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At least 847 records · Page 47Linked to original sources
PubMed2000

Drug-resistant Drosophila indicate glutamate-gated chloride channels are targets for the antiparasitics nodulisporic acid and ivermectin.

The fruit fly Drosophila melanogaster was used to examine the mode of action of the novel insecticide and acaricide nodulisporic acid. Flies resistant to nodulisporic acid were selected by stepwise increasing the dose of drug in the culture media. The resistant strain, glc(1), is at least 20-fold resistant to nodulisporic acid and 3-fold cross-resistant to the parasiticide ivermectin, and exhibited decreased brood size, decreased locomotion, and bang sensitivity. Binding assays using glc(1) head membranes showed a marked decrease in the affinity for nodulisporic acid and ivermectin. A combination of genetics and sequencing identified a proline to serine mutation (P299S) in the gene coding for the glutamate-gated chloride channel subunit DmGluClalpha. To examine the effect of this mutation on the biophysical properties of DmGluClalpha channels, it was introduced into a recombinant DmGluClalpha, and RNA encoding wild-type and mutant subunits was injected into Xenopus oocytes. Nodulisporic acid directly activated wild-type and mutant DmGluClalpha channels. However, mutant channels were approximately 10-fold less sensitive to activation by nodulisporic acid, as well as ivermectin and the endogenous ligand glutamate, providing direct evidence that nodulisporic acid and ivermectin act on DmGluClalpha channels.

Animals↗
PubMed2002

Distribution of ClC-2 chloride channel in rat and human epithelial tissues.

The ubiquitous ClC-2 Cl(-) channel is thought to contribute to epithelial Cl(-) secretion, but the distribution of the ClC-2 protein in human epithelia has not been investigated. We have studied the distribution of ClC-2 in adult human and rat intestine and airways by immunoblotting and confocal microscopy. In the rat, ClC-2 was present in the lateral membranes of villus enterocytes and was predominant at the basolateral membranes of luminal colon enterocytes. The expression pattern of ClC-2 in the human intestine differed significantly, because ClC-2 was mainly detected in a supranuclear compartment of colon cells. We found significant expression of ClC-2 at the apex of ciliated cells in both rat and human airways. These results show that the distribution of ClC-2 in airways is consistent with participation of ClC-2 channels in Cl(-) secretion and indicate that extrapolation of results from studies of ClC-2 function in rat intestine to human intestine is not straightforward.

Amino Acid Sequence↗
PubMed1995

Excision-activated chloride channels in cultured postnatal rat hippocampal neurons.

Chloride permeable intermediate conductance single channel events activated on patch excision were found in outside-out patches from cultured postnatal hippocampal neurons. A majority of the channels had a conductance of 83 +/- 2.1 pS when recorded in a symmetrical TEACl solution. Two other populations of channels with conductance values of 62 +/- 2.1 pS and 145 +/- 1.9 pS were also observed. The reversal potentials for these intermediate conductance Cl- channels coincided with that of the GABA activated channels. The channels characteristically appeared 5-15 min after patch excision, suggesting that these channels may be blocked by some diffusible factors under physiological conditions. Based on the measurements of channel burst durations while the channel was partially blocked, and the channel open times after complete relief from the block, the mechanism of blockade does not appear to be a simple open channel blockade. The high prevalence and its potential regulation by cytosolic factors suggest an important physiological role for these Cl- channels coupling neuronal excitability with cellular metabolism.

Animals↗
PubMed1997

EAAT4, a glutamate transporter with properties of a chloride channel, is predominantly localized in Purkinje cell dendrites, and forms parasagittal compartments in rat cerebellum.

Glutamate transporters play a pivotal role in terminating glutamatergic transmission by eliminating glutamate from the synaptic cleft. Four different glutamate transporter cDNAs have been isolated thus far, and their tissue distribution has been investigated using northern blot and immunohistochemical analysis. We raised site-directed antisera against a synthetic oligopeptide corresponding to the C-terminal of EAAT4, a recently cloned human glutamate transporter, and investigated the distribution of EAAT4 in rat cerebellum. Western blot analysis demonstrated that the affinity-purified antiserum SAE4 recognized specifically a single band (about 62 kDa) in the rat cerebellum, cerebrum and spinal cord. The SAE4-immunoreactivity was localized predominantly in the dendritic spines and distal dendrites of Purkinje cells. The intensity of the immunoreactivity was uneven among Purkinje cells, forming parasagittal compartments. Since EAAT4 also has the properties of a glutamate-gated chloride channel, it should be able to modulate the transmission at the parallel fiber-Purkinje cell synapses.

Amino Acid Transport System X-AG↗
PubMed2006

Genetic segregation of airway disease traits despite redundancy of calcium-activated chloride channel family members.

Complex airway diseases such as asthma and chronic obstructive pulmonary disease exhibit stereotyped traits (especially airway hyperreactivity and mucous cell metaplasia) that are variably expressed in each patient. Here, we used a mouse model for virus-induced long-term expression of these traits to determine whether individual traits can be genetically segregated and thereby linked to separate determinants. We showed that an F2 intercross population derived from susceptible and nonsusceptible mouse strains can manifest individual phenotypic extremes that exhibit one or the other disease trait. Functional genomic analysis of these extremes further indicated that a member of the calcium-activated chloride channel (CLCA) gene family designated mClca3 was inducible with mucous cell metaplasia but not airway hyperreactivity. In confirmation of this finding, we found that mClca3 gene transfer to mouse airway epithelium was sufficient to induce mucous cell metaplasia but not airway hyperreactivity. However, newly developed mClca3(-/-) mice exhibited the same degree of mucous cell metaplasia and airway hyperreactivity as wild-type mice. Bioinformatic analysis of the Clca locus led to the identification of mClca5, and gene transfer indicated that mClca5 also selectively drives mucous cell metaplasia. Thus, in addition to the capacity of CLCA family members to exhibit diverse functional activities, there is also preserved function so that more than one family member mediates mucous cell metaplasia. Nonetheless, Clca expression appears to be a selective determinant of mucous cell metaplasia so that shared homologies between CLCA family members may still represent a useful target for focused therapeutic intervention in hypersecretory airway disease.

Animals↗
PubMed1995

Cloning and characterization of CLCN5, the human kidney chloride channel gene implicated in Dent disease (an X-linked hereditary nephrolithiasis).

Dent disease, an X-linked familial renal tubular disorder, is a form of Fanconi syndrome associated with proteinuria, hypercalciuria, nephrocalcinosis, kidney stones, and eventual renal failure. We have previously used positional cloning to identify the 3' part of a novel kidney-specific gene (initially termed hClC-K2, but now referred to as CLCN5), which is deleted in patients from one pedigree segregating Dent disease. Mutations that disrupt this gene have been identified in other patients with this disorder. Here we describe the isolation and characterization of the complete open reading frame of the human CLCN5 gene, which is predicted to encode a protein of 746 amino acids, with significant homology to all known members of the ClC family of voltage-gated chloride channels. CLCN5 belongs to a distinct branch of this family, which also includes the recently identified genes CLCN3 and CLCN4. We have shown that the coding region of CLCN5 is organized into 12 exons, spanning 25-30 kb of genomic DNA, and have determined the sequence of each exon-intron boundary. The elucidation of the coding sequence and exon-intron organization of CLCN5 will both expedite the evaluation of structure/function relationships of these ion channels and facilitate the screening of other patients with renal tubular dysfunction for mutations at this locus.

Adult↗
PubMed1998

Functional and molecular expression of volume-regulated chloride channels in canine vascular smooth muscle cells.

1. We examined the possibility of functional and molecular expression of volume-regulated Cl- channels in vascular smooth muscle using the whole-cell patch-clamp technique and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) on cells from canine pulmonary and renal arteries. 2. Decreasing external osmolarity induced cell swelling, which was accompanied by activation of Cl--dependent outward-rectifying membrane currents with an anion permeability sequence of SCN- > I- > Br- > Cl- > aspartate-. These currents were sensitive to block by DIDS, extracellular ATP and the antioestrogen compound tamoxifen. 3. Experiments were performed to determine whether the molecular form of the volume-regulated chloride channel (ClC-3) is expressed in pulmonary and renal arteries. Quantitative RT-PCR confirmed expression of ClC-3 in both types of smooth muscle. ClC-3 expression was 76.4 % of beta-actin in renal artery and 48.0 % of beta-actin in pulmonary artery. 4. We conclude that volume-regulated Cl- channels are expressed in vascular smooth muscle cells and exhibit functional properties similar to those found in other types of cells, presumably contributing to the regulation of cell volume, electrical activity and, possibly, myogenic tone.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1990

Decrease in the function of the gamma-aminobutyric acid-coupled chloride channel produced by the repeated administration of pentylenetetrazol to rats.

The acute administration of pentylenetetrazol (PTZ; 25-75 mg/kg i.p.) failed to modify the specific binding of t-[35S]butylbicyclophosphorothionate ([35S]TBPS) to membrane preparations from the cerebral cortex of the rat. In contrast, the repeated administration of PTZ (30 mg/kg i.p., three times a week for 12 weeks) reduced by 26% the density of [35S]TBPS binding sites without modifying the dissociation constant. This effect was observed 3 days after the last PTZ administration. A parallel reduction of gamma-aminobutyric acid (GABA)-stimulated 36Cl- uptake was measured in the cerebral cortex of PTZ-treated rats 3 days after the last injection. The repeated administration of PTZ produced sensitization to the drug, or chemical kindling. In fact, no convulsions were observed in the first week of treatment, but all the animals became sensitized to PTZ by the 12th week. The results are consistent with the hypothesis that chronic treatment with PTZ at a subconvulsant dose causes a decrease in GABA-coupled chloride channel activity that may be related to the chemical kindling produced by this compound.

Animals↗
PubMed1996

The tyrosine kinase p60c-src regulates the fast gate of the cystic fibrosis transmembrane conductance regulator chloride channel.

The role of the tyrosine kinase p60c-src on the gating of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel was investigated with the cell-attached and excised patch clamp technique in conjunction with current noise analysis of recordings containing multiple channels per patch. Spectra of CFTR-generated current noise contained a low-frequency and a high-frequency Lorentzian noise component. In the cell-attached mode, the high-frequency Lorentzian was significantly dependent on the membrane potential, while the low-frequency Lorentzian was unaffected. Excision of forskolin-stimulated patches into ATP-containing solution significantly reduced the amplitude of the voltage-dependent high-frequency Lorentzian. Addition of the tyrosine kinase p60c-src to excised, active, CFTR-containing membrane patches increased mean currents by 54%, increased the corner frequency of the low-frequency Lorentzian, and recovered the high-frequency Lorentzian and its characteristics. Treatment with lambda-phosphatase inactivated src-induced currents and changes in gating. When active patches were excised under conditions in which patch-associated tyrosine phosphatases were blocked with sodium vanadate, the high-frequency gating remained relatively unchanged. The results suggest that CFTR's open probability and its voltage-dependent fast gate are dependent on tyrosine phosphorylation, and that membrane-associated tyrosine phosphatases are responsible for inactivation of the fast gate after patch excision.

3T3 Cells↗
PubMed2001

Dietary glycine prevents peptidoglycan polysaccharide-induced reactive arthritis in the rat: role for glycine-gated chloride channel.

Peptidoglycan polysaccharide (PG-PS) is a primary structural component of bacterial cell walls and causes rheumatoid-like arthritis in rats. Recently, glycine has been shown to be a potential immunomodulator; therefore, the purpose of this study was to determine if glycine would be protective in a PG-PS model of arthritis in vivo. In rats injected with PG-PS intra-articularly, ankle swelling increased 21% in 24 to 48 h and recovered in about 2 weeks. Three days prior to reactivation with PG-PS given intravenously (i.v.), rats were divided into two groups and fed a glycine-containing or nitrogen-balanced control diet. After i.v. PG-PS treatment joint swelling increased 2.1 +/- 0.3 mm in controls but only 1.0 +/- 0.2 mm in rats fed glycine. Infiltration of inflammatory cells, edema, and synovial hyperplasia in the joint were significantly attenuated by dietary glycine. Tumor necrosis factor alpha (TNF-alpha) mRNA was detected in ankle homogenates from rats fed the control diet but not in ankles from rats fed glycine. Moreover, intracellular calcium was increased significantly in splenic macrophages treated with PG-PS; however, glycine blunted the increase about 50%. The inhibitory effect of glycine was reversed by low concentrations of strychnine or chloride-free buffer, and it increased radiolabeled chloride influx nearly fourfold, an effect also inhibited by strychnine. In isolated splenic macrophages, glycine blunted translocation of the p65 subunit of NF-kappaB into the nucleus, superoxide generation, and TNF-alpha production caused by PG-PS. Further, mRNA for the beta subunit of the glycine receptor was detected in splenic macrophages. This work supports the hypothesis that glycine prevents reactive arthritis by blunting cytokine release from macrophages by increasing chloride influx via a glycine-gated chloride channel.

Animals↗
PubMed2005

Voltage-gated calcium and Ca2+-activated chloride channels and Ca2+ transients: voltage-clamp studies of perfused and intact cells of Chara.

The voltage-clamp technique was used to study Ca(2+) and Cl(-) transient currents in the plasmalemma of tonoplast-free and intact Chara corallina cells. In tonoplast-free cells [perfused medium with ethylene glycol bis(2-aminoethyl ether)tetraacetic acid] long-term inward and outward currents through Ca channels consisted of two components: with and without time-dependent inactivation. The voltage dependence of the Ca channel activation ratio was found to be sigmoid-shaped, with about -140-mV activation threshold, reaching a plateau at V>50 mV. As the voltage increased, the characteristic activation time decreased from approximately 10(3) ms in the threshold region to approximately 10 ms in the positive region. The positive pulse-activated channels can then be completely deactivated, which is recorded by the Ca(2+) tail currents, at below-threshold negative voltages with millisecond-range time constants. This tail current is used for fast and brief Ca(2+) injection into tonoplast-free and intact cells, to activate the chloride channels by Ca(2+) . When cells are perfused with EDTA-containing medium in the presence of excess Mg(2+), this method of injection allows the free submembrane Ca(2+) concentration, [Ca(2+)](c), to be raised rapidly to several tens of micromoles per liter. Then a chloride component is recorded in the inward tail current, with the amplitude proportional to [see text]. When Ca(2+) is thus injected into an intact cell, it induces an inward current in the voltage-clamped plasmalemma, having activation-inactivation kinetics qualitatively resembling that in EDTA-perfused cells, but a considerably higher amplitude and duration (approximately 10 A m(-2) and tau(inact)~0.5 s at -200 mV). Analysis of our data and theoretical considerations indicate that the [Ca(2+)](c) rise during cell excitation is caused mainly by Ca(2+) entry through plasmalemma Ca channels rather than by Ca(2+) release from intracellular stores.

Calcium↗
PubMed1994

Isolation and partial characterization of a chloride channel gene which is expressed in kidney and is a candidate for Dent's disease (an X-linked hereditary nephrolithiasis).

Dent's disease, an X-linked renal tubular disorder, is a form of Fanconi syndrome which is characterized by proteinuria, hypercalciuria, nephrocalcinosis, kidney stones and renal failure. Previous studies localised the gene responsible to Xp11.22, within a microdeletion involving the hypervariable locus DXS255. Further analysis using new probes which flank this locus indicate that the deletion is less than 515 kb. A 185 kb YAC containing DXS255 was used to screen a cDNA library from adult kidney in order to isolate coding sequences falling within the deleted region which may be implicated in the disease aetiology. We identified two clones which are evolutionarily conserved, and detect a 9.5 kb transcript which is expressed predominantly in the kidney. Sequence analysis of 780 bp of ORF from the clones suggests that the identified gene, termed hCIC-K2, encodes a new member of the CIC family of voltage-gated chloride channels. Genomic fragments detected by the cDNA clones are completely absent in patients who have an associated microdeletion. On the basis of the expression pattern, proposed function and deletion mapping, hCIC-K2 is a strong candidate for Dent's disease.

Amino Acid Sequence↗
PubMed1997

Cloning and expression of a complementary DNA encoding a molluscan octopamine receptor that couples to chloride channels in HEK293 cells.

A cDNA encoding a G-protein-coupled receptor was cloned from the central nervous system of the pond snail Lymnaea stagnalis. The predicted amino acid sequence of this cDNA most closely resembles the Drosophila tyramine/octopamine receptor, the Locusta tyramine receptor, and an octopamine receptor (Lym oa1) that we recently cloned from Lymnaea. After stable expression of the cDNA in HEK293 cells, we found that [3H]rauwolscine binds with high affinity to the receptor (KD = 6.2.10(-9) M). Octopamine appears to be the most potent naturally occurring agonist to displace the [3H]rauwolscine binding (Ki = 3.0.10(-7) M). Therefore, the receptor is considered to be an octopamine receptor and is consequently designated Lym oa2. The novel receptor shares little pharmacological resemblance with Lym oa1, indicating that the two receptors represent different octopamine receptor subfamilies. Octopaminergic stimulation of Lym oa2 does not induce changes in intracellular concentrations of cAMP or inositol phosphates. However, electrophysiological experiments indicate that octopamine is able to activate a voltage-independent Cl- current in HEK293 cells stably expressing Lym oa2. Although opening of this chloride channel most probably does not require the activation of either protein kinase A or C, it can be blocked by inhibition of protein phosphorylation.

Amino Acid Sequence↗
PubMed1997

Inactivation of calcium-activated chloride channels in smooth muscle by calcium/calmodulin-dependent protein kinase.

To determine the mechanisms responsible for the termination of Ca2+-activated Cl- currents (ICl(Ca)), simultaneous measurements of whole cell currents and intracellular Ca2+ concentration ([Ca2+]i) were made in equine tracheal myocytes. In nondialyzed cells, or cells dialyzed with 1 mM ATP, ICl(Ca) decayed before the [Ca2+]i decline, whereas the calcium-activated potassium current decayed at the same rate as [Ca2+]i. Substitution of AMP-PNP or ADP for ATP markedly prolonged the decay of ICl(Ca), resulting in a rate of current decay similar to that of the fall in [Ca2+]i. In the presence of ATP, dialysis of the calmodulin antagonist W7, the Ca2+/calmodulin-dependent kinase II (CaMKII) inhibitor KN93, or a CaMKII-specific peptide inhibitor the rate of ICl(Ca) decay was slowed and matched the [Ca2+]i decline, whereas H7, a nonspecific kinase inhibitor with low affinity for CaMKII, was without effect. When a sustained increase in [Ca2+]i was produced in ATP dialyzed cells, the current decayed completely, whereas in cells loaded with 5'-adenylylimidodiphosphate (AMP-PNP), KN93, or the CaMKII inhibitory peptide, ICl(Ca) did not decay. Slowly decaying currents were repeatedly evoked in ADP- or AMP-PNP-loaded cells, but dialysis of adenosine 5'-O-(3-thiotriphosphate) or okadaic acid resulted in a smaller initial ICl(Ca), and little or no current (despite a normal [Ca2+]i transient) with a second stimulation. These data indicate that CaMKII phosphorylation results in the inactivation of calcium-activated chloride channels, and that transition from the inactivated state to the closed state requires protein dephosphorylation.

Adenosine Triphosphate↗
PubMed1997

Effects of [3H]-BIDN, a novel bicyclic dinitrile radioligand for GABA-gated chloride channels of insects and vertebrates.

1. The radiolabelled bicyclic dinitrile, [3H]-3,3-bis-trifluoromethyl-bicyclo[2.2.1]heptane-2,2-dicarbonitrile ([3H]-BIDN), exhibited, specific binding of high affinity to membranes of the southern corn rootworm (Diabrotica undecimpunctata howardi) and other insects. A variety of gamma-aminobutyric acid (GABA) receptor convulsants, including the insecticides heptachlor (IC50, 35 +/- 3 nM) and dieldrin (IC50, 93 +/- 7 nM), displaced [3H]-BIDN from rootworm membranes. When tested at 100 microM, 1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]oct ane(EBOB), 4-t-butyl-2,6,7-trioxa-1-phosphabicy-clo[2.2.2]octane-1-thio ne (TBPS), 1-phenyl-4-t-butyl-2,6,7-trioxabicyclo[2.2.2]octane (TBOB) and picrotoxin failed to displace 50% of [3H]-BIDN binding to rootworm membranes indicating that the bicyclic dinitrile radioligand probes a site distinct from those identified by other convulsant radioligands. 2. Dissociation studies showed that dieldrin, ketoendrin, toxaphene, heptachlor epoxide and alpha and beta endosulphan displace bound [3H]-BIDN from rootworm membranes by a competitive mechanism. 3. Rat brain membranes were also shown to possess a population of saturable, specific [3H]-BIDN binding sites, though of lower affinity than in rootworm and with a different pharmacological profile. Of the insecticidal GABAergic convulsants that displaced [3H]-BIDN from rootworm, cockroach (Periplaneta americana) and rat brain membranes, many were more effective in rootworm. 4. Functional GABA-gated chloride channels of rootworm nervous system and of cockroach nerve and muscle were blocked by BIDN, whereas cockroach neuronal GABA(B) receptors were unaffected. 5. Expression in Xenopus oocytes of either rat brain mRNA, or cDNA-derived RNA encoding a GABA receptor subunit (Rdl) that is expressed widely in the nervous system of Drosophila melanogaster resulted in functional, homo-oligomeric GABA receptors that were blocked by BIDN. Thus, BIDN probes a novel site on GABA-gated Cl- channels to which a number of insecticidally-active molecules bind.

Animals↗