-4513 was observed as long as 8 days after withdrawal. Chronic exposure to ethanol did not alter [3H]SR 95531 ([2-(3'-carbethoxy-2'propyl)-3-amino-6-p-methoxyphenylpyridazinium bromide] binding to low-affinity GABAA receptors or muscimol stimulation of chloride flux; and did not alter [3H]Ro15-4513 or [3H]flunitrazepam binding to central BZ receptors or allosteric modulation of this binding by muscimol (i.e., muscimol-shift). These results suggest that chronic exposure to ethanol reduces coupling between BZ agonist sites and the chloride channel, and may be responsible for the development of cross-tolerance between ethanol and BZ agonists. In contrast, coupling between BZ inverse agonist sites and the chloride channel is increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Cloning and expression of a PKC-regulated chloride channel.

The cDNA of a protein kinase C-regulated chloride channel (CIC-3) was cloned. CIC-3 encodes 760 amino acids, which has significant amino acid identity with the previously cloned CIC chloride channels. CIC-3 cRNA elicited the chloride currents in Xenopus oocytes, which was completely blocked by activation of protein kinase C by 12-O-tetradecanoylphorbol 13-acetate (TPA). The most abundant expression of CIC-3 mRNA was observed in rat brain, and the moderate level of CIC-3 expression was also observed in various rat tissues including adrenal gland, kidney, and lung. These findings suggest that CIC-3 may have important roles in various types of cells with regulation by protein kinase C.

Animals↗

PubMed2005

Inhibition of ClC-2 chloride channels by a peptide component or components of scorpion venom.

ClC chloride channels play essential roles in membrane excitability and maintenance of osmotic balance. Despite the recent crystallization of two bacterial ClC-like proteins, the gating mechanism for these channels remains unclear. In this study we tested scorpion venom for the presence of novel peptide inhibitors of ClC channels, which might be useful tools for dissecting the mechanisms underlying ClC channel gating. Recently, it has been shown that a peptide component of venom from the scorpion L. quinquestriatus hebraeus inhibits the CFTR chloride channel from the intracellular side. Using two-electrode voltage clamp we studied the effect of scorpion venom on ClC-0, -1, and -2, and found both dose- and voltage-dependent inhibition only of ClC-2. Comparison of voltage-dependence of inhibition by venom to that of known pore blockers revealed opposite voltage dependencies, suggesting different mechanisms of inhibition. Kinetic data show that venom induced slower activation kinetics compared to pre-venom records, suggesting that the active component(s) of venom may function as a gating modifier at ClC-2. Trypsinization abolished the inhibitory activity of venom, suggesting that the component(s) of scorpion venom that inhibits ClC-2 is a peptide.

Animals↗

PubMed2001

Expression of chloride channel, ClC-5, and its role in receptor-mediated endocytosis of albumin in OK cells.

By using Western blot and RT-PCR analyses, the expression of ClC-5, a member of the ClC family of voltage-gated chloride channels, and its mRNA was detected in OK cells. The effect of chloride channel inhibitors on receptor-mediated endocytosis of albumin was examined in OK cells and compared to that of vacuolar H(+)-ATPase inhibitors. Accumulation of fluorescein-isothiocyanate (FITC)-albumin, a receptor-mediated endocytosis marker, was inhibited by 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), a chloride channel inhibitor, in a concentration-dependent fashion. In contrast, uptake of FITC-inulin, a fluid-phase endocytosis marker, was not affected by NPPB. Other chloride channel inhibitors, 4,4'-diisothiocyanatostilbene-2-2'-disulfonic acid and diphenylamine-2-carboxylic acid, also inhibited FITC-albumin uptake. NPPB, as well as a vacuolar H(+)-ATPase inhibitor bafilomycin A(1), caused a decrease in the affinity and in the maximal velocity of FITC-albumin uptake. These results suggest that chloride channel, most likely ClC-5, plays an important role in the receptor-mediated endocytosis of albumin in OK cells.

Albumins↗

PubMed1995

Gating of the voltage-dependent chloride channel CIC-0 by the permeant anion.

Chloride channels of the ClC family are important for the control of membrane excitability, cell volume regulation, and possibly transepithelial transport. Although lacking the typical voltage-sensor found in cation channels, gating of ClC channels is clearly voltage-dependent. For the prototype Torpedo channel ClC-0 (refs 11-15) we now show that channel opening is strongly facilitated by external chloride. Other less permeable anions can substitute for chloride with less efficiency. ClC-0 conductance shows an anomalous mole fraction behaviour with Cl-/NO3- mixtures, suggesting a multi-ion pore. Gating shows a similar anomalous behaviour, tightly linking permeation to gating. Eliminating a positive charge at the cytoplasmic end of domain D12 changes kinetics, concentration dependence and halide selectivity of gating, and alters pore properties such as ion selectivity, single-channel conductance and rectification. Taken together, our results strongly suggest that in these channels voltage-dependent gating is conferred by the permeating ion itself, acting as the gating charge.

Animals↗

PubMed2002

Photodegradation study of a new activator of the cystic fibrosis chloride channel, the 6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-07).

The photodegradation of 6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-07), a new activator of the transmembrane conductance regulator chloride channel, was studied in aqueous solutions exposed to artificial daylight (2300 Lux intensity). Various conditions of pH, concentration, and temperature were used. MPB-07 concentration was determined at regular time intervals by reversed-phase HPLC. MPB-07 stability was also studied at pH 7.4 in the dark. Results showed that in all the conditions tested MPB-07 underwent rapid photodegradation, apparently following first-order kinetics. Rate constants were dependent on the initial MPB-07 concentration, temperature, and pH. At pH 7.4, and for concentrations from 1 to 125 microM, half-lives ranged from 0.681 +/- 0.047 to 4.54 +/- 0.28 h. The Arrhenius plot was linear and activation energy was calculated to be 20.7 kJ x mol(-1). Analysis by chemical ionization-mass spectrometry showed that the chlorine atom of the MPB-07 molecule might be replaced by an OH group during the photodegradation process. In the dark, MPB-07 in solutions at pH 7.4 was found to be stable over a 6-week period. In conclusion, MPB-07 is a highly photolabile molecule that should be carefully protected from light when used.

Cystic Fibrosis Transmembrane Conductance Regulato↗

PubMed1985

The presence of voltage-gated sodium, potassium and chloride channels in rat cultured astrocytes.

Patch-clamp recording from the plasmalemma of rat cultured astrocytes reveals the presence of both voltage-dependent sodium and voltage-dependent potassium conductances. These conductances are similar but not identical to the corresponding conductances in the axolemma. Whereas the h infinity relation of the sodium channels has the same voltage dependence as in the nodal axolemma, the peak current-voltage relation is shifted by about 30 mV along the voltage axis in the depolarizing direction. It is speculated that the glial cells synthesize sodium and potassium channels for later insertion into the axolemma of neighbouring axons. The astrocytes also express a plasmalemmal voltage-dependent anion conductance that is turned on at about -40 mV (that is, near the resting potential of the cultured astrocytes). The channels involved are large enough to be just permeable to glutamate but not to ascorbate. It is suggested that the conductance of this channel for chloride plays a physiological role in the spatial buffering of potassium by glial cells.

Animals↗

PubMed2002

Mrp2 modulates the activity of chloride channels in isolated hepatocytes.

Adenosine triphosphate binding cassette family transport proteins are important organic ion transporters in hepatocytes but these molecules may also exhibit other functions. In the present study we have measured the effects of substrates of the canalicular organic ion transporter multidrug resistance associated protein 2 (Mrp2) on chloride channel activation and cell volume regulation. We found that substrates such as leukotriene D(4), 17-beta-estradiol glucuronide, and the leukotriene inhibitor MK-571 accelerated the activation of chloride channels by cell swelling and activated chloride channels in cytokine-pretreated hepatocytes. Two conjugated estrogens that are not Mrp2 substrates did not produce this effect. Hepatocytes derived from a strain of transport-deficient rats (TR(-)), which lack Mrp2 expression, showed none of these substrate effects. Coincident with their ability to activate channels, the Mrp2 substrates increased the rate of volume regulatory decrease by approximately 50% (P <.01), confirming that enhanced channel activation under this condition stimulated volume regulation. In TR-hepatocytes the Mrp2 substrate had no effect on volume regulation. In conclusion, Mrp2 plays a role in regulation of chloride channel function by reducing the lag time necessary for channel activation and consequently accelerating the process of cell volume regulation. Substrates of Mrp2 affect the ability of the protein to interact with chloride channels. These findings represent an alternative function of Mrp2 in hepatocytes.

Animals↗