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At least 613 records · Page 34Linked to original sources
PubMed2004

Basolateral ClC-2 chloride channels in surface colon epithelium: regulation by a direct effect of intracellular chloride.

BACKGROUND & AIMS: The principal function of the colon in fluid homeostasis is the absorption of NaCl and water. Apical membrane Na(+) channels, Na(+)/H(+), and Cl(-)/HCO(3)(-) exchangers have been postulated to mediate NaCl entry into colonocytes. The basolateral exit pathway for Cl(-) has recently been proposed to be via ClC-2 channels present in that membrane domain in surface epithelium. The aim of this report is to obtain functional data for a basolateral localization of ClC-2 and explore a possible direct regulation by intracellular Cl(-). METHODS: Guinea pig colon epithelium with the apical membrane perforated with nystatin in Ussing chambers is used to show a basolateral Cl(-) conductance. Gramicidin D perforated-patch configuration of the patch-clamp technique is used on isolated surface colonocytes. Heterologous expression of the recombinant channel and the whole-cell configuration are used to investigate a direct regulation by intracellular Cl(-). RESULTS: A basolateral membrane conductance with the characteristics of ClC-2 channels, including Cd(2+) sensitivity, selectivity, and inhibition by extracellular alkalinization, is present in distal colon epithelium. The effect of intracellular Cl(-) on this conductance suggests activation by the permeant anion. Using the recombinant ClC-2 channel, a strong dependence of its activity on intracellular Cl(-) is shown, with a shift of activation to more positive voltages as [Cl(-)](i) is increased. CONCLUSIONS: It is suggested that ClC-2 serves as an exit pathway for Cl(-) in the basolateral membranes of the distal colon and that its dependence on [Cl(-)](i) might provide a cross-talk mechanism to match fluxes at the apical and basolateral domains of these epithelial cells.

Animals↗
PubMed1995

Mutations in dominant human myotonia congenita drastically alter the voltage dependence of the CIC-1 chloride channel.

Autosomal dominant myotonia congenita (Thomsen's disease) is caused by mutations in the muscle chloride channel CIC-1. Several point mutations found in affected families (I29OM, R317Q, P480L, and Q552R) dramatically shift gating to positive voltages in mutant/WT heterooligomeric channels, and when measurable, even more so in mutant homooligomers. These channels can no longer contribute to the repolarization of action potentials, fully explaining why they cause dominant myotonia. Most replacements of the isoleucine at position 290 shift gating toward positive voltages. Mutant/WT heterooligomers can be partially activated by repetitive depolarizations, suggesting a role in shortening myotonic runs. Remarkably, a human mutation affecting an adjacent residue (E291K) is fully recessive. Large shifts in the voltage dependence of gating may be common to many mutations in dominant myotonia congenita.

Amino Acid Sequence↗
PubMed1987

The chloride channel blocking agent, t-butyl bicyclophosphorothionate, binds to the gamma-aminobutyric acid-benzodiazepine, but not to the glycine receptor in rodents.

The inhibitory neurotransmitters glycine and gamma-aminobutyric acid (GABA) both activate transmembrane chloride channels of similar physical characteristics. A common ion channel component has therefore been postulated for both the glycine and GABA receptor proteins. Different convulsant drugs as picrotoxin and t-butyl bicyclophosphorothionate (TBPS) have been reported as channel-blocking ligands of the GABA receptor. Here, we show that the distribution of [35S]TBPS binding sites parallels the binding of the GABA receptor ligand [3H]flunitrazepam, but not that of the glycine receptor antagonist [3H]strychnine. Binding was examined in membrane fractions from different regions of the rat CNS and of the mutant mouse spastic, an animal deficient in glycine receptors. Also, affinity purification of the glycine receptor on aminostrychnine-agarose resulted in almost complete removal of [35S]TPBS binding sites from the receptor preparation. It is concluded that TBPS selectively binds to the GABA, but not glycine, receptor chloride channel complex.

Animals↗
PubMed1996

Chloride channels in excised membrane patches from human platelets: effect of intracellular calcium.

Human platelets were studied by patch clamp recordings from inside-out membranes; there were formed by briefly dipping the platelet, in cell-attached mode, into silicone grease. At 20 degrees C in symmetrical 150 mM NaCl, spontaneous channel openings were rarely observed at negative potentials, whereas depolarised potentials (+ 60 to + 100 mV) elicited sustained channel activity in 38% of patches. The single channel conductance was 53 +A- 1 pS at + 80 mV (outward current), decreasing to 20 +/- 2 pS at -80 mV (inward current). Ion substitution experiments indicated that this channel conducts Cl- and not Na+. Furthermore, 5-nitro-2-(3-phenylpropylamino)benzoate (100 microM), a recognized inhibitor of anion channels, induced a reversible 'flickery' channel block. We estimate that each platelet possesses < or = 30 such channels. Kinetic analysis suggested at least two open channel states (tau = 0.8 +/- 0.2 ms, tau = 22 +/- 14 ms, n = 4) and two closed states (tau = 0.8 +/- 0.2 ms, tau = 12 +/- 0.6 ms, n = 4). Increasing [Ca2+]i to 10 microM, following channel activation by depolarisation, had no significant effect on channel kinetics or open probability, however, elevated [Ca2+]i (300 nM-10 microM) increased the number of anion channels activated by subsequent depolarisation. This study represents the first recordings of ionic currents in excised, inside-out membrane patches from human platelets, and provides further evidence for the existence of chloride channels in these cells.

Blood Platelets↗
PubMed1993

Insecticide action at the GABA-gated chloride channel: recognition, progress, and prospects.

Three billion (3 x 10(9)) pounds of hexachlorocyclohexanes, polychlorobornanes, and chlorinated cyclodienes (such as lindane, toxaphene, and endosulfan, respectively) were used to control pest insects before their mode of action was established as blocking the GABA-gated chloride channel. With the restricted use or demise of these polychlorocycloalkanes (each approximately 50 to approximately 75% by weight of chlorine), the GABAergic system is for now an underutilized target of insecticide action. Newer compounds with outstanding potency at this receptor and as toxicants to houseflies are suitably-substituted 2,6,7-trioxabicyclo[2.2.2]-octanes, particularly the bicycloorthobenzoates, and 1,3-dithianes, including those with no halogenated substituents. Picrotoxinin analogs and alkynylphenyl-silatranes also act at this target but are of lower insecticidal activity. [3H]n-Propyl-ethynylbicycloorthobenzoate ([3H]EBOB) is for now the best radioligand for this insecticidally-relevant binding site in insects. Macrocyclic lactones such as the avermectins and moxidectin act at a different binding site to disrupt chloride flux and they have a different spectrum of insecticidal activity and no cross resistance with cyclodienes in houseflies. The search for new insecticides has provided the incentive and probes for a better understanding of the insect GABAergic system.

Animals↗
PubMed2002

CFTR chloride channels are regulated by a SNAP-23/syntaxin 1A complex.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate membrane fusion reactions in eukaryotic cells by assembling into complexes that link vesicle-associated SNAREs with SNAREs on target membranes (t-SNAREs). Many SNARE complexes contain two t-SNAREs that form a heterodimer, a putative intermediate in SNARE assembly. Individual t-SNAREs (e.g., syntaxin 1A) also regulate synaptic calcium channels and cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial chloride channel that is defective in cystic fibrosis. Whether the regulation of ion channels by individual t-SNAREs is related to SNARE complex assembly and membrane fusion is unknown. Here we show that CFTR channels are coordinately regulated by two cognate t-SNAREs, SNAP-23 (synaptosome-associated protein of 23 kDa) and syntaxin 1A. SNAP-23 physically associates with CFTR by binding to its amino-terminal tail, a region that modulates channel gating. CFTR-mediated chloride currents are inhibited by introducing excess SNAP-23 into HT29-Cl.19A epithelial cells. Conversely, CFTR activity is stimulated by a SNAP-23 antibody that blocks the binding of this t-SNARE to the CFTR amino-terminal tail. The physical and functional interactions between SNAP-23 and CFTR depend on syntaxin 1A, which binds to both proteins. We conclude that CFTR channels are regulated by a t-SNARE complex that may tune CFTR activity to rates of membrane traffic in epithelial cells.

Animals↗
PubMed1997

Reconstitution of functional voltage-gated chloride channels from complementary fragments of CLC-1.

We investigated the effect of truncations on the human muscle chloride channel CLC-1 and studied the functional complementation from partial proteins. Almost complete deletion of the cytoplasmic amino terminus did not affect currents, but truncating the intracellular COOH terminus after Leu720 abolished function. Currents were restored by coexpressing this membrane-embedded part with the lacking cytoplasmic fragment that contains domain D13, the second of the two conserved cystathionine beta-synthase (CBS) motifs present in all eukaryotic CLC proteins. However, if the cut was after Gln597 before the first CBS domain, no functional complementation was seen. Complementation was also obtained with channels "split" between transmembrane domains D7 and D8 or domains D8 and D9, but not when split between D10 and D11. Specificity of currents was tested by inserting point mutations in NH2-terminal (G188A and G230E) or COOH-terminal (K585E) fragments. In contrast to G188A and K585E, split channels did not tolerate the D136G mutation, suggesting that it may impede association from nonlinked fragments. Duplication, but not a lack of domain D8 was tolerated in "split" channels. Membrane domains D9-D12 can insert into the membrane without adding a preceding signal peptide to ensure the extracellular amino terminus of D9. Eventually, we succeeded in reconstituting CLC-1 channels from three separate polypeptides: the amino-terminal part up to D8, D9 through CBS1, and the remainder of the cytoplasmic carboxyl terminus. In summary, several regions of CLC channels behave autonomously regarding membrane insertion and folding and mediate protein-protein interactions strong enough to yield functional channels without a direct covalent link.

Animals↗
PubMed1994

Amino acid residues lining the chloride channel of the cystic fibrosis transmembrane conductance regulator.

The cystic fibrosis transmembrane conductance regulator forms a chloride channel that is regulated by phosphorylation and intracellular ATP levels. The structure of the channel-forming domains is undetermined. To identify the residues lining this channel we substituted cysteine, one at a time, for 9 consecutive residues (91-99) in the M1 membrane-spanning segment. The cysteine substitution mutants were expressed in Xenopus oocytes. We determined the accessibility of the engineered cysteine to charged, sulfhydryl-specific methanethiosulfonate reagents added extracellularly. We assume that, among residues in membrane-spanning segments, only those lining the channel will be accessible to react with these hydrophilic reagents and that such a reaction would irreversibly alter conduction through the channel. Only the cysteines substituted for Gly-91, Lys-95, and Gln-98 were accessible to the reagents. We conclude that these residues are in the channel lining. The periodicity of these residues is consistent with an alpha-helical secondary structure.

Amino Acid Sequence↗
PubMed1994

Dual action of the cyclodiene insecticide dieldrin on the gamma-aminobutyric acid receptor-chloride channel complex of rat dorsal root ganglion neurons.

The gamma-aminobutyric acidA (GABAA) receptor-chloride channel complex is known to be the target site of dieldrin, a cyclodiene insecticide, and lindane. In order to elucidate the mechanisms of dieldrin interaction with the GABA system, whole-cell patch clamp experiments were performed with rat dorsal root ganglion neurons in primary culture. When co-applied with GABA, dieldrin exerted a dual effect on the GABA-induced chloride current. The chloride current induced by 10 microM GABA was greatly enhanced by the first 20-sec co-application with 1 microM dieldrin, but the enhancement subsided during repeated co-applications, and the current was eventually suppressed below the control level. No recovery occurred after a prolonged washing with dieldrin-free solution. Desensitization of the chloride current was accelerated by dieldrin. However, when the period of co-application was limited to 2 sec, which was short enought to avoid desensitization, no suppression of current was observed during repeated co-applications and recovery occurred after washing. The desensitization and suppression occurred with an EC50 of 92 nM, whereas the enhancement required a higher EC50 at 754 nM. The GABA-induced chloride current comprised two components, one with a high sensitivity to dieldrin suppression with an EC50 of 5 nM and the other with a lower sensitivity with an EC50 of 92 nM. Dieldrin exerted its inhibitory effect on the GABA-induced current regardless of the presence or absence of pentobarbital and chlordiazepoxide. However, its effect was attenuated by the presence of picrotoxin. Furthermore, dieldrin suppressed the GABA-induced chloride current in a noncompetitive manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2006

Recovery of mucosal barrier function in ischemic porcine ileum and colon is stimulated by a novel agonist of the ClC-2 chloride channel, lubiprostone.

Previous studies utilizing an ex vivo porcine model of intestinal ischemic injury demonstrated that prostaglandin (PG)E(2) stimulates repair of mucosal barrier function via a mechanism involving Cl(-) secretion and reductions in paracellular permeability. Further experiments revealed that the signaling mechanism for PGE(2)-induced mucosal recovery was mediated via type-2 Cl(-) channels (ClC-2). Therefore, the objective of the present study was to directly investigate the role of ClC-2 in mucosal repair by evaluating mucosal recovery in ischemia-injured intestinal mucosa treated with the selective ClC-2 agonist lubiprostone. Ischemia-injured porcine ileal mucosa was mounted in Ussing chambers, and short-circuit current (I(sc)) and transepithelial electrical resistance (TER) were measured in response to lubiprostone. Application of 0.01-1 microM lubiprostone to ischemia-injured mucosa induced concentration-dependent increases in TER, with 1 microM lubiprostone stimulating a twofold increase in TER (DeltaTER = 26 Omega.cm(2); P < 0.01). However, lubiprostone (1 microM) stimulated higher elevations in TER despite lower I(sc) responses compared with the nonselective secretory agonist PGE(2) (1 microM). Furthermore, lubiprostone significantly (P < 0.05) reduced mucosal-to-serosal fluxes of (3)H-labeled mannitol to levels comparable to those of normal control tissues and restored occludin localization to tight junctions. Activation of ClC-2 with the selective agonist lubiprostone stimulated elevations in TER and reductions in mannitol flux in ischemia-injured intestine associated with structural changes in tight junctions. Prostones such as lubiprostone may provide a selective and novel pharmacological mechanism of accelerating recovery of acutely injured intestine compared with the nonselective action of prostaglandins such as PGE(2).

Alprostadil↗
PubMed1999

Induction of mouse Ca(2+)-sensitive chloride channel 2 gene during involution of mammary gland.

To understand molecular mechanisms that regulate mammary gland involution, we identified involution-induced cDNA clones by suppression subtractive hybridization methods. Nucleotide sequencing of a clone revealed that it was 97% identical to Ca(2+)-sensitive chloride channel 1 (mCLCA1) gene that has been identified in lung tissue. We concluded that our clone was derived from different gene with mCLCA1 and named it mCLCA2. We confirmed that expression of mCLCA2 gene was predominant in mammary gland while mCLCA1 mRNA was mainly detected in lung tissues by RT-PCR. Northern analysis showed that the mCLCA2 gene was induced at involution phase compared to pregnant and lactating phases of mammary gland. Under serum starvation, HC11 mammary epithelial cells showed DNA fragmentation and induction of mCLCA2 expression.

Amino Acid Sequence↗
PubMed1995

Characterization of a human and murine gene (CLCN3) sharing similarities to voltage-gated chloride channels and to a yeast integral membrane protein.

We describe the isolation and characterization of a human gene (CLCN3) and its murine homologue (Clcn3) sharing significant sequence and structural similarities with all previously identified members of the voltage-gated chloride channel (ClC) family. This gene is expressed primarily in tissues derived from neuroectoderm. Within the brain, Clcn3 expression is particularly evident in the hippocampus, olfactory cortex, and olfactory bulb. CLCN3 encodes a 760-amino-acid protein that differs by only 2 amino acid residues from the protein encoded by Clcn3. CLCN3 protein also shows a high similarity with GEF1, an integral membrane protein of the yeast Saccharomyces cerevisiae known to be involved in respiration and iron-limited cell growth, and with the predicted protein product of a DNA sequence from the mold Septoria nodorum. This high degree of sequence conservation in very distantly related species such as human and yeast indicates that this gene has retained a fundamental function throughout evolution.

Amino Acid Sequence↗
PubMed1991

Chloride channel blockers inhibit ACTH secretion from mouse pituitary tumor cells.

The effect of several chemically related chloride channel blocking drugs was investigated on the adrenocorticotropic hormone (ACTH) secretory process in mouse clonal AtT-20 corticotrophs. When cells were simultaneously exposed to diphenylamine-2-carboxylate (DPC) or related substances (Hoechst compounds 131, 143, and 144) and the adenylate cyclase activator forskolin, ACTH secretion was inhibited by 76-95% [half-maximal inhibitory concentration (IC50) 450, 15, 84, and 32 microM, respectively]. All four compounds also blocked forskolin-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) synthesis in AtT-20 cells by 51-87% (IC50 190, 29, 100, and 130 microM for DPC and compounds 131, 143, and 144, respectively). Pertussis toxin pretreatment of cells caused a partial reversal of DPC-inhibited forskolin-stimulated cAMP formation. The toxin had no effect on inhibition of forskolin-stimulated ACTH secretion by DPC. Secretion of ACTH in response to cAMP-independent stimulants such as the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate or the calcium channel agonist BAY K 8644 were blocked by compound 131 as was the secretory response to 8-bromoadenosine 3',5'-cyclic monophosphate. These results suggest that phenylanthranilic acids have adenylate cyclase inhibiting action but that the postcyclase activity is more relevant to the ability of these compounds to block ACTH secretion. DPC also blocked 125I efflux (an index of Cl- secretion) from AtT-20 cells. Because an increase in osmotic strength of the culture media reduced forskolin-stimulated ACTH secretion, these data suggest that DPC and related compounds may negatively modulate chloride-dependent osmotically driven ACTH secretion from AtT-20 cells.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed2004

Electrostatics of ion stabilization in a ClC chloride channel homologue from Escherichia coli.

The structural determinants of electrostatics of ion stabilization within EcClC, a ClC-type chloride channel homologue from Escherichia coli, are studied using a continuum dielectric approximation. Specifically, the ion occupancy is investigated in the wild-type protein and a mutant thereof, and the contribution to the electrostatic binding free energy of local and non-local interactions is characterized at the single-residue level. This analysis shows that, in spite of the desolvation cost and the strong ion-ion repulsion, all previously reported binding sites can be occupied simultaneously. The stabilizing effect of the protein arises from hydrogen bonding as well as from longer-range favorable interactions, such as with the strictly conserved Lys131 side-chain. The latter is involved in the stabilization of the conserved GSGIP motif that delimits two of the binding sites. Interestingly, an additional low-affinity binding site, mediated by a structurally analogous motif including the side-chain of Arg340, can be identified on the extracellular side of the permeation pathway. Finally, it is shown that, in contrast to K-channels, and in analogy to the SBP/PBP sulfate/phosphate-binding proteins, the contribution of helix macrodipoles to chloride binding in EcClC is only marginal.

Amino Acid Sequence↗
PubMed1998

Block by picrotoxin of a GABAergic chloride channel expressed on crayfish muscle after axotomy.

Outside-out patches containing a gamma-aminobutyric acid (GABA)-activated chloride channel expressed after axotomy on crayfish deep extensor abdominal muscle were excised. GABA and the blocker picrotoxin (PTX) were applied to the patches using a liquid filament switch to study the effects of picrotoxin on the GABA-elicited currents. Coapplication of GABA and PTX resulted in a reduction of the current amplitude compared with that elicited by the same GABA concentration alone. This reduction of the amplitude was dependent on both the GABA and PTX concentrations. The rise time of the current decreased after coapplication of GABA and PTX. Evaluation of the single channel currents and off-currents in the presence of GABA and PTX showed a dramatic shortening of the burst duration of the channel. The open time distributions were not altered, whereas in the closed time distributions a new closed time was apparent in presence of PTX. Preincubation with PTX prior to the GABA pulse resulted in an increase of the rise time. This effect was dependent on the PTX concentration only. Possible mechanisms are discussed to explain the effects of PTX and are implemented into the existing molecular reaction scheme of the channel.

Animals↗
PubMed1999

Dynamic diversification from a putative common ancestor of scorpion toxins affecting sodium, potassium, and chloride channels.

Scorpions have survived successfully over millions of years without detectable changes in their morphology. Instead, they have developed an efficient alomonal machinery and a stinging device supporting their needs for prey and defense. They produce a large variety of polypeptidic toxins that bind and modulate ion channel conductance in excitable tissues. The binding site, mode of action, and chemical properties of many toxins have been studied extensively, but little is known about their genomic organization and diversity. Genes representing each of the major classes of Buthidae scorpion toxins, namely, "long" toxins, affecting sodium channels (alpha, depressant, and excitatory), and "short" toxins, affecting potassium and chloride channels, were isolated from a single scorpion segment and analyzed. Each toxin type was found to be encoded by a gene family. Regardless of toxin length, 3-D structure, and site of action, all genes contain A+T-rich introns that split, at a conserved location, an amino acid codon of the signal sequence. The introns vary in length and sequence but display identical boundaries, agree with the GT/AG splice junctions, and contain T-runs downstream of a putative branch point, 5'-TAAT-3'. Despite little sequence similarity among all toxin classes, the conserved gene organization, intron features, and common cysteine-stabilized alpha-helical (CSH) core connecting an alpha-helix to a three-stranded beta-sheet suggest, that they all evolved from an ancestral common progenitor. Furthermore, the vast diversity found among genomic copies, cDNAs, and their protein products for each toxin suggests an extensive evolutionary process of the scorpion "pharmaceutical factory," whose success is due, most likely, to the inherent permissiveness of the toxin exterior to structural alterations.

Amino Acid Sequence↗
PubMed2003

The chloride channel ClC-4 contributes to endosomal acidification and trafficking.

Mutations in the gene coding for the chloride channel ClC-5 cause Dent's disease, a disease associated with proteinuria and renal stones. Studies in ClC-5 knockout mice suggest that this phenotype is related to defective endocytosis of low molecular weight proteins and membrane proteins by the renal proximal tubule. In this study, confocal micrographs of proximal tubules and cultured epithelial cells revealed that the related protein ClC-4 is expressed in endosomal membranes suggesting that this channel may also contribute to the function of this organelle. In support of this hypothesis, specific disruption of endogenous ClC-4 expression by transfection of ClC-4 antisense cDNA acidified endosomal pH and altered transferrin trafficking in cultured epithelial cells to the same extent as the specific disruption of ClC-5. Both channels can be co-immunoprecipitated, arguing that they may partially contribute to endosomal function as a channel complex. These studies prompt future investigation of the role of ClC-4 in renal function in health and in Dent's disease. Future studies will assess whether the severity of Dent's disease relates not only to the impact of particular mutations on ClC-5 but also on the consequences of those mutations on the functional expression of ClC-4.

Animals↗
PubMed1999

Novel mutations in the muscle chloride channel CLCN1 gene causing myotonia congenita in Spanish families.

Mutations in the muscular voltage-dependent chloride channel gene (CLCN1), located at 7q35, lead to recessive and dominant myotonia congenita. We report four novel mutations identified in this gene, after clinical, electromyographic, and genetic studies performed on 13 unrelated families. Two of the four mutations (2512insCTCA and A218T) were identified in families with Thomsen's disease, one (Q658X) in a family with Becker's disease, and the fourth (R669C) in a presumably sporadic patient with the Becker phenotype. Although identification of the mutations allows us to establish some genotype/phenotype correlations, this does not wholly account for the clinical heterogeneity and the inheritance patterns of the disease.

Chloride Channels↗