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PubMed1990

Calcium-dependent regulation of airway epithelial chloride channels.

To determine how cell calcium ([Ca2+]c) regulates apical Cl- channels, we measured the rate of 125-Iodide (125I-) efflux to assay Cl- channel activity in intact cells and examined cell-free membrane patches from cultured canine tracheal epithelial cells. The Ca2+ elevating agonist bradykinin and the calcium ionophore A23187 increased 125I- efflux. This response did not require prostaglandin production. Under several conditions, changes in [Ca2+]c were temporally dissociated from changes in channel activation: a transient increase in [Ca2+]c caused a prolonged stimulation of 125I- efflux. Neither Cl- channel activation nor open-channel probability was affected by varying internal [Ca2+] in excised membrane patches. Adenosine 3',5'-cyclic monophosphate (cAMP)- and Ca2(+)-dependent channel activation may be independent: cAMP-stimulated 125I- efflux did not require an increase in [Ca2+]c, Ca2(+)-stimulated efflux did not require an increase in cAMP, and simultaneous addition of A23187 and isoproterenol produced additive effects on 125I- efflux. The data suggest that an increase in [Ca2+]c activates Cl- channels, however, the effect of Ca2+ appears to be indirect, not involving a ligand-type interaction with the channel.

Animals↗
PubMed1990

Double-barreled chloride channels of collecting duct basolateral membrane.

Microelectrode studies have shown that the basolateral membrane of the principal cells (PC) of the rabbit cortical collecting duct (CCD) contains Cl(-)-conductive pathways. To determine the properties of single Cl- channels we prepared the basolateral membrane for patch clamping by incubating the CCD in collagenase and/or tearing the basement membrane with a fine needle. When high concentrations of collagenase were used, only a small nonselective channel was observed. In low concentrations or the absence of collagenase, however, we identified a Cl- channel (g46) in both cell-attached and excised patches. The Cl- channel gated rapidly between three equally spaced substates of 0 (S0), 23 (S1), and 46 pS (S2) and slowly between states C (inactive) and S0. The conductance of each substate was not voltage dependent between pipette potentials from -60 to +60 mV (cell attached). The probability that the channel gated from C to S0 increased with hyperpolarizing potentials, but the probability that g46 was in substate S0 increased with depolarizing patch potentials. This channel was similar to that described by Miller for the nonexcitable membrane of the electric organ of Torpedo californica. Because g46 was the most frequently observed basolateral membrane channel and was voltage dependent at physiological potentials, it is probably the channel responsible for the dominant Cl- conductance of PC.

Animals↗
PubMed2004

The structural basis of ClC chloride channel function.

ClC channels constitute a large family of Cl(-)-selective ion channels that are present in diverse organisms. The channels have a complex gating behavior, in which channel opening and closing is tightly coupled to ion permeation. Recent success in the structure determination of bacterial channels has revealed the overall architecture of this family and provided important insight into ion selectivity and gating. The ClC channels are homodimers, with each subunit containing an ion conduction pore. In the narrow selectivity filter of each pore, Cl(-) are bound to several sites through electrostatic interactions with helix dipoles and the partial charges of protein residues. In the closed conformation of the channel, a conserved glutamate residue occupies one of the ion-binding sites; on channel opening, this glutamate residue moves out of the binding site, thereby making room for an additional Cl(-) ion.

Amino Acid Sequence↗
PubMed1989

The pharmacological properties of GABA receptor-coupled chloride channels using 36Cl-influx in cultured spinal cord neurons.

gamma-Aminobutyric acid (GABA) synaptic pharmacology was studied in cultured mouse spinal cord using various biochemical techniques. The intact cultures were used to study GABA uptake, benzodiazepine (BZ) binding and GABA-stimulated 36Cl-influx, all under similar physiological conditions. The cultures appear to contain a population of GABAergic neurons, since there was evidence of neuronal active transport of [3H]GABA by these cells. There also was a significant population of neuronal BZ receptors based on the finding of clonazepam-displaceable [3H]flunitrazepam (FLU) binding. In addition, the presence of GABA receptors coupled to the BZ receptors was shown by the enhancement of FLU binding by GABA (10-100 microM). GABAA and glycine-coupled Cl- channels were also demonstrated using 36Cl-influx. GABA-induced 36Cl-influx was specific for GABAA agonists, since GABAB receptor agonist, baclofen and other excitatory neurotransmitters did not alter the 36Cl-influx. Both GABA (Km = 9.1 microM) and muscimol (Km = 2.0 microM) produced concentration-dependent increases in 36Cl-influx in the cultures. The GABA-stimulated Cl-influx was blocked by the GABAA receptor antagonist (+)bicuculline (IC50 = 4.5 microM) and by the Cl- channel antagonist picrotoxinin (IC50 = 25 microM). These results indicate that spinal cord cultures are a useful model for comparing the binding characteristics of the GABA receptor complex to the activation of the coupled Cl- channels using the 36Cl-influx assay.

Animals↗
PubMed1992

Chloride channels in apical and basolateral membranes of CCD cells (RCCT-28A) in culture.

Previously, we found that isoproterenol activates whole cell Cl- conductance by a pathway involving adenosine 3',5'-cyclic monophosphate and protein kinase A (PKA) in a renal cell line (RCCT-28A) derived from the cortical collecting duct. The goal of the present study was to determine whether PKA activates Cl- channels in the apical and/or basolateral membrane. Using the patch clamp technique we found a 305-pS Cl- channel, described previously (22), located exclusively in the apical membrane and an outwardly rectifying Cl- channel (13/96 pS) located exclusively in the basolateral membrane. The outward rectifier was highly selective to Cl- versus cations, was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and 5-nitro-2-(3-phenylpropylamino)-benzoic acid, but was not regulated by cytoplasmic pH or Ca2+. Neither isoproterenol nor PKA activated the 305-pS Cl- channel. In contrast, PKA activated a subset of outwardly rectifying channels in inside-out patches. In another subset of outwardly rectifying channels, formation of the inside-out configuration increased channel activity. These channels, however, were not sensitive to PKA. In conclusion, these experiments show that isoproterenol increases the Cl- conductance of RCCT-28A cells by activating a subset of outwardly rectifying Cl- channels located in the basolateral membrane.

Animals↗
PubMed2005

ClC-5: a chloride channel with multiple roles in renal tubular albumin uptake.

ClC-5 is a chloride (Cl(-)) channel expressed in renal tubules and is critical for normal tubular function. Loss of function nonsense or missense mutations in ClC-5 are associated with Dent's disease, a condition in which patients present with low molecular weight (LMW) proteinuria (including albuminuria), hypercalciuria and nephrolithiasis. Several key studies in ClC-5 knockout mice have shown that the proteinuria results from defective tubular reabsorption of proteins. ClC-5 is typically regarded as an intracellular Cl(-) channel and thus the defect in this receptor-mediated uptake pathway was initially attributed to the failure of the early endosomes to acidify correctly. ClC-5 was postulated to play a key role in transporting the Cl(-) ions required to compensate for the movement of H(+) during endosomal acidification. However, more recent studies suggest additional roles for ClC-5 in the endocytosis of albumin. ClC-5 is now known to be expressed at low levels at the cell surface and appears to be a key component in the assembly of the macromolecular complex involved in protein endocytosis. Furthermore, mutations in ClC-5 affect the trafficking of v-H(+)-ATPase and result in decreased expression of the albumin receptor megalin/cubulin. Thus, the expression of ClC-5 at the cell surface as well as its presence in endosomes appears to be essential for normal protein uptake by the renal proximal tubule.

Albumins↗
PubMed2003

A conserved pore-lining glutamate as a voltage- and chloride-dependent gate in the ClC-2 chloride channel.

ClC-2 is a ubiquitously expressed, two-pore homodimeric Cl- channel opened by hyperpolarisation. Little is known about its gating mechanisms. Crystallographic and functional studies in other ClC channels suggest that a conserved glutamate residue carboxylate side-chain can close protopores by interacting with a Cl--binding site in the pore. Competition for this site is thought to provide the molecular basis for gating by extracellular Cl-. We now show that ClC-2 gating depends upon intra- but not extracellular Cl- and that neutralisation of E217, the homologous pore glutamate, leads to loss of sensitivity to intracellular Cl- and voltage. Experiments testing for transient activation by extracellular protons demonstrate that E217 is not available for protonation in the closed channel state but becomes so after opening by hyperpolarisation. The results suggest that E217 is a hyperpolarisation-dependent protopore gate in ClC-2 and that access of intracellular Cl- to a site normally occupied by its side-chain in the pore stabilises the open state. A remaining hyperpolarisation-dependent gate might correspond to that closing both pores simultaneously in other ClC channels.

Amino Acid Substitution↗
PubMed1991

Chloride channel regulation in the skeletal muscle of normal and myotonic goats.

External intercostal muscle biopsies from normal and congenitally myotonic goats were studied in vitro at 30 degrees C using a two-microelectrode square-pulse cable analysis assisted by computer. The resting chloride conductance (Gcl) was estimated from the difference between the mean membrane conductance in chloride-containing and chloride-free bathing media. The protein kinase C (PKC) activator, 4-beta-phorbol-12,13-dibutyrate. (0.1-2.0 microM) blocks a maximum of 76% of Gcl in normal goat fibers and induces myotonic hyperexcitability similar to that of congenitally myotonic goat fibers. The Gcl block was partially antagonized by pretreatment with the PKC inhibitor, staurosporine (10 microM). The "inactive" 4-alpha-phorbol-12,13-didecanoate had no effect at 50 microM, whereas the "active" 4-beta isomer blocked 41% Gcl at 1 microM. The nearly absent Gcl of congenitally myotonic goat fibers was not restored by treatment with high concentrations of the PKC inhibitors staurosporine, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), or tetrahydropapaveralone (THP). Also, forskolin and cholera toxin, which may increase cyclic adenosine monophosphate (cAMP) levels, or the R(+) clofibric acid enantiomers and taurine, which increase Gcl in normal fibers, were also unable to restore Gcl in myotonic goat fibers. The data suggest that PKC may be a chloride channel regulator in normal goat skeletal muscle fibers, however the molecular defect of congenitally myotonic fiber does not appear to be due to excessive activity of PKC.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1988

Does beta-alanine activate more than one chloride channel associated receptor?

In order to define which receptor-channel complexes are activated by beta-alanine, we performed cross-desensitization experiments between this amino acid and the classical inhibitory neurotransmitters, glycine and gamma-aminobutyric acid (GABA), all of which increase chloride conductances. Using the whole-cell patch-clamp technique and cultured chick spinal neurons, we found that beta-alanine completely desensitizes the glycine response, and only partially reduces GABA evoked currents. No interaction occurred between GABA and glycine at the level of their respective receptors. Conversely, both GABA and glycine reduced the beta-alanine response. These results indicate that beta-alanine activates the glycine receptor, and also suggest that it could be a partial agonist of GABAA receptor(s).

Alanine↗
PubMed1993

Volume-activated chloride channels in HeLa cells are blocked by verapamil and dideoxyforskolin.

The possible role of Cl- currents in regulatory volume decrease processes has been explored in HeLa cells using the whole-cell recording mode of the patch-clamp technique. Cells showed very small currents in voltage-clamp experiments performed with Cl(-)-rich, permeant-cation-free (N-methyl-D-glucamine replacement) intracellular and bathing solutions. Exposure of the cells to hypotonic solutions visibly swelled the cells and activated, reversibly, an outward rectifying Cl- current, which decayed at the most depolarised voltages used. Replacement of extracellular Cl- by a series of halide anions, SCN- and gluconate was consistent with an anion selectivity sequence: SCN- > I- > Br- > Cl- > F- > gluconate. The volume-regulated Cl- current was effectively inhibited by 100 microM 5-nitro-2-(3-phenyl-propylamino)-benzoic acid and by 100 microM 4,4'-diisothiocyanotostilbene-2,2-disulphonic acid, substances known to block Cl- channels in a variety of cells. Chloride current activation by hypotonicity was dependent on the presence of ATP in the intracellular solution and this requirement could be replaced by the non-hydrolysable analogue ATP[gamma S] and Mg(2+)-free ATP. The data suggest that the channels responsible for the current described are involved in the regulatory volume decrease in HeLa cells. The characteristics of this Cl- current are similar to those of the current associated with expression of multidrug resistance P-glycoprotein. Furthermore, the currents in HeLa cells were inhibited rapidly and reversibly by verapamil and 1,9-dideoxyforskolin, which are known to inhibit P-glycoprotein function.

Adenosine Triphosphate↗
PubMed2006

Development and validation of HTS flux assay for endogenously expressed chloride channels in a CHO-K1 cell line.

An atomic absorption spectroscopy-based detection system was employed to develop a new non-radioactive flux assay for chloride (Cl-) channels in a high throughput format. Cl- flux is assayed by measuring the extent to which Cl- precipitates an excess amount of silver ions (Ag+). A linear correlation was observed between theoretical and determined Cl- concentration with an r2 value of 0.996. The assay was found to be free from interference from various ions and proteins. The assay was used to study the physiology of endogenously expressed Cl- channels in a Chinese hamster ovary-K1 cell line. Cl- efflux was activated in response to an increased concentration of K+ (100 mM), Ca2+ (4 mM), and ionomycin (10 microM) as calcium ionophore. The efflux was also sensitive to pH as slightly higher efflux of Cl- was observed at an acidic pH of 3.2 in comparison to the neutral pH of 7.4. The Cl- efflux was inhibited by 100 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 500 microM 5-nitro-2-(3-phenylpropylamino) benzoate (NPPB) but not by tolbutamide, niflumic acid, or glybenclamide, indicating that the channel current is not sensitive to other cystic fibrosis transmembrane conductance regulator inhibitors. The 50% inhibitory concentration (IC50) values of DIDS at pH 7.4 and pH 3.2 were 17 microM and 19 microM, respectively. An IC50 of 26 microM was observed for NPPB. The assay had a Z' factor of 0.678.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

Central-type benzodiazepines modulate GABAA receptor chloride channels in cultured pituitary melanotrophs.

The effects of gamma-aminobutyric acid (GABA) and benzodiazepines on the electrical activity of cultured frog melanotrophs were studied using the patch-clamp technique. In the cell-attached configuration, the exposure to GABA caused a blockage of the spontaneous firing. In the whole-cell configuration, with physiological chloride concentrations, GABA evoked a hyperpolarization associated with a decrease of membrane resistance, generating an inward chloride current. Clonazepam, a central-type benzodiazepine agonist, potentiated the GABA-induced current and the resulting hyperpolarization. In addition, the benzodiazepine inverse agonist Ro 19-4603 totally abolished GABA-induced hyperpolarizing chloride current. Since the pars intermedia of the frog pituitary is composed of a 'pure' population of endocrine cells enriched with GABAA receptors, our results indicate that these cells represent a valuable model in which to investigate the electrophysiological effects of ligands for the GABAA benzodiazepine receptor complex.

Animals↗
PubMed1993

Small conductance chloride channels in acinar cells from the rat mandibular salivary gland are directly controlled by a G-protein.

This paper reports a 5pS Cl- channel in endpiece cells from the rat mandibular gland. The anion selectivity of the channel was SCN- > (Br- = I-) > NO3- > Cl-, which was similar to whole-cell recordings of a macroscopic anion conductance in these cells. The channel was activated by intracellular Ca2+ at depolarising potentials, with no significant activation when the membrane patch was hyperpolarised. The channel was directly activated by an Al/F complex and GTP gamma S, in separate experiments, applied to the cytosolic face of inside-out excised patches. These G-protein activators stimulated the Cl channel when the Ca2+ concentration was below 1nM, and suggests that this channel may be coupled directly to a G-protein in the apical membrane.

Aluminum↗
PubMed1985

[Ionic mechanisms of the effects of phenibut and GABA unrelated to changes in the function of chloride channels].

It is established that beta-phenyl-GABA (phenibut) and partly GABA elicit direct depolarization of the isolated spinal cord motoneurons. The depolarizing effect of phenibut and a depolarizing component of GABA action do not alter in the presence of picrotoxin (10(-5) mol/l) and in the chloride-deficient medium. This depolarizing phenibut effect which is not bound with activation of GABAA-receptors and chloride channels coupled with them does not alter in Na+-deficient medium, enhances in the medium with excess of K+ ions (10 mol/l) and in presence of imidazol (5 . 10(-4) mol/l) and is completely abolished in the Ca2+-deficient medium with 2 mmol/l of Mn2+ or in the presence of 10(-4) mol/l theophylline. It is supposed that phenibut and partly GABA diminish intracellular concentration of cAMP via GABAB-receptor activation and decrease functional activity of voltage-dependent Ca2+-ionic channels and Ca2+-activated outward K+-currents.

Animals↗
PubMed1989

Actions of anaesthetics and avermectin on GABAA chloride channels in mammalian dorsal root ganglion neurones.

1. The gamma-aminobutyric acid (GABA)-mimetic actions of some anaesthetics and the antehelminthic avermectin B1a were examined on freshly isolated mammalian dorsal root ganglion (DRG) neurones by use of suction electrodes and a single electrode voltage clamp. 2. Pentobarbitone (60 microM-3 mM), chloralose (600 microM-1 mM), etomidate (10-100 microM), alphaxalone (10-60 microM) and avermectin (10-60 microM) directly activated chloride channels in GABA-sensitive DRG neurones. The agonist action was sensitive to block by bicuculline and picrotoxinin. 3. Steady-state current-voltage (I-V) curves for the anaesthetics were either linear, or rectified in the opposite direction to steady-state I-V curves obtained with GABA. Current relaxations in response to voltage jumps were also of the opposite direction. An extra surge of current ('bounce') was commonly observed on washout of some of these agonists. 4. Pentobarbitone was ineffective as an agonist at alkali pH (10.4 and 9.4), but was approximately twice as effective at acid (5.4) than at normal (7.4) pH values. 5. These results suggest that some anaesthetics and avermectin are capable of 'blocking' GABA channels in addition to activating them.

Anesthetics↗
PubMed1996

Chloride-channel block inhibits T lymphocyte activation and signalling.

Both large- and small-conductance chloride (Cl-) channels have been found in human T lymphocytes; however, apart from possible roles in mediating regulatory volume decrease, their functions are not understood. We have used patch-clamp electrophysiology, Ca2+ spectrofluorometry, and Western blot assay for phosphotyrosine to investigate the effects of blocking Cl- channels on proliferation and on specific events in the activation of normal human T cells. Four chemically distinct Cl- channel blockers inhibited both the small-conductance Cl- channels and phytohemagglutinin (PHA)-induced lymphocyte proliferation in a similar dose-dependent manner; their order of potency was 5-nitro-2(3-phenylpropylamino)-benzoic acid (NPPB) > 4,4'-diisothiocyano-2,2'-disulfonic acid (DIDS) > flufenamic acid >> IAA-94. The Cl- channel blockers inhibited both the PHA-induced mobilization of Ca2+ and the rapid tyrosine phosphorylation of several polypeptides. Cell proliferation was not rescued by the Ca+ ionophore ionomycin or by addition of exogenous interleukin-2 (IL-2). Moreover, the blockers also inhibited phosphotyrosine expression in IL-2-treated, activated lymphoblasts. Thus, our results support a role for Cl- channels in early, PHA-evoked signalling and in later, II-2-dependent stages of lymphocyte activation and proliferation.

Blotting, Western↗