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PubMed2006

The intracellular region of ClC-3 chloride channel is in a partially folded state and a monomer.

In contrast to bacterial ClC chloride channels, all eukaryotic ClC chloride channels have a conserved long intracellular region that makes up of the carboxyl terminus of the protein and is necessary for channel functions as a channel gate. Little is known, however, about the molecular structure of the intracellular region of ClC chloride channels so far. Here, for the first time, we have expressed and purified the intracellular region of the rat ClC-3 chloride channel (C-ClC-3) as a water-soluble protein under physiological conditions, and investigated its structural characteristics and assembly behavior by means of circular dichroism (CD) spectroscopy, differential scanning calorimetry (DSC), size exclusion chromatography and analytical ultracentrifugation. The far-UV CD spectra of C-ClC-3 in the native state and in the presence of urea clearly show that the protein has a significantly folded secondary structure consisting of alpha-helices and beta-sheets, while the near-UV CD spectra and DSC experiments indicate the protein is deficient in well-defined tertiary packing. Its Stokes radius is larger than its expected size as a folded globular protein, as determined on size exclusion chromatography. Furthermore, the DisEMBL program, a useful computational tool for the prediction of disordered/unstructured regions within a protein sequence, predicts that the protein is in a partially folded state. Based on these results, we conclude that C-ClC-3 is partially folded. On the other hand, both size exclusion chromatography and sedimentation equilibrium analysis show that C-ClC-3 exists as a monomer in solution, not a dimer like the whole ClC-3 molecule.

Amino Acid Sequence↗
PubMed2004

Activating mutation of the renal epithelial chloride channel ClC-Kb predisposing to hypertension.

The chloride channel ClC-Kb is expressed in the basolateral cell membrane of the distal nephron and participates in renal NaCl reabsorption. Loss-of-function mutations of ClC-Kb lead to classic Bartter syndrome, a rare salt-wasting disorder. Recently, we identified the ClC-Kb(T481S) polymorphism, which confers a strong gain-of-function effect on the ClC-Kb chloride channel. The present study has been performed to explore the prevalence of the mutation and its functional significance in renal salt handling and blood pressure regulation. As evident from electrophysiological analysis with the 2-electrode voltage-clamp technique, heterologous expression of ClC-Kb(T481S) in Xenopus oocytes gave rise to a current that was 7-fold larger than the current produced by wild-type ClC-Kb. The prevalence of the mutant allele was significantly higher in an African population from Ghana (22%) than in whites (12%). As tested in 1 white population, carriers of ClC-Kb(T481S) were associated with significantly higher systolic (by approximately 6.0 mm Hg) and diastolic (by approximately 4.2 mm Hg) blood pressures and significantly higher prevalence (45% versus 25%) of hypertensive (> or =140/90 mm Hg) blood pressure levels. Individuals carrying ClC-Kb(T481S) had significantly higher plasma Na+ concentrations and significantly decreased glomerular filtration rate. In conclusion, the mutation ClC-Kb(T481S) of the renal epithelial Cl- channel ClC-Kb strongly activates ClC-Kb chloride channel function in vitro and may predispose to the development of essential hypertension in vivo.

Adult↗
PubMed1991

Bicarbonate permeability of epithelial chloride channels.

Bicarbonate permeability of epithelial chloride channels has been studied using the patch-clamp technique. The experiments were performed in excised inside-out oriented membrane patches from three different cultured cell types: (a) HT29 colon carcinoma cell line, (b) T84 colon carcinoma cell line, and (c) respiratory epithelial cells (REC) in primary culture. In all three preparations we observed outwardly rectifying chloride channels with similar conductances with 145 mmol/l NaCl solution in the pipette and in the bath (Cl- pipette/Cl- bath). When Cl- was replaced by HCO3- in the bath (Cl-/HCO3-) the conductance of the channel at negative clamp voltages was reduced significantly by 40% for HT29 (n = 6), 39% for T84 (n = 7), and 38% for REC (n = 6). Similarly, the zero-current potential (V1 = 0) was shifted from 0 mV (Cl-/Cl-) to negative values (Cl-/HCO3-) revealing permeability ratios PCl/PHCO3 of 2.4 +/- 0.1 for HT29 (n = 6), 2.0 +/- 0.1 for T84 (n = 7), and 1.8 +/- 0.1 for REC (n = 7). With NaHCO3 as the pipette solution and NaCl in the bath, the V1 = 0 was positive and a PCl/PHCO3 value of 2.3 +/- 0.1 was determined for HT29 (n = 5). Replacement of Cl- in the bath by HCO3- reduced V1 = 0 to values close to 0 mV. In another series of experiments, the pipette was filled with 145 mmol/l NaCl and the bath contained 35 mmol/l NaCl to which 35 mmol/l NaHCO3 were added. We found that neither the conductance for the inward current nor V1 = 0 was changed significantly with the addition of NaHCO3 (HT29, n = 6). We conclude that the HCO3- permeability and HCO3- conductance of these channels is about half of that for Cl-.

Bicarbonates↗
PubMed1988

Reconstitution and phosphorylation of chloride channels from airway epithelium membranes.

Airway epithelial chloride secretion is controlled by the apical-membrane chloride permeability. Purified apical-membrane vesicles from bovine tracheal epithelium have now been shown to contain functional chloride channels by using the planar-bilayer technique. Three types of chloride channels were observed; a voltage-dependent, calcium-independent, 71-picoSiemen (in 150 mM NaCl) channel accounted for more than 80 percent of the vesicular chloride conductance and was under strict control of phosphorylation. The channel underwent a fast rundown in less than 2 to 3 minutes of recording, and reactivation required in situ exposure to a phosphorylating "cocktail" containing the catalytic subunit of the adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase. Mean open time and open probability were increased after phosphorylation, whereas slope conductance remained unchanged. Thus, metabolic control of tracheal chloride single channels can now be studied in vitro.

Animals↗
PubMed1996

Identification of a Drosophila melanogaster glutamate-gated chloride channel sensitive to the antiparasitic agent avermectin.

Glutamate-gated chloride channels, members of the ligand-gated ion channel superfamily, have been shown in nematodes and in insects to be a target of the antiparasitic agent avermectin. Two subunits of the Caenorhabditis elegans glutamate-gated chloride channel have been cloned: GluCl-alpha and GluCl-beta. We report the cloning of a Drosophila melanogaster glutamate-gated chloride channel, DrosGluCl-alpha, which shares 48% amino acid and 60% nucleotide identity with the C. elegans GluCl channels. Expression of DrosGluCl-alpha in Xenopus oocytes produces a homomeric chloride channel that is gated by both glutamate and avermectin. The DrosGluCl-alpha channel has several unique characteristics not observed in C. elegans GluCl: dual gating by avermectin and glutamate, a rapidly desensitizing glutamate response, and a lack of potentiation of the glutamate response by avermectin. The pharmacological data support the hypothesis that the DrosGluCl-alpha channel represents the arthropod H-receptor and an important target for the avermectin class of insecticides.

Animals↗
PubMed2004

ClC-5 chloride channel alters expression of the epithelial sodium channel (ENaC).

ClC-5 chloride channels and epithelial sodium channels (ENaC) are present in many cell types including airway and retinal epithelia. Since ENaC activity is known to be affected by chloride transport, we co-injected Xenopus oocytes with cRNAs encoding ENaC and ClC-5 to investigate whether channel currents are impacted by heterologous co-expression of these proteins. ClC-5 currents were not detectably affected by co-expression with ENaC, whereas amiloride-sensitive ENaC currents were significantly lower compared to control oocytes expressing ENaC alone. Co-expression of ENaC with cRNA sequences encoding non-conducting fragments of ClC-5 revealed that the amino acid sequence region between positions 347 and 647 was sufficient for inhibition of ENaC currents. Co-expression of ENaC and another transport protein, the sodium dicarboxylate co-transporter (NaDC-1), did not affect ENaC currents. To test whether the inhibitory effects of ClC-5 were specific for ENaC, ClC-5 was also co-expressed with CFTR. CFTR currents were also inhibited by co-expression with ClC-5, whereas ClC-5 currents were unaffected. Western blot analysis of biotinylated oocyte surface membranes revealed that the co-expression of ClC-5 with ENaC, CFTR, or NaDC-1 decreased the abundance of these proteins at the surface membrane. We conclude that overexpression of ClC-5, specifically amino acids 347-647, can alter the normal translation or trafficking of ENaC and other ion transport proteins by a mechanism that is independent of the chloride conductance of ClC-5.

Amiloride↗
PubMed2002

CLC chloride channels: correlating structure with function.

CLC chloride channels form a large gene family that is found in bacteria, archae and eukaryotes. Previous mutagenesis studies on CLC chloride channels, combined with electrophysiology, strongly supported the theory that these channels form a homodimeric structure with one pore per subunit (a'double-barrelled' channel), and also provided clues about gating and permeation. Recently, the crystal structures of two bacterial CLC proteins have been obtained by X-ray diffraction analysis. They confirm the double-barrelled architecture, and reveal a surprisingly complex and unprecedented channel structure. At its binding site in the pore, chloride interacts with the ends of four helices that come from both sides of the membrane. A glutamate residue that protrudes into the pore is proposed to participate in gating. The structure confirms several previous conclusions from mutagenesis studies and provides an excellent framework for their interpretation.

Amino Acid Sequence↗
PubMed1995

Failure of P-glycoprotein (MDR1) expressed in Xenopus oocytes to produce swelling-activated chloride channel activity.

1. P-glycoprotein, the protein product of the multidrug resistance (MDR1) gene, has ATP-dependent transporter activity. It has been suggested that P-glycoprotein may also function as a volume-regulated chloride channel or chloride channel regulator. To assess the chloride channel function of P-glycoprotein, we examined swelling-activated chloride conductances in Xenopus oocytes injected with human MDR1 cRNA. 2. Functional expression of P-glycoprotein in Xenopus oocytes was confirmed using Western blot analysis and by assessing transport of the P-glycoprotein substrate, calcein AM. 3. Endogenous, swelling-activated chloride conductances were virtually absent by the time P-glycoprotein expression was confirmed. Thus, this expression system afforded the advantage of assessing putative MDR1-associated chloride currents in the absence of background currents. 4. The currents activated by hypotonic shock (50%) in both MDR1-injected and control (water-injected) oocytes were not significantly different. The swelling response was due in part to the activation of a potassium-selective conductance which could be inhibited by barium. No chloride-selective currents were activated by hypotonic shock in the presence or absence of barium. Therefore, we conclude that P-glycoprotein expression does not produce a swelling-activated chloride conductance in the Xenopus oocyte expression system.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

A chloride channel widely expressed in epithelial and non-epithelial cells.

Chloride channels have several functions, including the regulation of cell volume, stabilizing membrane potential, signal transduction and transepithelial transport. The plasma membrane Cl- channels already cloned belong to different structural classes: ligand-gated channels, voltage-gated channels, and possibly transporters of the ATP-binding-cassette type (if the cystic fibrosis transmembrane regulator is a Cl- channel). The importance of chloride channels is illustrated by the phenotypes that can result from their malfunction: cystic fibrosis, in which transepithelial transport is impaired, and myotonia, in which ClC-1, the principal skeletal muscle Cl- channel, is defective. Here we report the properties of ClC-2, a new member of the voltage-gated Cl- channel family. Its sequence is approximately 50% identical to either the Torpedo electroplax Cl- channel, ClC-0 (ref. 8), or the rat muscle Cl- channel, ClC-1 (ref. 9). Isolated initially from rat heart and brain, it is also expressed in pancreas, lung and liver, for example, and in pure cell lines of fibroblastic, neuronal, and epithelial origin, including tissues and cells affected by cystic fibrosis. Expression in Xenopus oocytes induces Cl- currents that activate slowly upon hyperpolarization and display a linear instantaneous current-voltage relationship. The conductivity sequence is Cl- greater than or equal to Br- greater than I-. The presence of ClC-2 in such different cell types contrasts with the highly specialized expression of ClC-1 (ref. 9) and also with the cloned cation channels, and suggests that its function is important for most cells.

Amino Acid Sequence↗
PubMed2000

Volume- and calcium-activated chloride channels in human umbilical vein endothelial cells.

AIM: To characterize the properties of chloride currents and its modulation in human umbilical vein endothelial cells (HUVEC). METHODS: Using whole-cell patch-clamp recording techniques. RESULTS: Exposure of HUVEC to 13.5% and 27% hypotonic solution (HTS) induced a current ICl, vol. This current was correlated with the changes in cell volume and showed a modest outward rectification. It was slowly inactivated at positive potential (> 50 mV), and it was time- and voltage-independent in kinetics. The current densities (pA/pF) were 20 +/- 3 (13.5% HTS) and 58 +/- 4 (27% HTS, n = 7), respectively at +100 mV test potential. Applying GTP gamma s (300 mumol.L-1) elicited a current similar to ICl, vol, while cAMP (0.5 mmol.L-1) had no effect on the current. Increase in [Ca2+]i, either by directly loading cells with high concentration of Ca2+ (CaCl2), or by perfusing vasoactive agonist ATP (10 mumol.L-1), activated ICl, Ca. The current density was only (23 +/- 5) pA/pF (n = 8 cells). Such current was slowly activated at positive potential, inactivated quickly at negative potential, and showed strong outward rectification. Both currents were inhibited by DIDS and verapamil. Challenging a cell with elevated [Ca2+]i and HTS activated ICl, vol on the top of ICl, Ca in the same cell, suggested co-existence of these two currents and that they were modulated by different ways. cAMP-regulated chloride channel and ClC (chloride channel family) channel were absent. CONCLUSION: HUVEC express two kinds of chloride channels, ICl, vol activated by change in cell volume and ICl, Ca by elevation of [Ca2+]i, respectively.

Adenosine Triphosphate↗
PubMed1995

Cloning, tissue distribution, and intrarenal localization of ClC chloride channels in human kidney.

Two kidney-specific chloride channels, ClC-K1 and ClC-K2, have been isolated from rat kidney. In the present study, we sought to isolate human homologue of rat ClC-K2 chloride channel that was present in the thick ascending limb of Henle's loop and collecting ducts. Human kidney cDNA library was screened with the whole rat ClC-K2 cDNA probe. Two highly homologous but not identical cDNAs were isolated and sequenced. Northern analysis showed that both clones were expressed only in kidney among various human tissues, demonstrating that kidney-specific ClC family members were also present in human kidney. Because both clones had almost the same nucleotide identity (approximately 80%) with rat ClC-K2, we could not determine by sequence alone which human clone corresponded to rat ClC-K2. Accordingly, we performed reverse transcription PCR using dissected human nephron segments and identified the site of expression of each clone in human nephron segments. One clone was only expressed in the thin limb of Henle's loop and the other was expressed in glomeruli, proximal tubules, and collecting ducts. We identified the latter clone as human ClC-K2 based on the localization of rat ClC-K1 and ClC-K2. Identification of human ClC-K2 clone will be of help in understanding the genetic involvement of chloride channel in disorders of chloride transport such as Bartter's syndrome.

Amino Acid Sequence↗
PubMed2000

Physiological role of CLC-K1 chloride channel in the kidney.

Although CLC chloride channels share a common structure, the sites of expression in the body and their intracellular localization are different among CLCs. CLC-K1 and -K2 are kidney-specific CLC chloride channels. We have clarified their localization in the plasma membranes of tubular cells by immunohistochemistry and proposed their roles in transepithelial chloride transport. Since there exit no good inhibitors for these channels, a gene knockout approach was the only way to establish their roles in kidney. While we were generating CLC-K knockout mice, Simon et al. reported that the mutations of CLC-K2 in human resulted in Bartter's syndrome. This had been anticipated since CLC-K2 is known to be present in the basolateral plasma membranes of the distal nephron where sodium-dependent chloride transporters are present in the apical membranes. Thus, CLC-K2 constitutes an important route for chloride reabsorption as an exit for chloride ions in the basolateral membrane. Another important finding in Simon's report was that no CLC-K (a human homologue of rat CLC-K1) mutation was found in patients with Bartter's syndrome. This suggested that CLC-K1 has a different role in kidney. We believed this to be true, based on the finding that the intrarenal localization of CLC-K1 and CLC-K2 are completely different. In the CLC-K1 knockout mice, we could clearly verify that (i) the high chloride permeability in the tAL was mediated by CLC-K1 and (ii) this chloride transport is necessary for urinary concentration. Further studies are necessary to elucidate the detailed mechanisms of the urinary-concentrating defect in Clcnk1 -/- mice. Exact clearance studies and measurements of osmolality and solute contents in the inner medulla will provide the answer to this question.

Animals↗
PubMed2004

Exploration of the basolateral chloride channels in the renal tubule using.

Chloride channels located on the basolateral membrane are known to be involved in chloride absorption in several parts of the renal tubule, and particularly in the thick ascending limb and distal convoluted tubule. The data available suggest that the ClC-K channels play the major role in this process. We provide here a description of the electrophysiological properties of these channels, still very incomplete at this stage, and we attempt to compare ClC-Ks to three chloride channels that we have identified in the basolateral membrane of microdissected fragments of the mouse renal tubule using the patch-clamp technique. Based on anion selectivity and dependence on external pH and calcium shown by the ClC-Ks, we propose candidate ClC-K1 and ClC-K2 in native tissue. We also discuss the possibility that chloride channels that do not belong to the ClC family may also be involved in the absorption of chloride across the cortical thick ascending limb.

Animals↗
PubMed1994

Kinin-stimulated chloride secretion in mouse colon requires the participation of CFTR chloride channels.

The effect of lysylbradykinin on electrogenic chloride secretion in the epithelium of the mouse colon has been investigated. The peptide was active only when applied to the basolateral surface and its effects were inhibited by the B2 receptor antagonist, Hoe 140, also applied to the same surface. The chloride channel blocker, niflumic acid, also inhibited the response to kinin when added apically. Cyclo-oxygenase inhibition with piroxicam attenuated the responses to kinin, indicating involvement of prostaglandins in the responses. It is concluded that lysylbradykinin increases chloride secretion by acting via B2 receptors and, as with other tissues, brings about secretion through the agency of multiple messengers. In colonic epithelia from cystic fibrosis (CF) mice lysylbradykinin was without effect, suggesting that the final effector process involves apically located cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels.

Animals↗
PubMed2004

Kinetic and pharmacological characterization of desensitizing and non-desensitizing glutamate-gated chloride channels in cockroach neurons.

Glutamate-gated chloride channels (GluCls) are found only in invertebrate nerve and muscle, where they mediate inhibitory synaptic transmission, and are important target sites of insecticides. Two GluCl subtypes have previously been distinguished in isolated cockroach CNS neurons based on differential pharmacology. The present study characterizes the kinetics and pharmacological properties of desensitizing and non-desensitizing GluCls. Both types of GluCls were sensitive to glutamate and ibotenic acid. The non-desensitizing GluCl subtype was elicited by glutamate with an EC(50) of 115.8 microM and a Hill coefficient of 2.6 and was also sensitive to the agonist ibotenic acid with an EC(50) of 42 microM and a Hill coefficient of 1.7. The desensitizing and non-desensitizing currents were carried by chloride ions, and occurred either separately or in combination in individual neurons. The GluCls were also found to coexist with and function independently of the GABA-activated chloride channels. The desensitizing and non-desensitizing GluCls exhibited different sensitivities to the ligand-gated channel blocker picrotoxinin. The desensitizing GluCls were blocked only 8% by 30 microM picrotoxinin, whereas the non-desensitizing GluCls were potently blocked by picrotoxinin with an IC(50) of 4.1 microM. The insecticides fipronil and dieldrin at 1 microM inhibited the desensitizing currents by 56 and 13%, respectively, and the non-desensitizing currents by 98 and 43%, respectively. It is concluded that the two types of GluCls found in cockroach neurons exhibit significantly different electrophysiological and pharmacological characteristics.

Animals↗
PubMed1999

Picrotoxin blockade of invertebrate glutamate-gated chloride channels: subunit dependence and evidence for binding within the pore.

Glutamate-gated chloride channels have been described in nematodes, insects, crustaceans, and mollusks. Subunits from the nematode and insect channels have been cloned and are phylogenetically related to the GABA and glycine ligand-gated chloride channels. Ligand-gated chloride channels are blocked with variable potency by the nonselective blocker picrotoxin. The first two subunits of the glutamate-gated chloride channel family, GluClalpha and GluClbeta, were cloned from the free living nematode Caenorhabditis elegans. In this study, we analyze the blockade of these novel channels by picrotoxin. In vitro synthesized GluClalpha and GluClbeta RNAs were injected individually or coinjected into Xenopus oocytes. The EC50 values for picrotoxin block of homomeric GluClalpha and GluClbeta were 59 microM and 77 nM, respectively. Picrotoxin block of homomeric GluClbeta channels was promoted during activation of membrane current with glutamate. In addition, recovery from picrotoxin block was faster during current activation by glutamate. A chimeric channel between the N-terminal extracellular domain of GluClalpha and the C-terminal membrane-spanning domain of GluClbeta localized the higher affinity picrotoxin binding site to the membrane-spanning domains of GluClbeta. A point mutation within the M2 membrane-spanning domain of GluClbeta reduced picrotoxin sensitivity >10,000-fold. We conclude that picrotoxin blocks GluCl channels by binding to a site accessible when the channel is open.

Amino Acid Sequence↗
PubMed2000

Chlorotoxin does not inhibit volume-regulated, calcium-activated and cyclic AMP-activated chloride channels.

It was the aim of this study to look for a high-affinity and selective polypeptide toxin, which could serve as a probe for the volume-regulated anion channel (VRAC) or the calcium-activated chloride channel (CaCC). We have partially purified chlorotoxin, including new and homologous short chain insectotoxins, from the crude venom of Leiurus quinquestriatus quinquestriatus (Lqq) by means of gel filtration chromatography. Material eluting between 280 and 420 min, corresponding to fractions 15-21, was lyophilized and tested on VRAC and CaCC, using the whole-cell patch-clamp technique. We have also tested the commercially available chlorotoxin on VRAC, CaCC, the cystic fibrosis transmembrane conductance regulator (CFTR) and on the glioma specific chloride channel (GCC). VRAC and the correspondent current, I(Cl,swell), was activated in Cultured Pulmonary Artery Endothelial (CPAE) cells by a 25% hypotonic solution. Neither of the fractions 16-21 significantly inhibited I(Cl,swell) (n=4-5). Ca(2+)-activated Cl(-) currents, I(Cl,Ca), activated by loading T84 cells via the patch pipette with 1 microM free Ca(2+), were not inhibited by any of the tested fractions (15-21), (n=2-5). Chlorotoxin (625 nM) did neither effect I(Cl,swell) nor I(Cl,Ca) (n=4-5). The CFTR channel, transiently transfected in COS cells and activated by a cocktail containing IBMX and forskolin, was not affected by 1.2 microM chlorotoxin (n=5). In addition, it did not affect currents through GCC. We conclude that submicromolar concentrations of chlorotoxin do not block volume-regulated, Ca(2+)-activated and CFTR chloride channels and that it can not be classified as a general chloride channel toxin.

Animals↗
PubMed1989

Regulation of chloride channels by protein kinase C in normal and cystic fibrosis airway epithelia.

Apical membrane chloride channels control chloride secretion by airway epithelial cells. Defective regulation of these channels is a prominent characteristic of cystic fibrosis. In normal intact cells, activation of protein kinase C (PKC) by phorbol ester either stimulated or inhibited chloride secretion, depending on the physiological status of the cell. In cell-free membrane patches, PKC also had a dual effect: at a high calcium concentration, PKC inactivated chloride channels; at a low calcium concentration, PKC activated chloride channels. In cystic fibrosis cells, PKC-dependent channel inactivation was normal, but activation was defective. Thus it appears that PKC phosphorylates and regulates two different sites on the channel or on an associated membrane protein, one of which is defective in cystic fibrosis.

Calcium↗