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At least 829 records · Page 46Linked to original sources
PubMed1997

Probing the structural and functional domains of the CFTR chloride channel.

The cystic fibrosis transmembrane conductance regulator (CFTR) forms an anion-selective channel involved in epithelial chloride transport. Recent studies have provided new insights into the structural determinants of the channel's functional properties, such as anion selectivity, single-channel conductance, and gating. Using the scanning-cysteine-accessibility method we identified 7 residues in the M1 membrane-spanning segment and 11 residues in and flanking the M6 segment that are exposed on the water-accessible surface of the protein; many of these residues may line the ion-conducting pathway. The pattern of the accessible residues suggests that these segments have a largely alpha-helical secondary structure with one face exposed in the channel lumen. Our results suggest that the residues at the cytoplasmic end of the M6 segment loop back into the channel, narrowing the lumen, and thereby forming both the major resistance to ion movement and the charge-selectivity filter.

ATP-Binding Cassette Transporters↗
PubMed1996

Characterization and molecular reaction scheme of a chloride channel expressed after axotomy in crayfish.

The nerve to the deep extensor abdominal muscle (DEAM) in crayfish species Astacus astacus, containing four excitatory and one inhibitory motor axons, was cut in the third segment on one side of the animal. The distal axon stump was not subject to phagocytosis but was present for months after the axotomy. The two lateral bundles of the DEAM were prepared 4-6 weeks after the axotomy. The gamma-aminobutyric-acid-(GABA-) activated chloride channel of these bundles was characterized by applying pulses of GABA to outside-out patches of the muscle membrane and measuring the responses. Based on the dose/response relationship of the peak current and of the rise time as well as on single-channel kinetics, a detailed molecular scheme for the reaction of the channel with GABA was derived. This scheme contains four binding steps of the agonist to the receptor and two open states. Simulations of the dose/response relationships with this model resulted in a set of rate constants which generate proper fits. In comparison to the channels present in innervated muscles, the channels of denervated muscles have a higher affinity for GABA, a lower single-channel conductance, four versus five binding steps, and non-cooperative binding. The first three of these adaptations of denervated muscles correspond to similar changes in denervated vertebrate muscles.

Abdominal Muscles↗
PubMed2002

Chloride channel 7 (CLCN7) gene mutations in intermediate autosomal recessive osteopetrosis.

Osteopetrosis is a heterogeneous group of inherited disorders that includes a malignant autosomal recessive form, an intermediate autosomal recessive form and autosomal dominant forms of the disease. Most malignant osteopetroses have been ascribed to mutations in the OC116 gene encoding the human a3 subunit of vacuolar H(+)-ATPase. Few cases of autosomal recessive malignant osteopetrosis have been ascribed to mutations in the chloride channel 7 gene (CLCN7), which accounts for all autosomal dominant type II cases reported to date. Up until now, however, nothing has been known regarding the molecular basis of the intermediate form of osteopetrosis (IARO). Our study of two Portuguese IARO families shows that homozygosity for ClCN7 mutations also accounts for this form of osteopetrosis. The two patients presented with spontaneous fractures in the first years of life and generalised increase of bone density. Direct sequencing of the ClCN7 gene in both patients revealed homozygosity for two mutations (G203D and P470Q). We conclude therefore that ClCN7 mutations not only account for some dominant and malignant forms but also for intermediate forms of osteopetrosis.

Alleles↗
PubMed1998

Identification of two mutations and a polymorphism in the chloride channel CLCN-1 in patients with Becker's generalized myotonia.

Myotonia congenita is an inherited muscle disorder characterized by muscle stiffness and hypertrophy. Its clinical phenotype depends, in part, on whether it is inherited as a dominant or recessive trait, respectively designated Thomsen's disease or Becker's generalized myotonia (BGM). In either case, it is associated with abnormalities in the muscle currents that are linked to the gene (CLCN-1) on human chromosome 7q35 encoding the skeletal muscle chloride channel. Single-strand conformation polymorphism analysis was used to screen two families with the BGM for mutations in the CLCN-1 gene. Two new mutations were found (G 201ins and A317Q). The latter mutation has been previously described in Thomsen's disease.

Amino Acid Substitution↗
PubMed2005

Glutamate-gated chloride channels and the mode of action of the avermectin/milbemycin anthelmintics.

The macrocyclic lactones are the biggest selling and arguably most effective anthelmintics currently available. They are good substrates for the P-glycoproteins, which might explain their selective toxicity for parasites over their vertebrate hosts. Changes in the expression of these pumps have been implicated in resistance to the macrocyclic lactones, but it is clear that they exert their anthelmintic effects by binding to glutamate-gated chloride channels expressed on nematode neurones and pharyngeal muscle cells. This effect is quite distinct from the channel opening induced by glutamate, the endogenous transmitter acting at these receptors, which produces rapidly opening and desensitising channels. Ivermectin-activated channels open very slowly but essentially irreversibly, leading to a very long-lasting hyperpolarisation or depolarisation of the neurone or muscle cell and therefore blocking further function. Molecular and genetic studies have shown that there are multiple GluCl isoforms in both free-living and parasitic nematodes: the exact genetic make-up and functions of the GluCl may vary between species. The known expression patterns of the GluCl explain most of the observed biological effects of treatment with the macrocyclic lactones, though the reason for the long-lasting inhibition of larval production in filarial species is still poorly understood.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2006

Expression of CLC-K chloride channels in the rat cochlea.

Current models of the lateral K+ recycling pathway in the mammalian cochlea include two multicellular transport networks separated from one another by three interstitial gaps. The first gap is between outer hair cells and Deiters cells, the second is between outer sulcus cells and type II spiral ligament fibrocytes and the third is between intermediate and marginal cells in the stria vascularis. K+ taken up by cells bordering these interstitial spaces is accompanied by Cl-. Maintaining appropriate electrolyte balance and membrane potentials in these cells requires a mechanism for exit of the resorbed Cl-. One possible candidate for regulating this Cl- efflux is ClC-K, a chloride channel previously thought to be kidney specific. Here, we demonstrate the expression of both known isoforms of ClC-K in the organ of Corti, spiral ligament and stria vascularis of the rat cochlea by immunohistochemical, Western blot and RT-PCR analysis. These results indicate a role for ClC-K in mediating Cl- recycling in the cochlea. The widespread expression of both ClC-K isoforms in the cochlea may help to explain the symptoms of Bartter's syndrome Type III, a mutation in the hClC-Kb gene (human homologue of ClC-K2), which results in renal salt wasting without deafness. These data support the hypothesis that both isoforms of ClC-K are co-expressed in some cell membranes and account for the preservation of hearing in the presence of a mutation in only one channel isoform.

Animals↗
PubMed1995

GTP- and GDP-analogues modulate an inwardly rectifying chloride channel in cultured hippocampal neurons.

Three different GABA-insensitive Cl- channels could be resolved in cultured hippocampal neurons using the inside-out patch clamp configuration. The most commonly observed channel revealed an inward rectification with a chord conductance of 40 pS in symmetrical Cl- solutions at a membrane potential of -50 mV and had voltage sensitive gating kinetics. Channel openings were not observed in cell-attached patch, and after excision, several minutes of perfusion of the cytoplasmic side were required before detecting the first openings. The open state probability was increased by guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S 10(-4) M) and reduced by guanosine 5'-O-(2-thiophosphate) (GDP-beta-S 10(-4) M) suggesting its regulation by G proteins. This new identified chloride channel may account for the previously described voltage-sensitive, inward-rectifying whole cell Cl- current which was enhanced by adenosine in a pertussis toxin-sensitive manner.

Animals↗
PubMed1987

Benzodiazepine receptor modulation of [35S]TBPS binding to the chloride channel. Noncompetitive inhibition of classical benzodiazepines and competitive inhibition of the partial agonist, CGS 9895, by CGS 8216.

Benzodiazepine receptor ligands are known to modulate the binding of [35S]TBPS to the chloride channel via an allosteric action. In the cases of diazepam, zopiclone and CGS 9895, the enhanced binding of [35S]TBPS induced by these benzodiazepine agonists was antagonized by CGS 8216. This antagonism was characterized both by a shift to the right and a decrease in the maximal stimulation, for the dose-response curves of diazepam and zopiclone. In the case of CGS 9895, the maximal response was not decreased. These data indicate that CGS 8216 is a noncompetitive antagonist of classical benzodiazepine receptor agonists but is a competitive inhibitor of the partial agonist, CGS 9895.

Animals↗
PubMed1995

cAMP-activated chloride channels in a CFTR-transfected pancreatic adenocarcinoma-derived cell line, pANS6.

Pancreatic adenocarcinoma cell lines rarely express the CFTR gene, despite the high levels of CFTR protein that are present in primary pancreatic duct cells. We have attempted to generate a non-CF pancreatic adenocarcinoma cell line that stably produces high levels of CFTR mRNA and protein by transfecting a vector containing the CFTR cDNA, driven by a strong mammalian promoter, into the poorly differentiated pancreatic adenocarcinoma cell line, Panc-1. The pANS6 pancreatic duct cell line expresses substantial levels of CFTR mRNA, but little CFTR protein. Despite this we were able to detect low conductance chloride channels in 40% of patches, stimulated with cAMP, that have similar biophysical properties to CFTR.

Adenocarcinoma↗
PubMed2000

Tyrosine kinases open lymphocyte chloride channels.

Osmotic swelling of lymphocytes opens outwardly rectifying Cl(-) channels (ORCC) through the src-like kinase p56(lck). The central role of this tyrosine protein kinase has been shown by genetic and pharmacologic manipulation of the enzyme. Furthermore, p56(lck) activates ORCC independently of cell volume increase. ORCC in lymphocytes and epithelial cells from cystic fibrosis (CF) patients are resistant to activation by cAMP. However, osmotic swelling as well as intracellular purified p56(lck) can activate ORCC in CF lymphocytes. In non-CF lymphocytes ORCC is opened by either, intracellular cAMP, p56(lck) or by osmotic swelling. Osmotic activation of ORCC can be blocked by the tyrosine kinase inhibitor lavendustin in both cell types. Regulation of ORCC by p56(lck) thus represents an alternative pathway of stimulating membrane chloride conductance that is left functional in cystic fibrosis. In addition to osmoregulation these mechanisms could play a major role when cells actively change their volume, i.e. during proliferation and apoptosis. Activation of the tyrosine kinase p56(lck) is an important regulatory step for opening of chloride channels in lymphocytes.

Animals↗
PubMed2002

Identification of HLA-A*0201-restricted T cell epitopes derived from the novel overexpressed tumor antigen calcium-activated chloride channel 2.

Vaccination against tumor Ags may become a promising treatment modality especially in cancer types where other therapeutic approaches fail. However, diversity of tumors requires that a multitude of Ags become available. Differential expression in normal vs cancerous tissues, both at the mRNA and the protein level, may identify Ag candidates. We have previously compared transcripts from squamous cell lung cancer and normal lung tissue using differential display analysis, and found a transcript that was overexpressed in malignant cells and was identical with the calcium-activated chloride channel 2 (CLCA2) gene. We have now selected HLA-A2-restricted peptides from CLCA2, and have generated T cell lines against the CLCA2-derived KLLGNCLPTV, LLGNCLPTV, and SLQALKVTV peptides using in vitro priming. Specificity of T cells was ascertained in ELISPOT assays. The primed T cells also recognized allogeneic tumor cells in an Ag-specific and HLA-restricted fashion. Moreover, peptide LLGNCLPTV was also independently recognized by CD8(+) T cells expanded from pancreatic carcinoma/T cell cocultures. CLCA2-specific CD8(+) T cells were absent from the peripheral blood of healthy donors. These data indicate that an immune response can be induced against CLCA2, which thus may become an important Ag for anti-tumor vaccination approaches.

Alleles↗
PubMed2001

Characterization of a calcium-activated chloride channel as a shared target of Th2 cytokine pathways and its potential involvement in asthma.

Interleukin (IL)-9 is a T helper (Th) 2 cytokine recently implicated as an essential factor in determining susceptibility to asthma. Transgenic mice overexpressing IL-9 exhibit many features that are characteristic of human asthma. To better understand the mechanism by which IL-9 mediates the various biologic activities in asthma, we performed suppressive subtraction hybridization with whole lung from IL-9 transgenic and control mice. Here we report the identification of mCLCA3, a calcium-activated chloride channel that was specifically induced in the lung epithelium of IL-9 transgenic mice. Expression of mCLCA3 could also be induced by intratracheal administration of IL-9 or other Th2 cytokines (IL-4, IL-13), but not by interferon-gamma. Moreover, expression of mCLCA3 was induced in the lung of antigen-exposed mice, and this induction could be suppressed by neutralizing IL-9 antibody treatment, indicating IL-9 is both necessary and sufficient to induce mCLCA3 in this experimental model of asthma. Finally, we demonstrate that hCLCA1 is the human counterpart to mCLCA3 and is also induced in vitro in human primary lung cells by Th2 cytokine treatment. Together, these data strongly implicate the involvement of mCLCA3 (in mice) and hCLCA1 (in humans) in the pathogenesis of Th2 cytokine-mediated asthmatic disorders.

Amino Acid Sequence↗
PubMed1999

A familial syndrome due to Arg648Stop mutation in the X-linked renal chloride channel gene.

We describe a familial syndrome in two brothers who were investigated after the casual discovery of tubular proteinuria in their 1st month of life. During a follow-up of 20 and 11 years, respectively, the two children grew well and were asymptomatic, but developed the same biochemical abnormalities, i.e., tubular proteinuria and hyperphosphaturia, progressive decrease in serum phosphorus below the normal values for age, and an increase in serum 1,25-dihydroxyvitamin D levels over normal values. Moreover, hyperabsorptive hypercalciuria and systemic osteopenia developed and progressively worsened. In both children, at a different age, medullary nephrocalcinosis appeared. The oldest boy suffered a progressive decrease in urinary concentration ability and in glomerular filtration rate. Oral phosphate supplementation led to reversal of all biochemical abnormalities, with the exception of decreased phosphate tubular reabsorption and tubular proteinuria. With long-term phosphate supplementation, a normal bone mass was reached, while progression of nephrocalcinosis was arrested and impairment of renal function was slowed down. In a family study (siblings and parents), the only detectable abnormality was microglobinuria in the mother, thus suggesting a X-linked inheritance of this disorder. In the two probands a mutation within the renal chloride channel gene (CLCN5) was discovered.

Child↗
PubMed2005

A ClC chloride channel homolog and ornithine-containing membrane lipids of Rhizobium tropici CIAT899 are involved in symbiotic efficiency and acid tolerance.

Rhizobium tropici CIAT899 is highly tolerant to several environmental stresses and is a good competitor for nodule occupancy of common bean plants in acid soils. Random transposon mutagenesis was performed to identify novel genes of this strain involved in symbiosis and stress tolerance. Here, we present a genetic analysis of the locus disrupted by the Tn5 insertion in mutant 899-PV9, which lead to the discovery of sycA, a homolog of the ClC family of chloride channels and Cl-/H+ exchange transporters. A nonpolar deletion in this gene caused serious deficiencies in nodule development, nodulation competitiveness, and N2 fixation on Phaseolus vulgaris plants, probably due to its reduced ability to invade plant cells and to form stable symbiosomes, as judged by electron transmission microscopy. A second gene (olsC), found downstream of sycA, is homologous to aspartyl/asparaginyl beta-hydroxylases and modifies two species of ornithine-containing lipids in vivo, presumably by hydroxylation at a still-unknown position. A mutant carrying a nonpolar deletion in olsC is symbiotically defective, whereas overexpressed OlsC in the complemented strain provokes an acid-sensitive phenotype. This is the first report of a ClC homolog being essential for the establishment of a fully developed N2-fixing root nodule symbiosis and of a putative beta-hydroxylase that modifies ornithine-containing membrane lipids of R. tropici CIAT899, which, in turn, are contributing to symbiotic performance and acid tolerance.

Amino Acid Sequence↗
PubMed1993

Patch-clamp and chloride channels in Ascaris suum.

Single-channel electrophysiology is an invaluable tool fo the study of ion channels. However, it is a technique that has failed to attract widespread use by parasitologists. Here, Diane Dixon and Richard Martin outline the principles undelrying single channel recording and highlight its uses in the discovery of a new and unusual chloride channel in the musculature of Ascaris suum.

Journal Article↗