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At least 253 records · Page 14Linked to original sources
PubMed2006

Apical Maxi-chloride channel from human placenta: 12 years after the first electrophysiological recordings.

The Maxi-chloride channel was the first ion channel described by electrophysiological methods in placenta. Because it is difficult to access a complex epithelium such as the placenta for electrophysiological procedures, the studies of ion channels from placental membranes have been performed only very recently. It was only in 1993 that a direct demonstration of a high-conductance chloride channel in apical membranes of intact trophoblastic epithelium was mentioned, and two years later, the description of this channel was reported from purified placental apical membranes reconstituted into artificial lipid membranes suitable for patch-clamp recordings. This brief review comments on the work done with regard to the electrophysiological characterization and regulation of the large-conductance or "Maxi" chloride channel and its contribution to the development of a cellular model for syncytiotrophoblast ion transport.

Chloride Channels↗
PubMed1999

CLC chloride channels in Caenorhabditis elegans.

The genome of the nematode Caenorhabditis elegans encodes six putative chloride channels (CeCLC-1 through CeCLC-6) that represent all three known branches of the mammalian CLC gene family. Using promoter fragments to drive the expression of the green fluorescent protein, CeCLC-2, -3, and -4 expression was studied in transgenic C. elegans. CeCLC-4 was specifically expressed in the large H-shaped excretory cell, where it was co-expressed with CeCLC-3, which is also expressed in other cells, including neurons, muscles, and epithelial cells. Also, CeCLC-2 was expressed in several cells of the nervous system, intestinal cells, and vulval muscle cells. Similar to mammalian CLC proteins, only two nematode CLC channels elicited detectable plasma membrane currents in Xenopus oocytes. CeCLC-3 currents were inwardly rectifying and were activated by positive prepulses. Its complex gating behavior can be explained by two gates, at least one of which depends on extracellular anions. In this respect it resembles some mammalian chloride channels with which it also shares a preference of chloride over iodide. C. elegans thus provides new opportunities to understand common mechanisms underlying structure and function in CLC channels and will allow for a genetic dissection of chloride channels in this simple model organism.

Animals↗
PubMed1988

Asymmetrical activation of GABA-gated chloride channels in cerebral cortex.

A pronounced left/right asymmetry of GABA-gated chloride channels was observed in rat cerebral cortex. This asymmetry was manifest as a higher apparent affinity and density of binding sites for the "cage" convulsant [35S]t-butylbicyclophosphorothionate (TBPS) in the right compared to left cortex. Asymmetries in [35S]TBPS binding were not observed in other brain areas, and were restricted to the occipital and entorhinal/pyriform areas of cerebral cortex. Evaluation of other components of the benzodiazepine/GABA receptor chloride ionophore complex in cerebral cortex suggests that this asymmetry is not present in either benzodiazepine receptors or that population of GABA receptors linked to benzodiazepine receptors. Brief restraint-stress significantly increased both the apparent affinity and number of [35S]TBPS binding sites in cortex, but the left/right asymmetry was no longer apparent. These findings indicate a time-dependent, asymmetric response of cortical GABA-gated chloride channels to stress rather than an anatomical hemispheric difference, and suggest that GABA-gated chloride channels may be involved in a differential processing of stressful stimuli by the cerebral hemispheres.

Animals↗
PubMed2004

Molecular and functional analyses of two new calcium-activated chloride channel family members from mouse eye and intestine.

Two new calcium-activated chloride channel (CLCA) family members, mCLCA5 and mCLCA6, have been cloned from mouse eye and intestine, respectively. mCLCA5 is highly homologous to hCLCA2, and mCLCA6 is highly homologous to hCLCA4. mCLCA5 is widely expressed with strong expression in eye and spleen, whereas mCLCA6 is primarily expressed in intestine and stomach. mCLCA6 is also expressed as a splice variant lacking exon 8 and part of exon 10 in intestine and stomach. Transfection of tsA201 cells with enhanced green fluorescent protein-tagged versions of the three cDNAs reveals protein products of 155 and 65 kDa for mCLCA5 and mCLCA6 and 145 and 65 kDa for the mCLCA6 splice variant. In vitro translation of mCLCA5 generates a 90-kDa protein that does not appear to be glycosylated. mCLCA6 also generates a 90-kDa protein that is glycosylated to a 110-kDa product, whereas the mCLCA6 splice variant generates an 80-kDa product that is 100 kDa after glycosylation. Treatment of enhanced green fluorescent protein-tagged mCLCA6 with PNGase F (peptide: N-glycosidase F) to remove N-linked glycosyl groups shows a reduction in size of the 65 kDa product to 60 kDa. Consistent with the hypothesis that mCLCA5, mCLCA6, and its splice variant encode calcium-activated chloride channels, in HEK293 cells expressing CLCAs ionomycin-evoked increases in intracellular calcium stimulated a current that reversed near Cl(-) equilibrium potential, E(Cl). Furthermore, these currents were inhibited by the chloride channel blocker niflumic acid. Given the prominent role of hCLCA2 in cancer cell adhesion and the unique high level of expression of hCLCA4 in brain, the identification of their murine counterparts presents the opportunity to clarify the role of CLCAs in disease and normal cell physiology.

Amino Acid Sequence↗
PubMed2002

Novel animal-health drug targets from ligand-gated chloride channels.

The world's three best-selling veterinary antiparasitic drugs ('parasiticides') act on ligand-gated ion channels. The sequencing of the complete genomes of the invertebrate genetic model organisms Caenorhabditis elegans and Drosophila melanogaster has led to the recent cloning of new subunits of 5-hydroxytryptamine-gated and histamine-gated chloride channels. Together with L-glutamate-gated chloride channels, which are important targets of known parasiticides, and acetylcholine-gated chloride channels, these new classes of ligand-gated chloride channels, which are known only from invertebrates, add to our understanding of inhibitory neural signalling. They could offer the prospect of being targets for a new generation of selective drugs to control nematode and insect parasites.

Amino Acid Sequence↗
PubMed2000

Chloride channel expression in cultured human fetal RPE cells: response to oxidative stress.

PURPOSE: The human fetal cell line RPE 28 SV4 has been useful for studies of oxidative stress and apoptosis in retinal pigmented epithelium. This cell model is now assessed in functional investigations of chloride channel activity. The study aims to determine the presence of specific chloride channels, including CFTR and ClC channels, to identify the properties of membrane chloride currents and to assess their modulation by hydrogen peroxide, cAMP, and other agents. METHODS: Channel expression was determined using RT-PCR and cDNA cloning and biochemical and immunocytochemical methods. Membrane currents were analyzed using whole-cell, patch-clamp techniques. RESULTS: RT-PCR results confirmed the presence of ClC-5 mRNA, and a full-length clone encoding ClC-3 was isolated from a cDNA library for RPE 28 SV4 cells. Specific staining for CFTR and several ClC channels was detected by immunocytochemistry. Whole-cell chloride currents (under conditions of symmetrical chloride concentrations) averaged 16.9 +/- 3.4 pA/pF (at +100 mV; n = 8), showed outward rectification, and had an anion permeability sequence of Cl(-) > I(-) > cyclamate. Currents were stimulated by cAMP cocktail (250 microM cAMP, 100 microM IBMX, and 25 microM forskolin) and were inhibited by 1 mM DIDS. The oxidative agent hydrogen peroxide (100 microM) decreased the current by 34% +/- 10% (n = 4). CONCLUSIONS. These findings suggest that RPE 28 SV4 cells possess regulated chloride channels including CFTR and members of the ClC chloride channel family. The inhibition of chloride currents by H(2)O(2) suggests that this cell line may be advantageous for studies of chloride channel modulation by oxidative stress.

1-Methyl-3-isobutylxanthine↗
PubMed1993

Molecular cloning of a chloride channel that is regulated by dehydration and expressed predominantly in kidney medulla.

Complementary DNA encoding a rat kidney chloride channel (CIC-K1) was isolated by a polymerase chain reaction (PCR) cloning strategy. We designed degenerate primers, based on the regions where previously cloned chloride channels (CIC-0, -1, and -2) possess significant amino acid identity, and performed reverse transcription PCR with whole kidney mRNA. The 686-amino acid protein encoded by CIC-K1 is about 40% identical to the previously cloned chloride channels and has a similar hydropathy profile. Expression of CIC-K1 in Xenopus oocytes induced Cl- currents that activate instantaneously upon hyperpolarization and depolarization, and displayed a slightly outwardly rectifying current-voltage relationship. The message for CIC-K1 was 2.4 kilobases and was found predominantly in kidney, especially in the inner medulla. Reverse transcription PCR technique using micro-dissected nephron segments revealed that the main site of expression in kidney was the thin ascending limb of Henle's loop, which has the highest Cl- permeability among the nephron segments and is thought to be involved in a counter-current system for urine concentration in the inner medulla. The abundance of CIC-K1 mRNA in kidney increased about 4-fold as rats became dehydrated by deprivation of water for 5 days. The site of expression and the regulation by dehydration suggest that CIC-K1 function may be important in urinary concentrating mechanisms.

Amino Acid Sequence↗
PubMed1994

Two isoforms of a chloride channel predominantly expressed in thick ascending limb of Henle's loop and collecting ducts of rat kidney.

Complementary DNAs encoding rat kidney chloride channels (ClC-K2L and ClC-K2S) were isolated by a polymerase chain reaction cloning strategy. Degenerate primers were designed based on the significant amino acid identity of the previously cloned chloride channels (ClC-0, -1, -2, and -K1). The 687-amino acid protein encoded by ClC-K2L is about 80% identical to rat ClC-K1 and about 40% identical to ClC-0, -1, and -2. ClC-K2S encodes a 632-amino acid protein in which 55 amino acids containing the putative second membrane-spanning domain of ClC-K2L are deleted. Chloride currents induced by both clones were very similar in terms of inhibitor sensitivity and anion selectivity (Br- > I- > Cl- >> cyclamate-). Northern blot with total ClC-K2L as a probe under high stringency revealed its message predominantly in kidney, especially in the outer and inner medulla. Reverse transcription polymerase chain reaction technique using microdissected nephron segments revealed that the main site of expression of both clones in kidney was the thick ascending limb of Henle's loop and collecting ducts, where the existence of a variety of chloride channels and their importance for maintaining body fluid homeostasis have been demonstrated. These results suggest that ClC-K2L and -K2S are chloride channels in the thick ascending limb and collecting ducts and may be important routes for transcellular chloride transport like ClC-K1.

Amino Acid Sequence↗
PubMed2003

[Expressions of chloride channel ClC-2 and ClC-3 in human nasal polyps].

OBJECTIVE: To investigate the role of chloride channel ClC-2 and ClC-3 in human nasal polyps. METHOD: Two chloride channels ClC-2 and ClC-3 were studied in 27 cases of nasal polyps with immunochemistric staining. RESULT: Nasal polyps revealed strong positive staining of ClC-2 and ClC-3, the ClC-2 and ClC-3 proteins localized mainly in epithelial cells, sub-epithelial mucous glands without significant distinction. The number of positive cells of ClC-2 and ClC-3 are 17.6 +/- 6.33 and 23.49 +/- 6.14. CONCLUSION: The chloride channels ClC-2 and ClC-3 may play a role in the formation of nasal polyps.

Adult↗
PubMed1998

The yeast CLC chloride channel functions in cation homeostasis.

A defect in the yeast GEF1 gene, a CLC chloride channel homolog leads to an iron requirement and cation sensitivity. The iron requirement is due to a failure to load Cu2+ onto a component of the iron uptake system, Fet3. This process, which requires both Gef1 and the Menkes disease Cu2+-ATPase yeast homolog Ccc2, occurs in late- or post-Golgi vesicles, where Gef1 and Ccc2 are localized. The defects of gef1 mutants can be suppressed by the introduction of Torpedo marmorata CLC-0 or Arabidopsis thaliana CLC-c and -d chloride channel genes. The functions of Gef1 in cation homeostasis provide clues to the understanding of diseases caused by chloride channel mutations in humans and cation toxicity in plants.

Cations↗
PubMed1996

A family of putative chloride channels from Arabidopsis and functional complementation of a yeast strain with a CLC gene disruption.

We have cloned four novel members of the CLC family of chloride channels from Arabidopsis thaliana. The four plant genes are homologous to a recently isolated chloride channel gene from tobacco (CLC-Nt1; Lurin, C., Geelen, D., Barbier-Brygoo, H., Guern, J., and Maurel, C. (1996) Plant Cell 8, 701-711) and are about 30% identical in sequence to the most closely related CLC-6 and CLC-7 putative chloride channels from mammalia. AtCLC transcripts are broadly expressed in the plant. Similarly, antibodies against the AtCLC-d protein detected the protein in all tissues, but predominantly in the silique. AtCLC-a and AtCLC-b are highly homologous to each other ( approximately 87% identity), while being approximately 50% identical to either AtCLC-c or AtCLC-d. None of the four cDNAs elicited chloride currents when expressed in Xenopus oocytes, either singly or in combination. Among these genes, only AtCLC-d could functionally substitute for the single yeast CLC protein, restoring iron-limited growth of a strain disrupted for this gene. Introduction of disease causing mutations, identified in human CLC genes, abolished this capacity. Consistent with a similar function of both proteins, the green fluorescent protein-tagged AtCLC-d protein showed the identical localization pattern as the yeast ScCLC protein. This suggests that in Arabidopsis AtCLC-d functions as an intracellular chloride channel.

Amino Acid Sequence↗
PubMed2004

Characterization of three myotonia-associated mutations of the CLCN1 chloride channel gene via heterologous expression.

Two novel mutations of the human CLCN1 chloride channel gene, c.592C>G (p.L198V) and c.2255A>G (p.K752R), are described, occurring coincidentally in the one myotonic patient. These individual mutations and a construct with both mutations in the one cDNA were transcribed and expressed in Xenopus oocytes where channel gating parameters were extracted from chloride currents recorded under voltage clamp. We found that the p.L198V mutation has its major effects on the common (or slow) gate of the chloride channel, as do other dominant ClC-1 mutations, and may therefore be causative of the patient's symptoms (when co-expressed with wild-type human ClC-1, the p.L198V mutation exerts a dominant negative effect on common gating) but the p.K752R mutation appears to be innocuous and may be a benign polymorphism. A third mutant, the recently described c.2795C>T (p.P932L), was expressed in HEK 293 cells. Despite the severity of the disease associated with this mutation, chloride currents in cells expressing p.P932L were not significantly different from those of cells expressing wild-type ClC-1.

Black or African American↗
PubMed2002

Inhibition of gap junction hemichannels by chloride channel blockers.

Electrophysiological methods were used to assess the effect of chloride-channel blockers on the macroscopic and microscopic currents of mouse connexin50 (Cx50) and rat connexin46 (Cx46) hemichannels expressed in Xenopus laevis oocytes. Oocytes were voltage-clamped at -50 mV and hemichannel currents (ICx50 or ICx46) were activated by lowering the extracellular Ca2+ concentration ([Ca2+]o) from 5 mM to 10 microM. Ion-replacement experiments suggested that ICx50 is carried primarily (>95%) by monovalent cations (PK : PNa : PCl = 1.0 : 0.74 : 0.05). ICx50 was inhibited by 18beta-glycyrrhetinic acid (apparent Ki, 2 microM), gadolinium (3 microM), flufenamic acid (3 microM), niflumic acid (11 microM), NPPB (15 microM), diphenyl-2-carboxylate (26 microM), and octanol (177 microM). With the exception of octanol, niflumic acid, and diphenyl-2-carboxylate, the above agents also inhibited ICx46. Anthracene-9-carboxylate, furosemide, DIDS, SITS, IAA-94, and tamoxifen had no inhibitory effect on either ICx50 or ICx46. The kinetics of ICx50 inhibition were not altered at widely different [Ca2+]o (10-500 microM), suggesting that drug-hemichannel interaction does not involve the Ca2+ binding site. In excised membrane patches, application of flufenamic acid or octanol to the extracellular surface of Cx50 hemichannels reduced single channel-open probability without altering the single-channel conductance, but application to the cytoplasmic surface had no effect on the channels. We conclude that some chloride-channel blockers inhibit lens-connexin hemichannels by acting on a site accessible only from the extracellular space, and that drug-hemichannel interaction involves a high-affinity site other than the Ca2+ binding site.

Animals↗
PubMed2002

Oxidant and antioxidant modulation of chloride channels expressed in human retinal pigment epithelium.

Retinal pigment epithelium (RPE) possesses regulated chloride channels that are crucial for transepithelial fluid and ion transport. At present, little is known about the molecular nature of chloride channels in human adult RPE (haRPE) or the effects of oxidative stress on membrane conductance properties. In the present study, we assessed ClC channel and cystic fibrosis transmembrane conductance regulator (CFTR) expression and membrane chloride conductance properties in haRPE cells. ClC-5, ClC-3, ClC-2, and CFTR mRNA expression was confirmed with RT-PCR analysis, and protein expression was detected with Western blot analysis and immunofluorescence microscopy. Whole cell recordings of primary cultures of haRPE showed an outwardly rectifying chloride current that was inhibited by the oxidant H(2)O(2). The inhibitory effects of H(2)O(2) were reduced in cultured human RPE cells that were incubated with precursors of glutathione synthesis or that were stably transfected to overexpress glutathione S-transferase. These findings indicate a possible role for ClC channels in haRPE cells and suggest possible redox modulation of human RPE chloride conductances.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1993

Tissue expression of mRNA of chloride channel from MDCK cells and its regulation by protein kinases.

A new chloride channel has recently been identified by expression cloning (Paulmichl, M., et al. Nature 356: 238). To date there is no information available on the distribution of this channel in mammalian tissues. We cloned rat homologue of this Cl channel and found 92% identity in deduced amino-acid sequence. We studied the tissue distribution of its mRNA and regulation by protein kinases. Its mRNA was expressed in all 17 bovine tissues we studied, most abundantly in the brain. In the kidney, it was expressed more in the medulla than cortex. Dehydration up to 5 days did not change its mRNA level in the rat kidney. Both phorbol myristate acetate (PMA) and forskolin down regulated its expression in MDCK cells, suggesting that both PKC and PKA modulated its expression. The physiological role of this chloride channel remains to be clarified.

Amino Acid Sequence↗
PubMed1993

Specific inhibitors distinguish the chloride channel and drug transporter functions associated with the human multidrug resistance P-glycoprotein.

Expression of the human multidrug resistance P-glycoprotein is associated with two activities, active drug transport and a volume-regulated chloride channel. In this study we define four classes of compound, based on their differential effects on these two activities. Class I compounds are substrates transported by P-glycoprotein. They also prevent channel activation when added to the cytoplasmic face of the membrane. Class II compounds include reversers of multidrug resistance such as verapamil. These compounds inhibit drug transport and block the chloride channel when added to the outer face of the membrane. Class III compounds include conventional channel blockers which block the chloride channel but do not influence drug transport. Class IV compounds, for example cyclosporin A, appear to inhibit drug transport but do not affect chloride channel activity. These findings have implications for the relationship between the channel and transporter functions associated with P-glycoprotein expression, and for the development of clinical agents which reverse multidrug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Characterisation of renal chloride channel, CLCN5, mutations in hypercalciuric nephrolithiasis (kidney stones) disorders.

Mutations of the renal-specific chloride channel (CLCN5) gene, which is located on chromosome Xp11.22, are associated with hypercalciuric nephrolithiasis (kidney stones) in the Northern European and Japanese populations. CLCN5 encodes a 746 amino acid channel (CLC-5) that has approximately 12 transmembrane domains, and heterologous expression of wild-type CLC-5 in Xenopus oocytes has yielded outwardly rectifying chloride currents that were markedly reduced or abolished by these mutations. In order to assess further the structural and functional relationships of this recently cloned chloride channel, additional CLCN5 mutations have been identified in five unrelated families with this disorder. Three of these mutations were missense (G57V, G512R and E527D), one was a nonsense (R648Stop) and one was an insertion (30:H insertion). In addition, two of the mutations (30:H insertion and E527D) were demonstrated to be de novo, and the G57V and E527D mutations were identified in families of Afro-American and Indian origin, respectively. The G57V and 30:H insertion mutations represent the first CLCN5 mutations to be identified in the N-terminus region, and the R648Stop mutation, which has been observed previously in an unrelated family, suggests that this codon may be particularly prone to mutations. Heterologous expression of the mutations resulted in a marked reduction or abolition of the chloride currents, thereby establishing their functional importance. These results help to elucidate further the structure-function relationships of this renal chloride channel.

Adult↗
PubMed2003

17beta-estradiol and tamoxifen regulate a maxi-chloride channel from human placenta.

Steroid hormones have been implicated in the modulation of several transport processes, including conductive chloride transport in epithelial cells. Micromolar concentrations of these hormones have been determined in blood of pregnant women. The purpose of this work was to explore the effects of 17beta-Estradiol, a steroid hormone, on the biophysical properties of the Maxi chloride channel present in apical membranes from human placental syncytiotrophoblast. Apical membrane chloride channels from human term placentas were reconstituted in giant liposomes suitable for electrophysiologic studies by the patch-clamp method. Low micromolar concentrations of 17beta-Estradiol inhibit the Maxi chloride channels in excised patches in a potential-dependent manner. The addition of 1 mM 17beta-Estradiol to the bath solution decreased the total current in the patch from 100% control to 71% at -40 mV holding potential and the current was not affected by 17beta-Estradiol at + 40 mV. However, the presence of the hormone did not affect the single-channel conductance, therefore its effect must be due to modulation of its open probability (Po). Interestingly, 17alpha-Estradiol did not change the total current in the patch. Tamoxifen, an antiestrogen, also showed inhibition, but in a voltage-independent manner. Our results suggest that the Maxi Cl- channel from human term placenta may be regulated by direct interaction of both compounds with the channel. From a functional point of view, the control of these channels by steroid hormones may be of great importance in placental physiology and their regulation may help to unravel their possible role in transplacental transport.

Cell Membrane↗