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PubMed2000

Molecular distribution of volume-regulated chloride channels (ClC-2 and ClC-3) in cardiac tissues.

The molecular identification of cardiac chloride channels has provided probes to investigate their distribution and abundance in heart. In this study, the molecular expression and distribution of volume-regulated chloride channels ClC-2 and ClC-3 in cardiac tissues were analyzed and quantified. Total RNA was isolated from atria and ventricles of several species (dog, guinea pig, and rat) and subjected to a quantitative RT-PCR strategy. ClC-2 and ClC-3 mRNA expression were calculated relative to beta-actin expression within these same tissues. The transcriptional levels of ClC-3 mRNA were between 1.8 and 10.2% of beta-actin expression in atria and between 3.4 and 8.6% of beta-actin in ventricles (n = 3 for each tissue). The levels of ClC-2 in both atria and ventricles were significantly less than those measured for ClC-3 (n = 3; P < 0.05). ClC-2 mRNA levels were between 0.04-0.08% and 0.03-0.18% of beta-actin expression in atria and ventricles, respectively (n = 3 for each tissue). Immunoblots of atrial and ventricular wall protein extracts demonstrated ClC-2- and ClC-3-specific immunoreactivity at 97 and 85 kDa, respectively. Immunohistochemical localization in guinea pig cardiac muscle demonstrates a ubiquitous distribution of ClC-2 and ClC-3 channels in the atrial and ventricular wall. Confocal analysis detected colocalization of ClC-2 and ClC-3 in sarcolemmal membranes and distinct ClC-3 immunoreactivity in cytoplasmic regions. The molecular expression of ClC-2 and ClC-3 in cardiac tissue is consistent with the proposed role of these chloride channels in the regulation of cardiac cell volume and the modulation of cardiac electrical activity.

Actins↗
PubMed1990

Studies on human porin. III. Does the voltage-dependent anion channel "Porin 31HL" form part of the chloride channel complex, which is observed in different cells and thought to be affected in cystic fibrosis?

"Porin 31HL", of known primary structure, is an integral protein of the plasmalemma of human B cells (Thinnes, F.P. et al. (1989) This Journal 370, 1253-1264; Kayser, H. et al. (1989) This Journal 370, 1265-1278). Purified "Porin 31HL" from human B lymphocytes was reconstituted into lipid bilayer membranes, where it formed defined voltage-dependent channels. Five minutes preincubation with 100 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, potent inhibitor of chloride transport, altered the channel-forming properties of the protein, so that it now showed small irregular channels instead of distinct steps. In addition, the voltage-dependence of the channel was abolished by the action of 4,4'-diisothiocyanatostilbene-2,2'-disulfonate. Functional and structural similarities between "Porin 31HL" and porin preparations from other human tissues and from other species suggest that this protein may be part of the chloride channel complex, which is defective in cystic fibrosis.

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

A high-throughput assay for modulators of ligand-gated chloride channels.

Invertebrate glutamate-gated chloride channels (GluCls) are important targets for anthelmintics and insecticides such as ivermectin. To facilitate screening for novel GluCl modulators, the Caenorhabditis elegans GluCl alpha2beta channel was chosen as a surrogate for parasite channels not yet cloned, and an inducible stable human embryonic kidney cell line was generated. Functional expression of the alpha2 and beta subunits was confirmed by whole-cell voltage clamp assays. Using this cell line, a high-throughput assay was developed that detects membrane potential changes associated with the activation of GluCls. In this assay, membrane depolarization was quantified via changes in fluorescence resonance energy transfer between two membrane-associated dyes. Robust and reproducible signals were detected in response to addition of glutamate or ivermectin. This assay was used for the screening of over 180,000 samples from natural and synthetic sources.

Animals↗
PubMed1997

Analysis of a protein region involved in permeation and gating of the voltage-gated Torpedo chloride channel ClC-0.

1. The chloride channel from the Torpedo electric organ, ClC-0, is controlled by two distinct ('fast' and 'slow') voltage-dependent gates. Here we investigate the effects of mutations in a region after putative transmembrane domain D12. A mutation in this region has previously been shown to change fast gating and permeation. 2. We used a combination of site-directed mutagenesis with two-electrode voltage-clamp and patch-clamp measurements. 3. Most conservative substitutions have minor effects, while more drastic mutations change kinetics and voltage dependence of fast gating, as well as ion selectivity and rectification. 4. While ClC-0 wild-type (WT) channels deactivate fully in two-electrode voltage clamp at negative voltages, channels do not close completely in patch-clamp experiments. Open probability is increased by intracellular chloride in a concentration- but not voltage-dependent manner. 5. In several mutants, including K519R, the minimal macroscopic open probability of fast gating is larger than in WT. Mutant channels fluctuate at negative potentials between open and closed conformations. Open probability is much more effectively increased by intracellular chloride than in WT. The observations support the idea that permeating ions inside the pore stabilize the open state. 6. Besides effects on permeation and gating of single protopores, some mutations affect 'slow' gating. In summary, the region after D12 participates in fast as well as in slow gating; mutations additionally influence permeation properties.

Animals↗
PubMed2002

Altered GABAergic function accompanies hippocampal degeneration in mice lacking ClC-3 voltage-gated chloride channels.

Mice lacking ClC-3 chloride channels, encoded by the Clcn3 gene, undergo neurodegeneration of the hippocampal formation and retina [Neuron, 29 (2001) 185-196; Genes Cells, 7 (2002) 597-605]. We independently created a mouse lacking the Clcn3 gene which demonstrated similar central nervous system abnormalities, including early postnatal degeneration of retinal photoreceptors. However, we observed a characteristic spatial-temporal sequence of hippocampal neurodegeneration that differs from the pattern previously reported. Anterior-to-posterior degeneration and astrogliosis of the dentate gyrus and hippocampus progressed over months. Sequential loss of hippocampal neuronal subpopulations began in the dentate gyrus and progressed to CA3, followed by CA1 neurons. Projection neurons of the entorhinal cortex degenerated, secondary to the loss of their synaptic targets within the hippocampal formation. Other characteristics of the Clcn3(-/-) mice included an abnormal gait, kyphosis, and absence of hindlimb escape extension upon tail elevation. Spontaneous seizures were observed in four adult Clcn3(-/-) mice, and one mouse died during the event. We hypothesized that neuronal injury may be related to recurrent seizures. Clcn3(-/-) mice had normal serum electrolytes and pH, and exhibited neither hyperglycemia nor rebound hypoglycemia following a glucose load. They displayed a greatly reduced susceptibility to pentylenetetrazole-induced seizures and an abnormally prolonged sedation to benzodiazepines. There was no change in vulnerability to kainic acid-induced seizures. Immunostaining revealed a progressive loss of GABA synthesizing cells in the dentate gyrus. The death of these cells was preceded by increased GABA(A) receptor immunoreactivity. These data suggest that GABA(A) inhibitory neurotransmission is altered in Clcn3(-/-) mice. The increase in GABA(A) receptor density may represent a compensatory response either to chronic excessive excitatory stimuli or reduced inhibitory input from local GABAergic interneurons within the dentate gyrus.

Animals↗
PubMed2003

The interacting binding domains of the beta(4) integrin and calcium-activated chloride channels (CLCAs) in metastasis.

CLCA (chloride channel, calcium-activated) proteins are novel pulmonary vascular addresses for blood-borne, lung-metastatic cancer cells. They facilitate vascular arrest of cancer cells via adhesion to beta4 integrin and promote early, intravascular, metastatic growth. Here we identify the interacting binding domains of endothelial CLCA proteins (e.g. hCLCA2, mCLCA5, mCLCA1, and bCLCA2) and beta4 integrin. Endothelial CLCAs share a common beta4-binding motif (beta4BM) in their 90- and 35-kDa subunits of the sequence F(S/N)R(I/L/V)(S/T)S, which is located in the second extracellular domain of the 90-kDa CLCA and near the N terminus of the 35-kDa CLCA, respectively. Using enzyme-linked immunosorbent, pull-down, and adhesion assays, we showed that glutathione S-transferase fusion proteins of beta4BMs from the 90- and 35-kDa CLCA subunits bind to the beta4 integrin in a metal ion-dependent manner. Fusion proteins from fibronectin and the integrins beta1 and beta3 served as negative controls. beta4BM fusion proteins competitively blocked the beta4/CLCA adhesion and prevented lung colonization of MDA-MB-231 breast cancer cells. A disrupted beta4BM in hCLCA1, which is not expressed in endothelia, failed to interact with beta4 integrin. The corresponding CLCA-binding domain of the beta4 integrin is localized to the specific determining loop (SDL). Again enzyme-linked immunosorbent, pull-down, and adhesion assays were used to confirm the interaction with CLCA proteins using a glutathione S-transferase fusion protein representing the C-terminal two-thirds of beta4 SDL (amino acids 184-203). A chimeric beta4 integrin in which the indicated SDL sequence had been replaced with the corresponding sequence from the beta1 integrin failed to bind hCLCA2. The dominance of the CLCA ligand in beta4 activation and outside-in signaling is discussed in reference to our previous report that beta4/CLCA ligation elicits selective signaling via focal adhesion kinase to promote metastatic growth.

Amino Acid Motifs↗
PubMed2004

Chloride channel gene expression in the rabbit cornea.

PURPOSE: The maintenance of stromal hydration by the corneal endothelium relies on active transendothelial anion transport, with bicarbonate and chloride the major anions carrying the current. However, the ion transport pathways that operate to maintain stromal hydration have yet to be fully elucidated. METHODS: We used RT-PCR to identify the gene expression profile of members of the ClC family of chloride channels in freshly isolated samples of rabbit corneal endothelium, stroma, and epithelium. The expression of a separate group of genes was also examined to confirm the purity of the sample collection protocol. The expression of the ClC-2 and ClC-3 channel protein in the cornea was also evaluated by light and electron microscopic immunolabelling. RESULTS: The mRNA for ClC-2, ClC-3, ClC-5, ClC-6, and ClC-7 were expressed in both the corneal epithelium and endothelium, and in the stroma. The mRNA for the skeletal muscle specific channel ClC-1 and the kidney specific chloride channel ClC-Ka were not detectable. ClC-4 mRNA was not detected in any rabbit tissue examined. The expression pattern of the mRNAs for collagens V, VI, VII, and VIII demonstrated the absence of contamination in epithelial and endothelial samples. ClC-2 and ClC-3 immunolabelling confirmed the presence of these proteins in corneal endothelium, stroma, and epithelium. CONCLUSIONS: Together with cystic fibrosis transmembrane conductance regulator (CFTR) and calcium activated chloride channel-1 (CLCA1), these results bring the number of chloride channel genes known to be expressed in the corneal endothelium and epithelium to seven. These channels are likely to be important for the maintenance of corneal transparency.

Animals↗
PubMed1999

Cloning of ClC-2 chloride channel from murine duodenum and its presence in CFTR knockout mice.

We report the cloning of a murine ClC-2 chloride channel cDNA from duodenal epithelium by reverse transcriptase-polymerase chain reaction (RT-PCR) using degenerate primers and by rapid amplification of cDNA ends (RACE)-PCR. Other than CFTR, this represents the first cloned chloride channel from intact intestine. The ClC-2 cDNA predicts encoding of a 908 amino acid polypeptide with a calculated M(r) of 99,373. The amino acid sequence of the murine ClC-2 chloride channel is over 94% identical to the ClC-2 chloride channel proteins of other species. Of interest is the finding that the ClC-2 mRNA is expressed about the same level in duodena from both CFTR knockout and wild-type mice. This is in keeping with the suggestion that ClC-2 might be a therapeutic target in cystic fibrosis.

Amino Acid Sequence↗
PubMed2000

Isolation and characterization of a Ca(2+)-activated chloride channel from human corneal epithelium.

PURPOSE: Transparency of the cornea is maintained through the activity of secretory mechanisms in the epithelium and endothelium, which offset the tendency of the stroma to imbibe fluid and swell. These secretory mechanisms establish osmotic gradients thereby providing the osmotic driving forces for coupled fluid transport from the stroma into both the tears and the anterior chamber. To further characterize the mechanism of epithelial Cl secretion, we cloned a cDNA encoding a Ca(2+)-dependent chloride channel, an abundant mRNA in human corneal epithelium. We investigated the abundance of all known human chloride channels in corneal epithelium to identify those responsible for regulating chloride conductance in this tissue. METHODS: For the isolation of a full-length cDNA clone, a probe was selected from a set of expressed sequenced tag (EST) clones classified as unique to corneal epithelium (http://bodymap. ims.u-tokyo.ac.jp). The expression patterns of the corresponding gene encoding novel chloride channel gene in human cornea and other tissues were examined by reverse transcription-polymerase chain reaction (RT-PCR). Quantitative PCR was performed to clarify the expression level of the novel chloride channel gene in cornea relative to that in other human tissues. RESULTS: We cloned a new Ca(2+)-activated chloride channel, CLCA2, from corneal epithelium. The full length cDNA clone encoded 943 amino acids with 62% identity to bovine Ca(2+)-activated chloride channel. The CLCA2 gene mapped to human chromosome 1p32. Quantitative expression analysis by RT-PCR showed that it is the most abundant chloride channel in corneal epithelium. CONCLUSION: High and tissue specific expression of the CLCA2 gene in human corneal epithelium implies an important role in corneal transparency maintenance.

Amino Acid Sequence↗
PubMed2000

New paradigms of CFTR chloride channel regulation.

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel controls salt and water transport across epithelial tissues. Alterations in the activity of this ion channel lead to two major human diseases: cystic fibrosis (low CFTR activity) and secretory diarrhea (excessive CFTR activity). The goal of this article is to review recent developments in our understanding of two aspects of CFTR biology: (i) interactions between CFTR domains (intramolecular interactions) that control the gating of this epithelial chloride channel and (ii) interactions between CFTR and other proteins (intermolecular interactions) that couple the activity of this ion channel to additional cellular processes in epithelial cells (e.g. membrane traffic). Clarifying the nature of these interactions may lead to the development of novel strategies for treating diseases that involve the CFTR chloride channel.

Animals↗
PubMed1990

Electrophysiological study of tert-butylbicyclophosphorothionate-induced block of spontaneous chloride channels.

The action of TBPS (tert-butylbicyclophosphorothionate) on spontaneous chloride channels recorded from porcine pars intermediate lobe cells in primary culture has been studied. This compound, which binds specifically to the gamma-aminobutyric acidA (GABAA) receptor complex, is known as a channel-gating (non-competitive) GABA antagonist. The present results show that TBPS reduces spontaneous chloride channel activity in a dose-dependent manner, with an IC50 equal to 55 nM, which is a value comparable to its affinity for the GABAA binding sites. Single-channel analysis revealed that TBPS affects neither the amplitude nor the open time of these spontaneous channels but prolongs the longer closed times, resulting in a dramatic decrease in opening probability.

Animals↗
PubMed2002

Expression of swelling- and/or pH-regulated chloride channels (ClC-2, 3, 4 and 5) in human leukemic and normal immune cells.

Chloride channels on immune cells reportedly play important roles in cell volume regulation, cell proliferation and immune functions, but they are not well characterized at the molecular level. We examined the expression of swelling-and/or pH-regulated chloride channels (ClC-2, 3, 4 and 5) in human leukemic cell lines [Jurkat and Hut-78 (T cells), Raji and Daudi (B cells), K-562 and HL-60 (myeloid cells)] and T cells, B cells and neutrophils from 8 normal subjects to clarify the difference of their expression among different cell types and maturity. Semi-quantitative RT-PCR and Northern blot analysis showed that ClC-3 was most abundantly expressed in all cells regardless of the cell types and maturity, while expression of ClC-2 was weak in these cells. Expression of ClC-4 was observed mainly in leukemic B cell lines, and in B cells and neutrophils from normal subjects. ClC-5 was expressed in all cell lines, while it was observed in only T and B cells but not in neutrophils from normal subjects. Thus, these chloride channels (ClC-2, 3, 4 and 5) showed distinct distribution among human immune cells, suggesting that they have specific roles in these cells. Molecular identification of chloride channels in leukocytes of different types and maturity may provide a new approach for the treatment of leukemia.

Adult↗
PubMed1988

Evidence for apical chloride channels in rabbit mandibular salivary glands. A chloride-selective microelectrode study.

Double-barrelled, chloride-selective microelectrodes were used to study mandibular gland acinar cells at rest and during cholinergic stimulation. At rest, intracellular chloride activity was five times the expected equilibrium activity. During sustained stimulation with acetylcholine, chloride activity fell to three times the expected equilibrium activity. Thus, the gradient for chloride exit was reduced in the stimulated cell. These results lead to the conclusion that stimulation increases the permeability of the acinar cell to chloride. Experiments in which extracellular chloride was removed provided evidence that the permeability increase was due to opening of chloride channels located principally in the apical membrane of the acinar cell.

Acetylcholine↗
PubMed1997

Chloride channel blockers inhibit myogenic tone in rat cerebral arteries.

1. We have investigated the role of chloride channels in pressure-induced depolarization and contraction of cerebral artery smooth muscle cells. 2. Two chloride channel blockers, indanyloxyacetic acid (IAA-94) and 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), caused hyperpolarizations (10-15 mV) and dilatations (up to 90%) of pressurized (80 mmHg), rat posterior cerebral arteries. Niflumic acid, a blocker of calcium-activated chloride channels, did not affect arterial tone. 3. Dilatations to IAA-94 and DIDS were unaffected by potassium channel blockers, but were prevented by elevated potassium. IAA-94 and DIDS had no effect on membrane potential or diameter of arteries at low intravascular pressure, where myogenic tone is absent. Reduction of extracellular chloride (60 mM Cl-) increased the pressure-induced contractions. Removal of extracellular sodium did not affect the pressure-induced responses. 4. Our results suggest that intravascular pressure activates DIDS- and IAA-94-sensitive chloride channels to depolarize arterial smooth muscle, thereby contributing to the myogenic constriction.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2001

Drastic reduction of the slow gate of human muscle chloride channel (ClC-1) by mutation C277S.

1. Single channel measurements suggest that the human muscle chloride channel ClC-1 presumably has a double barrelled structure, with a fast single protopore gate and a slow common pore gate similar to that of ClC-0, the chloride channel from Torpedo. The single point mutation C212S has been shown to abolish the slow gating of ClC-0 locking the slow gate in the open state. In order to test the hypothesis that the slow gating process found in ClC-1 corresponds to the well characterised slow gate found in ClC-0 we investigated the gating effects in ClC-1 of the homologous mutation corresponding to C212S, C277S. 2. We found that the mutation C277S strongly reduced the slow component of macroscopic gating relaxations at negative and at positive voltages. 3. Time constants of the fast gating relaxations were not affected by the mutation but the minimal open probability of the fast gate at negative voltages was slightly reduced to 0.08 compared with the WT value of 0.22. 4. Additionally, we characterised the block of WT ClC-1 and mutant C277S by the S(-) enantiomer of CPB (2-(p-chlorophenoxy) butyric acid), and found that the block is practically unaffected by the mutation suggesting that CPB does not interact with the slow gate of ClC-1. 5. We conclude that the slow and fast gating processes of ClC-1, respectively, reflect the slow common pore gate and the single protopore gate of the double-barrelled ClC-1 channel.

Animals↗
PubMed1998

Evidence for the intracellular location of chloride channel (ClC)-type proteins: co-localization of ClC-6a and ClC-6c with the sarco/endoplasmic-reticulum Ca2+ pump SERCA2b.

Chloride channel protein (ClC)-6a and ClC-6c, a kidney-specific splice variant with a truncated C-terminus, are proteins that belong structurally to the family of voltage-dependent chloride channels. Attempts to characterize functionally ClC-6a or ClC-6c in Xenopus oocytes have so far been negative. Similarly, expression of both ClC-6 isoforms in mammalian cells failed to provide functional information. One possible explanation of these negative results is that ClC-6 is an intracellular chloride channel rather than being located in the plasma membrane. We therefore studied the subcellular location of ClC-6 isoforms by transiently transfecting COS and CHO cells with epitope-tagged versions of ClC-6a and ClC-6c. Confocal imaging of transfected cells revealed for both ClC-6 isoforms an intracellular distribution pattern that clearly differed from the peripheral location of CD2, a plasma-membrane glycoprotein. Furthermore, dual-labelling experiments of COS cells co-transfected with ClC-6a or -6c and the sarco/endoplasmic-reticulum Ca2+ pump (SERCA2b) indicated that the ClC-6 isoforms co-localized with the SERCA2b Ca2+ pump. Thus ClC-6a and ClC-6c are intracellular membrane proteins, most likely residing in the endoplasmic reticulum. In view of their structural similarity to proven chloride channels, ClC-6 isoforms are molecular candidates for intracellular chloride channels.

Animals↗
PubMed1991

Primary structure and functional expression of a developmentally regulated skeletal muscle chloride channel.

Skeletal muscle is unusual in that 70-85% of resting membrane conductance is carried by chloride ions. This conductance is essential for membrane-potential stability, as its block by 9-anthracene-carboxylic acid and other drugs causes myotonia. Fish electric organs are developmentally derived from skeletal muscle, suggesting that mammalian muscle may express a homologue of the Torpedo mamorata electroplax chloride channel. We have now cloned the complementary DNA encoding a rat skeletal muscle chloride channel by homology screening to the Cl- channel from Torpedo. It encodes a 994-amino-acid protein which is about 54% identical to the Torpedo channel and is predominantly expressed in skeletal muscle. Messenger RNA amounts in that tissue increase steeply in the first 3-4 weeks after birth, in parallel with the increase in muscle Cl- conductance. Expression from cRNA in Xenopus oocytes leads to 9-anthracene-carboxylic acid-sensitive currents with time and voltage dependence typical for macroscopic muscle Cl- conductance. This and the functional destruction of this channel in mouse myotonia suggests that we have cloned the major skeletal muscle chloride channel.

Amino Acid Sequence↗
PubMed1987

Involvement of neuronal chloride channels in ethanol intoxication, tolerance, and dependence.

Studies of the role of neuronal chloride channels in ethanol action have focused on chloride channels coupled to gamma-aminobutyric acid (GABA) receptors. Ethanol intoxication is hypothesized to result from enhancement of GABA action, leading to increased chloride conductance and decreased neuronal excitability. Chronic ethanol treatment is suggested to produce a subsensitivity to GABA, leading to decreased action of ethanol and hyperexcitability on withdrawal of ethanol. Behavioral, electrophysiological, and biochemical studies of acute and chronic ethanol treatments on the GABA-regulated chloride channels of brain are reviewed.

Alcoholic Intoxication↗