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PubMed1997

Chloride channels in the luminal membrane of rat pancreatic acini.

Pancreatic acini secret Na+, Cl- and H2O in response to secretagogues such as acetylcholine. Cl- channels in the luminal membrane are a prerequisite for this secretion. The properties of the corresponding conductance have previously been examined using whole-cell recordings. The present study attempts to examine the properties of the single channels in cell-attached and cell-free excised patches from the luminal membrane. To this end the pipettes were filled with an N-methyl-D-glucamine (NMDG+) chloride-gluconate solution. The voltage-clamp range was chosen to be pipette positive (cell negative, -60 to -130 mV) in order to increase the driving force for outward Cl- currents. Under resting conditions cell attached luminal patches had very few single-channel currents (12 out of 45 experiments). Their incidence was sharply increased by carbachol (CCH, 1 micromol/l) in 41 out of 45 experiments. The single-channel conductance of these channels was 1.97 +/- 0.05 pS. The properties of these channels in excised patches were examined further: their single-channel conductance was 2.2 +/- 0.07 pS (n = 59) and their conductance selectivity was I- > Br- > Cl- >> gluconate. None of the typical Cl- channel blockers (DIDS, NPPB, glibenclamide 100 micromol/l) blocked these channels. It is concluded that the luminal membrane of the rat pancreatic acinus possesses Cl- channels with very low conductance which are activated by carbachol.

Animals↗
PubMed2001

Synthetic chloride channel restores glutathione secretion in cystic fibrosis airway epithelia.

Cystic fibrosis (CF), an inherited disease characterized by defective epithelial Cl- transport, damages lungs via chronic inflammation and oxidative stress. Glutathione, a major antioxidant in the epithelial lung lining fluid, is decreased in the apical fluid of CF airway epithelia due to reduced glutathione efflux (Gao L, Kim KJ, Yankaskas JR, and Forman HJ. Am J Physiol Lung Cell Mol Physiol 277: L113-L118, 1999). The present study examined the question of whether restoration of chloride transport would also restore glutathione secretion. We found that a Cl- channel-forming peptide (N-K4-M2GlyR) and a K+ channel activator (chlorzoxazone) increased Cl- secretion, measured as bumetanide-sensitive short-circuit current, and glutathione efflux, measured by high-performance liquid chromatography, in a human CF airway epithelial cell line (CFT1). Addition of the peptide alone increased glutathione secretion (181 +/- 8% of the control value), whereas chlorzoxazone alone did not significantly affect glutathione efflux; however, chlorzoxazone potentiated the effect of the peptide on glutathione release (359 +/- 16% of the control value). These studies demonstrate that glutathione efflux is associated with apical chloride secretion, not with the CF transmembrane conductance regulator per se, and the defect of glutathione efflux in CF can be overcome pharmacologically.

Cell Line, Transformed↗
PubMed2000

Inhibition of volume-regulated and calcium-activated chloride channels by the antimalarial mefloquine.

We have used the whole-cell patch-clamp technique to study the effect of mefloquine (Lariam), a commonly used antimalarial drug, on the volume-regulated anion channel (VRAC) in cultured bovine pulmonary artery endothelial cells. We also examined its effects on other Cl(-) channels, i.e., the Ca(2+)-activated Cl(-) channel and the cystic fibrosis transmembrane conductance regulator, to assess the specificity of this compound for VRAC. At pH 7.4 mefloquine induced a fast and reversible block of the volume-sensitive chloride current (I(Cl,swell)), with an IC(50) value of 1.19 +/- 0.07 microM. The blocking efficiency increased with increasing extracellular pH (IC(50) value for pH 8.8 was 0.15 +/- 0.01 microM), indicating that this effect is mediated by the uncharged form of mefloquine. Ca(2+)-activated Cl(-) currents, I(Cl,Ca), activated by loading T84 cells via the patch pipette with 1 microM free Ca(2+) also were inhibited by mefloquine (IC(50) value 3.01 +/- 0.17 microM at pH 7.4). The cystic fibrosis transmembrane conductance regulator channel, transiently transfected in cultured bovine pulmonary artery endothelial cells, was not affected by 10 microM of the drug. This study describes for the first time effects of mefloquine on anion channels. Our data reveal a potent block of VRAC and Ca(2+)-activated Cl(-) channel at therapeutic concentrations. These results may contribute to a better understanding of the actions and side effects of this widely used antimalarial drug.

Animals↗
PubMed1988

Ca2+-mediated suppression of the GABA-response through modulation of chloride channel gating in frog sensory neurones.

A suppressant effect of intracellular free Ca2+ on the gamma-aminobutyric acid (GABA)-induced chloride inward current (ICI(GABA)) was studied in isolated frog sensory neurones under whole cell voltage clamp. Voltage-dependent Ca2+ influx elicited during the steady state of ICI(GABA)-induced a fast, slowly recovering current relaxation in the outward direction, the amplitude of which was dependent on total Ca2+ influx. This suppressant effect showed specificity for different divalent cations, suggesting action at a specific intracellular effector. Single channel recording revealed a Ca2+-dependent decrease in the duration of the open time of the GABA-gated Cl- channel without change in single channel conductance.

Animals↗
PubMed1987

A high-conductance calcium-dependent chloride channel in Ascaris suum muscle.

The properties of a high-conductance Ca-dependent Cl channel in the body muscle membrane of the nematode parasite Ascaris suum were examined with the patch-clamp technique. In Ascaris Ringer solution the current-voltage relationships of the channel appeared linear during hyperpolarization with a slope conductance of 200 pS (in symmetrical 175 mM-Cl): during depolarization the slope conductance reduced. Cation and anion replacement experiments showed this channel to be permeable to Cl ions. In artificial perienteric fluid (in symmetrical 78 mM-Cl) the channel conductance was found to be 114 pS. Subconductance states were seen when the patch was depolarized. The channel conductance showed saturation with increasing Cl concentrations. The channel was voltage sensitive: the probability of the channel being open and the mean open time of the channel reduced when the patch was depolarized. Mean channel open times between 2.4 and 215 ms were observed. Frequency histograms of the distributions of open and closed times were fitted with two and three exponentials respectively; this suggests complex channel kinetics with multiple open and closed states. Ca applied to the intracellular surface of the patch increased in a dose-dependent manner the number and probability of the channels being open. These channels were seen in about 20% of all patches and occasionally up to 15 channels were seen in a single patch. The distributions of the probabilities of seeing N channels open in multichannel patch records were not not always well fitted by the binomial distribution: it is suggested that adjacent channels could have different probabilities of being open. The possible functions of this channel are discussed.

Animals↗
PubMed1984

gamma-Aminobutyric acid-gated chloride channels in cultured cerebral neurons.

gamma-Aminobutyric acid (GABA), the most common inhibitory neurotransmitter in the vertebrate brain, acts by increasing the conductance of the neuronal membrane to chloride ions. The addition of GABA to monolayer cultures of chick cerebral neurons produced a 3-fold increase in the uptake of 36Cl-. This stimulation was maximal during the first 20 s after GABA addition but declined rapidly thereafter. The GABA-dependent uptake activity was doubled by increasing the external K+ concentration from 5.5 to 40 mM. The dependence of the 36Cl- entry rate on the external concentrations of GABA (K0.5 = 6 microM; Vmax = 4.4 nmol/mg of cell protein/s) and Cl-(Km = 105 mM; Vmax = 9.9 nmol mg-1 s-1) followed Michaelis-Menten kinetics. The GABA analog, muscimol, produced a similar response (K0.5 = 8 microM; Vmax = 5.2 nmol mg-1 s-1). While 50 microM 3-aminopropane sulfonate also stimulated 36Cl- uptake, 2,4-diaminobutyrate, taurine, and glycine were without effect. Bicuculline (Ki = 3.5 microM) was a noncompetitive inhibitor of GABA-dependent Cl- entry, while the inhibition by picrotoxin (Ki = 1.0 microM) was uncompetitive with GABA. Nearly one-half of the basal activity, observed in the absence of GABA, was blocked by the anion transport inhibitors, furosemide or 4-acetamido-4'-isothiocyano-2,2'-stilbene sulfonate, but these compounds gave no significant inhibition of the GABA-dependent activity. These results indicate that the basal route for 36Cl- entry into cerebral neurons involves electroneutral processes while the GABA-dependent influx occurs via specific ligand-gated Cl-channels.

Animals↗
PubMed1997

Localized changes in GABA receptor-gated chloride channel in rat brain after long-term haloperidol: relation to vacuous chewing movements.

Several lines of evidence suggest that the GABAergic system may be involved in dyskinetic side effects of long-term neuroleptic treatment. In this study, [35S]TBPS autoradiography was used to investigate changes in the GABA receptor-gated chloride ionophore in rats showing vacuous chewing movements (VCMs) after 21 weeks of treatment with haloperidol decanoate (HAL). A significant decrease in [35S]TBPS binding was observed in the globus pallidus of HAL-treated rats, compared to vehicle-treated controls (+23%, P < 0.001). However, this was equally observed in rats showing high VCM levels (> 70 counts/5 min) and those showing low VCM levels (< 30 counts/5 min), suggesting that this pallidal change cannot account for the differential development of VCMs after long-term HAL. In contrast, a small but significant increase in [35S]TBPS binding in the ventrolateral caudate-putamen was seen in animals in the high VCM group (+16%, P < 0.03), but not in those in the low VCM group, when compared to vehicle-treated controls. No significant alterations were found in other basal ganglia regions and related structures, including the substantia nigra, subthalamic nucleus, entopeduncular nucleus, and thalamic nuclei. The results are consistent with the notion of altered striatopallidal output as a result of chronic HAL treatment. They also suggest the possibility that alterations in GABA receptor-linked Cl channels in the ventrolateral caudate-putamen may contribute to the development of dyskinetic syndromes after long-term neuroleptic treatment.

Animals↗
PubMed2002

BN52021, a platelet activating factor antagonist, is a selective blocker of glycine-gated chloride channel.

We have found that the platelet activating factor antagonist (BN52021) is an effective blocker of the glycine (Gly) receptor-mediated responses in the hippocampal pyramidal neurons of rat. Using the whole-cell voltage clamp and concentration clamp recording techniques, we investigated the mechanism underlying the inhibitory action of this terpenoid on the glycine-induced chloride current. BN52021 selectively and reversibly inhibits glycine current in a non-competitive and voltage-dependent fashion. The antagonistic effect of this substance is more pronounced at positive membrane potentials. At holding potential -70mV and in the presence of 200 microM glycine IC50 value for the blocking action of BN52021 was 270+/-10nM. Repetitive applications of BN52021 reveal the use-dependence of its blocking action. When co-applied with strychnine (STR), a competitive glycine receptor antagonist, BN52021 does not alter the IC50 value for strychnine. The inhibitory effect of BN52021 on gamma-aminobutyric acid (GABA) current is at least 25 times less potent than the effect on glycine current. This substance fails to affect AMPA and NMDA responses. It may be concluded that BN52021 inhibits glycine-gated Cl- channels by interacting with the pore region and does not compete for the strychnine-binding centre.

Animals↗
PubMed2002

Amelioration of myocardial global ischemia/reperfusion injury with volume-regulatory chloride channel inhibitors in vivo.

BACKGROUND: Recently, the apoptotic volume decrease was suggested to be regulated by volume regulatory Cl- channels in cultured cell lines. We thus examined whether inhibition of volume-regulatory Cl- channels is cardioprotective, like caspase inhibition, by hindering the apoptosis of cardiomyocytes induced by global ischemia/reperfusion (I/R) in vivo. METHODS: We performed global ischemia for 8 min at 37 degrees C or 4 degrees C in isolated rat hearts, followed by 24-hr reperfusion via heterotopic heart transplantation. The heart tissue was examined by means of the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method, genomic DNA electrophoresis, and caspase-3 activity. Two blockers of volume-regulatory Cl- channels, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), and a broad-spectrum caspase inhibitor, benzoyloxycarbonyl-Asp-CH2OC(O)-2,6-dichlorobenzene (Z-Asp-DCB), were administered intravenously. Triphenyltetrazolium chloride (TTC) staining and ultrasound cardiography were performed to examine myocardial viability. The TTC-unstained region was assessed by means of horseradish peroxidase (HRP) infiltration and the TUNEL method. RESULTS: The transplanted hearts showed TUNEL-positivity and DNA laddering with a peak at 24 hr during reperfusion after ischemia at 37 degrees C, but not at 4 degrees C. NPPB and DIDS were as potent as Z-Asp-DCB for recovery of cardiac function and for blocking the appearance of TUNEL-positivity, DNA laddering, caspase 3 activity, and a TTC-unstained area. TTC-unstained areas were composed of either TUNEL- and slightly HRP-positive or TUNEL-negative and strongly HRP-positive cardiomyocytes. CONCLUSION: The present results demonstrated that myocardial DNA fragmentation, caspase activation, and loss of cardiac function after global I/R were blocked by NPPB and DIDS, similar to in the case of Z-Asp-DCB. These results suggest that inhibition of volume-regulatory Cl- channels is also effective for preventing cardiac I/R injury.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1990

A chloride channel from lobster walking leg nerves. Characterization of single-channel properties in planar bilayers.

A novel, small conductance of Cl- channel was characterized by incorporation into planar bilayers from a plasma membrane preparation of lobster walking leg nerves. Under conditions of symmetrical 100 mM NaCl, 10 mM Tris-HCl, pH 7.4, single Cl- channels exhibit rectifying current-voltage (I-V) behavior with a conductance of 19.2 +/- 0.8 pS at positive voltages and 15.1 +/- 1.6 pS in the voltage range of -40 to 0 mV. The channel exhibits a negligible permeability for Na+ compared with Cl- and displays the following sequence of anion permeability relative to Cl- as measured under near bi-ionic conditions: I- (2.7) greater than NO3- (1.8) greater than Br- (1.5) greater than Cl- (1.0) greater than CH3CO2- (0.18) greater than HCO3- (0.10) greater than gluconate (0.06) greater than F- (0.05). The unitary conductance saturates with increasing Cl- concentration in a Michaelis-Menten fashion with a Km of 100 mM and gamma max = 33 pS at positive voltage. The I-V curve is similar in 10 mM Tris or 10 mM HEPES buffer, but substitution of 100 mM NaCl with 100 mM tetraethylammonium chloride on the cis side results in increased rectification with a 40% reduction in current at negative voltages. The gating of the channel is weakly voltage dependent with an open-state probability of 0.23 at -75 mV and 0.64 at +75 mV. Channel gating is sensitive to cis pH with an increased opening probability observed for a pH change of 7.4 to 11 and nearly complete inhibition for a pH change of 7.4 to 6.0. The lobster Cl- channel is reversibly blocked by the anion transport inhibitors, SITS (4-acetamido, 4'-isothiocyanostilbene-2,2'-disulfonic acid) and NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid). Many of these characteristics are similar to those previously described for small conductance Cl- channels in various vertebrate cells, including epithelia. These functional comparisons suggest that this invertebrate Cl- channel is an evolutionary prototype of a widely distributed class of small conductance anion channels.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed2004

P2Y purinergic receptor regulation of CFTR chloride channels in mouse cardiac myocytes.

The intracellular signalling pathways and molecular mechanisms responsible for P2-purinoceptor-mediated chloride (Cl(-)) currents (I(Cl,ATP)) were studied in mouse ventricular myocytes. In standard NaCl-containing extracellular solutions, extracellular ATP (100 microm) activated two different currents, I(Cl,ATP) with a linear I-V relationship in symmetrical Cl(-) solutions, and an inwardly rectifying cation conductance (cationic I(ATP)). Cationic I(ATP) was selectively inhibited by Gd(3+) and Zn(2+), or by replacement of extracellular NaCl by NMDG; I(Cl,ATP) was Cl(-) selective, and inhibited by replacement of extracellular Cl(-) by Asp(-); both currents were prevented by suramin or DIDS pretreatment. In GTPgammaS-loaded cells, I(Cl,ATP) was irreversibly activated by ATP, but cationic I(ATP) was still regulated reversibly. GDPbetaS prevented activation of the I(Cl,ATP,) even though pertussis toxin pretreatment did not modulate I(Cl,ATP). These results suggest that activation of I(Cl,ATP) occurs via a G-protein coupled P2Y purinergic receptor. The I(Cl,ATP) persistently activated by GTPgammaS, was inhibited by glibenclamide but not by DIDS, thus exhibiting known pharmacological properties of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels. In ventricular cells of cftr(-/-) mice, extracellular ATP activated cationic I(ATP), but failed to activate any detectable I(Cl,ATP). These results provide compelling evidence that activation of CFTR Cl(-) channels in mouse heart are coupled to G-protein coupled P2Y purinergic receptors.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2005

A novel chloride channel in Drosophila melanogaster is inhibited by protons.

A systematic analysis of the Drosophila genome data reveals the existence of pHCl, a novel member of ligand-gated ion channel subunits. pHCl shows nearly identical similarity to glutamate-, glycine-, and histamine-gated ion channels, does however not belong to any of these ion channel types. We identified three different sites, where splicing generates multiple transcripts of the pHCl mRNA. The pHCl is expressed in Drosophila embryo, larvae, pupae, and the adult fly. In embryos, in situ hybridization detected pHCl in the neural cord and the hindgut. Functional expression of the three different splice variants of pHCl in oocytes of Xenopus laevis and Sf9 cells induces a chloride current with a linear current-voltage relationship that is inhibited by extracellular protons and activated by avermectins in a pH-dependent manner. Further, currents through pHCl channels were induced by a raise in temperature. Our data give genetic and electrophysiological evidence that pHCl is a member of a new branch of ligand-gated ion channels in invertebrates with, however, a hitherto unique combination of pharmacological and biophysical properties.

Alternative Splicing↗
PubMed1992

Chloride channels in cultured human skeletal muscle are regulated by G proteins.

The regulation of Cl- channels in human myoballs by G proteins was studied using whole-cell and inside-out patch recordings. After perfusion of the cell with 0.1 mM GTP[gamma S], the specific Cl- conductance, GCl, at standard resting potential (-85 mV) was increased from 5.9 microS/cm2 to 103 microS/cm2, and the kinetics upon stepping the potential to positive values was changed from an activating current with very slow inactivation to a fast inactivating current with no potential-dependent activation. These effects were not affected by the simultaneous blockade of several signal cascades involving G proteins. Addition of the protein kinase blockers PKI (25 microM), H8 (10 microM), or of the phospholipase-A2-blocking agent quinacrine (10 microM), had not much influence on these GTP[gamma S] effects. Buffering of the intracellular Ca2+ concentration (0.1 microM) or addition of the Ca2+/calmodulin antagonist trifluoperazine (50 microM) was also without effect. Pre-incubation of the cells with pertussis toxin or with cholera toxin did not change GCl. In excised inside-out patches voltage-clamped at -85 mV, application of GTP[gamma S] influenced the "intermediate" Cl- channel, the Cl- channel type having the highest density in these cells, by increasing the number of transitions in a half-conductance state. The probability of the channel being in one of the two conducting states rose from 0.015 to 0.67, and the kinetics of the single-channel currents was changed so that, on average, it was similar to the whole-cell current kinetics seen after application of GTP[gamma S]. It is concluded that a G protein is directly interacting with these channels.

Chlorides↗
PubMed1992

Effect of thiol group modification on ion flux and ligand binding properties of the GABAA-benzodiazepine receptor chloride channel complex.

Agents that modify thiol groups have been shown to alter ligand binding at a variety of receptor sites. In addition, alkylation of sulfhydryls has been shown to block ion channel conductance. We studied the effects of thiol reagents on gamma-aminobutyric acid (GABA)-activated chloride flux (36Cl-) and [3H]-diazepam binding in mouse brain membrane preparation (microsacs). Incubation of microsacs in the presence of: mercuric chloride (HgCl2), p-chloromercuriphenylsulfonic acid (pCMBS), hydroxymercuribenzoate (HMB), n-ethylmaleimide (NEM), or iodoacetic acid (IAA) attenuated GABA-stimulated Cl- uptake. The thiol reagents reduced both maximal stimulation and the potency of GABA to induce Cl- uptake. Thiol reagent treatment decreased the affinity of high-affinity [3H]-muscimol equilibrium binding. Supernatant prepared from microsacs treated with pCMBS stimulated Cl- uptake in the absence of GABA agonist in microsacs unexposed to thiol reagents. The supernatant taken from pCMBS-treated microsacs also stimulated [3H]-diazepam binding. This effect was blocked by the addition of the GABA receptor antagonist bicuculline. The concentration of endogenous GABA in supernatant from pCMBS-treated microsacs was sixfold greater than that in supernatant from control microsacs. This increase in levels of endogenous GABA by thiol reagents was due to both an increase in GABA release and a decrease in high-affinity GABA uptake.

Animals↗
PubMed1996

A novel chloride channel in Vicia faba guard cell vacuoles activated by the serine/threonine kinase, CDPK.

Calcium-Dependent Protein Kinases (CDPKs) in higher plants contain a C-terminal calmodulin-like regulatory domain. Little is known regarding physiological CDPK targets. Both kinase activity and multiple Ca2+-dependent signaling pathways have been implicated in the control of stomatal guard cell movements. To determine whether CDPK or other protein kinases could have a role in guard cell signaling, purified and recombinant kinases were applied to Vicia faba guard cell vacuoles during patch-clamp experiments. CDPK activated novel vacuolar chloride (VCL) and malate conductances in guard cells. Activation was dependent on both Ca2+ and ATP. Furthermore, VCL activation occurred in the absence of Ca2+ using a Ca2+-independent, constitutively active, CDPK* mutant. Protein kinase A showed weaker activation (22% as compared with CDPK). Current reversals in whole vacuole recordings shifted with the Nernst potential for Cl-and vanished in glutamate. Single channel recordings showed a CDPK-activated 34 +/- 5 pS Cl- channel. VCL channels were activated at physiological potentials enabling Cl- uptake into vacuoles. VCL channels may provide a previously unidentified, but necessary, pathway for anion uptake into vacuoles required for stomatal opening. CDPK-activated VCL currents were also observed in red beet vacuoles suggesting that these channels may provide a more general mechanism for kinase-dependent anion uptake.

Adenosine Triphosphate↗