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PubMed2003

Modeling study of human renal chloride channel (hCLC-5) mutations suggests a structural-functional relationship.

BACKGROUND: Dent's disease, a renal tubular disorder characterized by low-molecular-weight proteinuria, hypercalciuria, and nephrolithiasis, is due to inactivating mutations in the X-linked renal-specific chloride channel, hCLC-5. The x-ray crystal structures of two bacterial chloride channels (CLCs) have recently been established, thereby allowing us to construct a model for hCLC-5 and further examine the role of its mutations. METHODS: The data regarding 49 hCLC-5 mutations were reviewed. Thirty-four mutations that predicted absent or truncated channels were excluded. The remaining 15 mutations (one in-frame insertion and 14 missense mutations), 12 of which have been studied electrophysiologically, were assessed. The hCLC-5 sequence was aligned with the Salmonella typhimurium and Escherichia coli sequences and used to map the hCLC-5 mutations onto a three-dimensional model. RESULTS: hCLC-5 is a homodimeric protein, with each subunit consisting of 18 helices. None of the missense mutations involved the chloride (Cl-) selectivity filter, but 12 of the 15 mutations were found to be clustered at the interface of the two subunits. Six of these mutations occurred in two of the helices that either form part of the interface or lie in close proximity to the interface, and three other mutations that did not lead to complete loss of Cl- conductance were at the edge of the interface. CONCLUSION: These results demonstrate a crucial role for the interaction between the two subunits at the interface of the homodimeric hCLC-5.

Amino Acid Sequence↗
PubMed2004

AP-3-dependent mechanisms control the targeting of a chloride channel (ClC-3) in neuronal and non-neuronal cells.

Adaptor protein (AP)-2 and AP-3-dependent mechanisms control the sorting of membrane proteins into synaptic vesicles. Mouse models deficient in AP-3, mocha, develop a neurological phenotype of which the central feature is an alteration of the luminal synaptic vesicle composition. This is caused by a severe reduction of vesicular levels of the zinc transporter 3 (ZnT3). It is presently unknown whether this mocha defect is restricted to ZnT3 or encompasses other synaptic vesicle proteins capable of modifying synaptic vesicle contents, such as transporters or channels. In this study, we identified a chloride channel, ClC-3, whose level in synaptic vesicles and hippocampal mossy fiber terminals was reduced in the context of the mocha AP-3 deficiency. In PC-12 cells, ClC-3 was present in transferrin receptor-positive endosomes, where it was targeted to synaptic-like microvesicles (SLMV) by a mechanism sensitive to brefeldin A, a signature of the AP-3-dependent route of SLMV biogenesis. ClC-3 was packed in SLMV along with the AP-3-targeted synaptic vesicle protein ZnT3. Co-segregation of ClC-3 and ZnT3 to common intracellular compartments was functionally significant as revealed by increased vesicular zinc transport with increased ClC3 expression. Our work has identified a synaptic vesicle protein in which trafficking to synaptic vesicles is regulated by AP-3. In addition, our findings indicate that ClC-3 and ZnT3 reside in a common vesicle population where they functionally interact to determine vesicle luminal composition.

Animals↗
PubMed2006

Cloning and characterization of genes encoding alpha and beta subunits of glutamate-gated chloride channel protein in Cylicocyclus nassatus.

The invertebrate glutamate-gated chloride channels (GluCls) are receptor molecules and targets for the avermectin-milbemycin (AM) group of anthelmintics. Mutations in GluCls are associated with ivermectin resistance in the soil dwelling nematode Caenorhabditis elegans and the parasitic nematode Cooperia oncophora. In this study, full-length cDNAs encoding alpha and beta subunits of GluCl were cloned and sequenced in Cylicocyclus nassatus, a common and important cyathostomin nematode parasite of horses. Both genes possess the sequence characteristics typical of GluCls, and phylogenetic analysis confirms that these genes are evolutionarily closely related to GluCls of other nematodes and flies. Complete coding sequences of C. nassatus GluCl-alpha and GluCl-beta were subcloned into pTL1 mammalian expression vector, and proteins were expressed in COS-7 cells. Ivermectin-binding characteristics were determined by incubating COS-7 cell membranes expressing C. nassatus GluCl-alpha and GluCl-beta proteins with [(3)H]ivermectin. In competitive binding experiments, fitting the data to a one site competition model, C. nassatus GluCl-alpha was found to bind [(3)H]ivermectin with a high amount of displaceable binding (IC(50)=208 pM). Compared to the mock-transfected COS-7 cells, the means of [(3)H]ivermectin binding were significantly different for C. nassatus GluCl-alpha and the Haemonchus contortus GluCl (HcGluCla) (p=0.018 and 0.023, respectively) but not for C. nassatus GluCl-beta (p=0.370). This is the first report of orthologs of GluCl genes and in vitro expression of an ivermectin-binding protein in a cyathostomin species. These data suggest the likelihood of a similar mechanism of action of AM drugs in these parasites, and suggest that mechanisms of resistance may also be similar.

Amino Acid Sequence↗
PubMed1988

gamma-Aminobutyric acid-activated chloride channels: relationship to genetic differences in ethanol sensitivity.

We demonstrated recently that low concentrations of ethanol enhanced the muscimol-stimulated chloride influx in cerebellar membranes from long sleep (LS-ethanol sensitive) mice, but had no effect on membranes from short sleep (SS-ethanol resistant) mice. The LS and SS were selected from a heterogeneous stock (HS) of mice for differential sensitivity to the hypnotic effects of ethanol as measured by the duration of the loss of the righting reflex (sleep time). In the present study, we tested 100 HS for ethanol sleep time. The mice with the shortest sleep time (HS-SS) and the mice with the longest sleep time (HS-LS) were selected and tested for the effect of ethanol and muscimol on chloride flux in cerebellum. The effects of ethanol and muscimol on both cerebellar and cortical chloride flux were also examined in rats from the 7th generation selected for differential sensitivity to the hypnotic effects of ethanol (high acute ethanol sensitive rats-HAS and low acute ethanol sensitive rats-LAS). Low concentrations of ethanol (10-30 mM) potentiated muscimol stimulation of 36Cl- uptake in both cortical and cerebellar membranes prepared from ethanol-sensitive animals (HS-LS and HAS). None of the ethanol concentrations tested altered stimulated chloride uptake in ethanol-resistant animals (HS-SS and LAS). No differences in muscimol stimulation of chloride uptake were observed between the pairs of selected lines. These findings strongly suggest that genetic differences in ethanol hypnosis are related to differences in the sensitivity of gamma-aminobutyric acid-operated chloride channels to ethanol.

Animals↗
PubMed1999

Molecular cloning and characterization of a mitogen-activated protein kinase-associated intracellular chloride channel.

ERK7, a member of the mitogen-activated protein kinase family, has a carboxyl-terminal tail that is required for ERK7 activation, cellular localization, and its ability to inhibit DNA synthesis. To identify proteins that interact with ERK7, we utilized a yeast two-hybrid screen with the COOH-terminal tail of ERK7 as bait and isolated the cDNA for a novel protein termed CLIC3. The interaction between CLIC3 and ERK7 in mammalian cells was confirmed by co-immunoprecipitation. CLIC3 has significant homology to human intracellular chloride channels 1 (NCC27/CLIC1) and 2 and bovine kidney chloride channel p64. Like NCC27/CLIC1, CLIC3 is predominantly localized in the nucleus and stimulates chloride conductance when expressed in cells. Taken together, these results suggest that CLIC3 is a new member of the human CLIC family. The observed interaction between CLIC3 and ERK7 is the first demonstration of a stable complex between a protein that activates chloride ion transport and a member of the mitogen-activated protein kinase family of signal transducers. The specific association of CLIC3 with the COOH-terminal tail of ERK7 suggests that CLIC3 may play a role in the regulation of cell growth.

Amino Acid Sequence↗
PubMed1993

High-conductance chloride channels in BC3H1 myoblasts.

The existence of a high-conductance voltage sensitive chloride channel in BC3H1 myoblasts is documented. The conductance of the channel in symmetrical 150 mmol/l sodium chloride is around 400 pS. The probability of the channel being in the open state decreases with increasing of the imposed voltage from holding potential (0 mV) in both the depolarizing and the hyperpolarizing direction respectively. The bell-shaped open probability plot is asymmetrical, and can be fitted by two Boltzmann equations with different Vh and kn constants; the fitted values were -53 mV and -8.4 respectively for the negative side and +41 mV and +10.2 for the right side. When the unit Cl currents to rectangular pulses are summated the resulting total ionic Cl current shows relaxation, which increases with the amplitude of the pulse. The activation as well as the shape of the current can be significantly influenced by varying the amplitude and the direction of prepulses or holding potential. The high-conductance Cl channel shows several substrates (at least four with amplitudes of around 100 pS); frequently accompanied by flickerings.

Animals↗
PubMed1999

Identification of three novel members of the calcium-dependent chloride channel (CaCC) family predominantly expressed in the digestive tract and trachea.

Three novel human sequences showing striking homology to the recently described bovine Ca2+/calmodulin kinase II-dependent epithelial chloride channel bCaCC have been identified in an expressed sequence tags database. Full-length clones were isolated using a 5' RACE approach. The encoded predicted proteins display 65% overall homology to bCaCC. Tissue expression patterns of the corresponding genes, designated as hCaCC-1, -2 and -3, appear to be highly restricted, with the first two genes primarily expressed in the digestive tract. Another original feature as compared to the CaCC family members is the fact that hCaCC-2 also shows expression in the brain. Taken together these findings demonstrate the existence of several CaCC-like genes in humans, some of which display distinct tissue specificity patterns within the CaCC subfamily of chloride channels.

Amino Acid Sequence↗
PubMed2001

Expression of a CIC chloride channel in Caenorhabditis elegans gamma-aminobutyric acid-ergic neurons.

In this study, we report the complete cDNA sequence, genomic organization and expression pattern of the Caenorhabditis elegans ClC chloride channel gene, clh-6. Two different types of reporter gene fusions suggest that clh-6 expression is restricted to two gamma-aminobutyric acid-ergic RME neurons. These results are in striking contrast with the wide tissue distribution of messenger RNA for the related mammalian isoforms, CIC-6 and CIC-7. The restricted expression pattern of clh-6 provides a unique opportunity to study the biological function of a neuronal CIC chloride channel.

Amino Acid Sequence↗
PubMed2002

The target of Drosophila photoreceptor synaptic transmission is a histamine-gated chloride channel encoded by ort (hclA).

By screening Drosophila mutants that are potentially defective in synaptic transmission between photoreceptors and their target laminar neurons, L1/L2, (lack of electroretinogram on/off transients), we identified ort as a candidate gene encoding a histamine receptor subunit on L1/L2. We provide evidence that the ort gene corresponds to CG7411 (referred to as hclA), identified in the Drosophila genome data base, by P-element-mediated germ line rescue of the ort phenotype using cloned hclA cDNA and by showing that several ort mutants exhibit alterations in hclA regulatory or coding sequences and/or allele-dependent reductions in hclA transcript levels. Other workers have shown that hclA, when expressed in Xenopus oocytes, forms histamine-sensitive chloride channels. However, the connection between these chloride channels and photoreceptor synaptic transmission was not established. We show unequivocally that hclA-encoded channels are the channels required in photoreceptor synaptic transmission by 1) establishing the identity between hclA and ort and 2) showing that ort mutants are defective in photoreceptor synaptic transmission. Moreover, the present work shows that this function of the HCLA (ORT) protein is its native function in vivo.

Amino Acid Sequence↗
PubMed2002

The vitelliform macular dystrophy protein defines a new family of chloride channels.

Vitelliform macular dystrophy (VMD/Best disease; MIM*153700) is an early-onset autosomal dominant disorder in which accumulation of lipofuscin-like material within and beneath the retinal pigment epithelium is associated with a progressive loss of central vision. Bestrophin, the protein product of the VMD gene, has four predicted transmembrane domains. There are multiple bestrophin homologues in the human, Drosophila, and Caenorhabditis elegans genomes, but no function has previously been ascribed to these proteins, and they show no detectable homology to other proteins of known function. Using heterologous expression, we show here that human, Drosophila, and C. elegans bestrophins form oligomeric chloride channels, and that human bestrophin is sensitive to intracellular calcium. Each of 15 missense mutations asscociated with VMD greatly reduces or abolishes the membrane current. Four of these mutant bestrophins were coexpressed with the wild type and each dominantly inhibited the wild-type membrane current, consistent with the dominant nature of the disease. These experiments establish the existence of a new chloride channel family and VMD as a channelopathy.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2007

Patients with biallelic mutations in the chloride channel gene CLCNKB: long-term management and outcome.

BACKGROUND: Little information on the management and long-term follow-up of patients with biallelic mutations in the chloride channel gene CLCNKB is available. METHODS: Long-term follow-up was evaluated from 5.0 to 24 years (median, 14 years) after diagnosis in 13 patients with homozygous (n = 10) or compound heterozygous (n = 3) mutations. RESULTS: Medical treatment at last follow-up control included supplementation with potassium in 12 patients and sodium in 2 patients and medical treatment with indomethacin in 9 patients. At the end of follow-up, body height was 2.0 standard deviation score or less in 6 patients; 2 of these patients had growth hormone deficiency. Body weight (<or=2.0 standard deviation score in 6 patients) significantly increased (P < 0.05) at the end of follow-up in comparison to values at diagnosis. Nonpostural persistent proteinuria was present in 6 patients, and 4 patients had a glomerular filtration rate less than 75 mL/min/1.73 m(2) (<1.25 mL/s). CONCLUSION: These data show that some patients with biallelic mutations in the chloride channel gene CLCNKB tend to present with pathological proteinuria and impaired kidney function after a median follow-up of 14 years, and growth retardation is common and sometimes related to growth hormone deficiency in these patients.

Adolescent↗
PubMed2001

Chloride channels and the reactions of cells to topography.

The reactions of rat epitenon cells to substratum topography on the micrometric and nanometric scale such as groove-ridge structures include cell extension, elongation and orientation reactions. In this paper we report that stretch-sensitive chloride channels may be involved in the earliest stages of these reactions in epitenon fibroblast-like cells. We report that rat epitenon-cells can develop appreciable lateral mechanical tension that could stretch both the force generating cells themselves and those nearby. We show that cells in medium in which more than 80% of the chloride has been replaced by nitrate show little reaction to topography. Spreading of the cells takes place but is much reduced along the direction of the groove-ridge topography but enhanced across the topography. The chloride channel inhibitors NPPB (5-Nitro-2- (3phenylpropylamino) benzoicacid) 4,4'-disothiocyanostilbene-2, 2' sulphonic acid (DIDS) and Chlorotoxin produce similar results which are further accentuated when these inhibitors are presented in low chloride medium. An antibody against ClC3, which has close homology to ClC5/6 also, blocked reaction to topography. These treatments have no significant effect on cell spreading on planar surfaces nor do they lead to changes in internal pH in the cells. There is a slight inhibition of rates of cell movement. Experiments using antisense oligoribonucleotides to ClC-5 or ClC-6 channel m-RNA also inhibit topographic reactions, which provides further confirmation of the hypothesis. Since the ClC-3,4 and 5 share considerable sequence similarities in the genes and in their proteins it has not been possible to make an unambigous determination of which precise chloride channel(s) is (are) involved.

Journal Article↗
PubMed1988

Enhancement of t-[35S]butylbicyclophosphorothionate and [3H]strychnine binding by monovalent anions reveals similarities between gamma-aminobutyric acid- and glycine-gated chloride channels.

The characteristics of [3H]strychnine and t-[35S]-butylbicyclophosphorothionate ([35S]TBPS) binding to sites associated with glycine- and gamma-aminobutyric acid (GABA)-gated chloride channels were compared in the presence of a series of anions with known permeabilities through these channels. Good correlations were found between (a) the potencies (EC50) of these anions to stimulate radioligand binding and their permeabilities relative to chloride; (b) the affinities (KD) of these radioligands in the presence of fixed concentrations of these anions and their relative permeabilities; (c) the potencies (EC50) of these anions to stimulate [35S]TBPS and [3H]strychnine binding; and (d) the affinities (KD) of [3H]strychnine and [35S]TBPS measured at a fixed concentration of these anions. These studies support electrophysiological and biochemical observations demonstrating similarities between glycine- and GABA-gated chloride channels, and suggest that anions enhance [3H]strychnine and [35S]TBPS binding through specific anion binding sites located at the channels.

Animals↗
PubMed1990

Large unit conductance voltage chloride channels are expressed in mouse neural crest cells and embryonic dorsal root ganglion cells.

The early expression of voltage-activated chloride channels of large unitary conductance (450 pS in symmetrical 140 mM KCl) was demonstrated using patch-clamp techniques in two preparations: (i) neural crest cells isolated from 9-day-old (E9) mouse embryos and (ii) acutely isolated dorsal root ganglion cells isolated from E12 mouse embryos. Properties of these ionic channels have been analyzed using single channel recordings and the group mean of these single channels.

Animals↗
PubMed2004

Annexin 6 modulates the maxi-chloride channel of the apical membrane of syncytiotrophoblast isolated from human placenta.

The syncytiotrophoblast separates the maternal and fetal blood and constitutes the primary barrier for maternal-fetal transport. The Maxi-chloride channel from the apical membrane of the syncytiotrophoblast plays a role in the chloride conductance. Annexins can play an important role in the regulation of membrane events. In this study we evaluate the role of annexin 6 in the Maxichloride channel properties. The results showed that annexin 6 is bound in the apical placenta membranes in a calcium-dependent phospholipid-binding manner but also in a calcium-independent fashion. The neutralization of annexin 6 decreased the total current by 39 +/- 1.9% in the range of +/-80 mV, and the currents decrease with the time. The single-channel slope conductance was decreased from 253 +/- 7.4 pS (control) to 105 +/- 13 pS, and the amplitude decreased by 50%. The open probability was also affected when higher voltage steps were used, changes in either the positive or negative direction induced the channel to close, and the open probability (P(o)) did not decrease. In channels with neutralized annexin 6, it was maintained at 1 at +/-40 mV and at +/-80 mV. These results suggest that endogenous annexin 6 could regulate the Maxi-chloride channel. The results obtained with normal placentae, in which annexin 6 was neutralized, are similar to those described for the Maxi-chloride channel isolated from pre-eclamptic placenta. Together these data suggest that annexin 6 could play an important role in ion transport of the placenta.

Annexin A6↗
PubMed1991

Reconstitution of an epithelial chloride channel. Conservation of the channel from mudpuppy to man.

We have previously shown that monoclonal antibody E12 (MAb E12), one of several such antibodies raised against theophylline-treated Necturus gallbladder (NGB) epithelial cells, inhibits the chloride conductance in the apical membrane of that tissue. Since chloride channels are critical to the secretory function of epithelia in many different animals, we have used this antibody to determine whether the channels are conserved, and in an immunoaffinity column to isolate the channel protein. We now demonstrate that MAb E12 cross-reacts with detergent-solubilized extracts of different tissues from various species by enzyme-linked immunosorbent assay (ELISA). Western blot analysis shows that this monoclonal antibody recognizes proteins of Mr 219,000 in NGB, toad gallbladder, urinary bladder, and small intestine, A6 cells, rat colon, rabbit gastric mucosa, human lymphocytes, and human nasal epithelial cells, and inhibits the chloride conductance in toad gallbladder, rat colon, and human nasal epithelium. Detergent-solubilized protein eluted from an immunoaffinity column and then further purified via FPLC yields a fraction (Mr 200,000-220,000) which has been reconstituted into a planar lipid bilayer. There it behaves as a chloride-selective channel (PCl/PNa = 20.2 in a 150/50 mM trans-bilayer NaCl gradient) whose unit conductance is 62.4 +/- 4.6 pS, and which is blocked in the bilayer by the antibody. The gating characteristics of this channel indicate that it can exist as aggregates or as independent single channels, and that the antibody interferes with gating of the aggregates, leaving the unit channels unchanged. From these data we conclude that the protein of Mr 219,000 recognized by this monoclonal antibody is an important component of an epithelial chloride channel, and that this channel is conserved across a wide range of animal species.

Animals↗
PubMed1993

Effects of intracellular magnesium on calcium, potassium and chloride channels.

Intracellular free magnesium activity approximates 0.6-0.8 mM. Variations in concentration around this physiologic range profoundly affect current flow through calcium, potassium and chloride channels. These effects are exerted on both inwardly and outwardly directed currents and appear to be independent of the state of phosphorylation of the channel. In the heart, these effects are manifested in changes in action potential duration, which may help to explain postulated links between hypomagnesemia and arrhythmias. In the kidney, effects of magnesium on ion channels may subserve a protective role and may explain how potassium balance is altered in magnesium depletion syndromes.

Animals↗