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PubMed1998

Pore stoichiometry of a voltage-gated chloride channel.

Ion channels allow ions to pass through cell membranes by forming aqueous permeation pathways (pores). In contrast to most known ion channels, which have single pores, a chloride channel belonging to the CIC family (Torpedo CIC-0) has functional features that suggest that it has a unique 'double-barrelled' architecture in which each of two subunits forms an independent pore. This model is based on single-channel recordings of CIC-0 that has two equally spaced and independently gated conductance states. Other CIC isoforms do not behave in this way, raising doubts about the applicability of the model to all CIC channels. Here we determine the pore stoichiometry of another CIC isoform, human CIC-1, by chemically modifying cysteines that have been substituted for other amino acids located within the CIC ion-selectivity filter. The CIC-1 channel can be rendered completely susceptible to block by methanethiosulphonate reagents when only one of the two subunits contains substituted cysteines. Thiol side chains placed at corresponding positions in both subunits can form intersubunit disulphide bridges and coordinate Cd2+, indicating that the pore-forming regions from each subunit line the same conduction pathway. We conclude that human CIC-1 has a single functional pore.

Cadmium↗
PubMed1996

Characteristics of skeletal muscle chloride channel C1C-1 and point mutant R304E expressed in Sf-9 insect cells.

Using the baculovirus system, the skeletal muscle chloride channel, CIC-1 (rat), and a point mutant replacing arginine 304 with glutamic acid were expressed at high levels in cultured Sf-9 insect cells. Whole-cell patch-clamping revealed large inwardly rectifying currents with maxima up to 15 nA inward and 2.5 nA outward. Saturation was evident at voltage steps positive to +40 mV whilst steps negative to -60 mV produced inactivating currents made up of a steady state component and two exponentially decaying components with tau 1 = 6.14+/- 0.92 ms, tau 2 = 36.5+/- 3.29 ms (S.D) n = 7 for steps to -120 mV. Currents recorded in the outside-out patch configuration were often unexpectedly large and up to 5% of whole-cell currents obtained in the same cell, suggesting an uneven channel distribution in the plasmalemma of Sf-9 cells. The pharmacology of a number of chloride channel blockers, including anthracene-9-carboxylate (A9C), niflumate, and perrhenate, was investigated and showed for the first time that perrhenate is an effective blocker of C1C-1 and that it has a complex mechanism of action. Further, the potency of A9C was found to be dependent on external chloride concentration. As in studies on muscle cells themselves, blockade was rapidly effective and easily reversible, except when applying the indanyloxyacetate derivative, IAA94/95, which took up to 10 min to act, and, consistent with an intracellular site of action, was difficult to reverse by washing. Mutation of the highly conserved arginine at position 304 to a glutamic acid did not significantly alter the behaviour of the channel.

Animals↗
PubMedDate not supplied

Distinct actions of alcohols, barbiturates and benzodiazepines on GABA-activated chloride channels.

Similarities and differences in the actions of alcohols, barbiturates and benzodiazepines on GABA-activated chloride channels are reviewed. This question has been approached using behavioral measures, the uptake of 36Cl- by isolated brain membranes and the function of GABA-activated chloride channels expressed in Xenopus oocytes using brain mRNA. Although drugs from all three classes augment GABA-dependent chloride conductances, there are a number of differences between these drugs. Studies of genetic differences in sensitivity to sedative drugs and development of tolerance and cross-tolerance between these drugs indicate that actions of ethanol are similar (but not identical) to those of benzodiazepines but are quite different from those of pentobarbital.

Alcohols↗
PubMed1999

Renal chloride channel, CLCN5, mutations in Dent's disease.

Dent's disease is an X-linked renal tubular disorder characterized by low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis, and renal failure. Patients with Dent's disease may also suffer from rickets and other features of the renal Fanconi Syndrome. Patients may have mutations in the X-linked renal chloride channel gene, CLCN5, which encodes a 746-amino-acid protein with 12-13 transmembrane domains. We have investigated the 11 coding exons of CLCN5 for mutations in eight unrelated patients with Dent's disease. Leukocyte DNA was used for the polymerase chain reaction amplification of CLCN5 and the products analyzed for single-stranded conformational polymorphisms (SSCPs). Abnormal SSCPs were sequenced and revealed eight mutations. These consisted of three nonsense mutations (Arg34Stop, Arg648Stop, Arg704Stop), four deletions involving codons 40, 86, 157, and 241, and one acceptor splice consensus sequence mutation tgcag --> tgaag. The mutations were confirmed either by restriction endonuclease or sequence-specific oligonucleotide hybridization analysis. In addition, an analysis of 110 alleles from 74 unrelated normal individuals demonstrated that the DNA sequence changes were not common polymorphisms. All of the mutations predict truncated chloride channels that are likely to result in a functional loss. Thus, our findings expand the spectrum of CLCN5 mutations associated with Dent's disease and the results will help to elucidate further the functional domains of this novel chloride channel.

Amino Acid Sequence↗
PubMed2004

Apoptotic volume decrease, pH acidification and chloride channel activation during apoptosis requires CD45 expression in HPB-ALL T cells.

Mitochondrial-perturbating agents such as toxic coumponds induce apoptosis. We note that the loss of CD45 expression in the lymphoblastic leukemia cell line HPB-ALL (HPB45.0) leads to an inhibition of nuclear apoptosis. Our hypothesis is that the absence of CD45 disturbs protein function regulated by a proto-oncogene of the Src family playing a significant role in nuclear apoptosis. In this work we explore the importance of a chloride efflux on DNA fragmentation. The role of tyrosine kinase in the function and regulation of the chloride channels was determined. Our results showed a disturbance of ionic homeostasis in CD45 deficient lymphocytes (CD45-) in contrast to normal lymphocytes (CD45+). The phosphorylation levels of the chloride channels are considerably inhibited in CD45-, while the expression levels of these channels are similar in the two types of cells. A hypertonic medium inhibits DNA fragmentation in CD45+ while a hypotonic medium increases DNA fragmentation in CD45-. Thus CD45 plays a significant role in nuclear apoptosis by the regulation of the chloride channels responsible for ionic homeostasis of the cell essential for the DFF40 activation.

Antigens, CD↗
PubMed2004

Tetrandrine inhibits Ca2+-activated chloride channel in cultured human umbilical vein endothelial cells.

AIM: To characterize the electrophysiological and kinetic properties of Ca2+-activated chloride channel (CaCC) in cultured human umbilical vein endothelial cell line (HUVEC), and test the inhibitory effects of tetrandrine (Tet) on CaCC. METHODS: Ca2+-activated Cl- currents (I(Cl,Ca)) were recorded by patch-clamp whole cell configurations. [Ca2+]i was measured via intracellular Fura-2 fluorescence intensities. RESULTS: I(Cl,Ca) was activated by increasing [Ca2+]i via direct elevation of intracellular calcium. I(Cl,Ca) showed an apparent outward rectification properties, and it was activated in a voltage- and calcium-dependent mode. Tet dose-dependently inhibited I(Cl,Ca), the IC50 was (5.2+/-0.4) micromol/L (n=8 cells). Tet suppressed both voltage-dependent and calcium-dependent activation of I(Cl,Ca). The activation time constant was (326+/-12) ms [in the presence of 10 micromol/L Tet, compared to control (175+/-17) ms, at +100 mV], and Ca2+ concentration for half maximal activation was (387+/-61) nmol/L for Tet (compared to control (287+/-36) nmol/L. CONCLUSIONS: Tet effectively blocked I(Cl,Ca), and such effects might be due to its inhibitory effects on the activation process of Ca2+-activated chloride channel.

Alkaloids↗
PubMed1991

Evidence for a cytosolic inhibitor of epithelial chloride channels.

It has been known for several years that the outwardly rectifying 30-pS chloride channel, the regulation of which has been reported to be defective in cystic fibrosis, can be activated by excision of a membrane patch from a cell. This suggested that the cytosol contains an inhibitory factor, which diffuses away after excision, thereby releasing the channel block. To test for such an inhibitor we have isolated cytosol from two epithelial cell lines, and in larger quantities from pig kidney cortex. Kidney cortex was chosen because published and unpublished evidence suggested that proximal tubular cells might also have a tonically suppressed Cl- conductance in the brush-border membrane, which is activated during isolation of membrane vesicles. The inhibitory effect of the cytosol preparations was assessed by: (a) measuring conductive Cl- fluxes on renal proximal tubular brush-border membrane vesicles preloaded with or without cytosol, and (b) recording single Cl- channel currents from excised membrane patches of nasal polyp epithelia and CFPAC-1 cells in the presence and absence of cytosol. All cytosol preparations tested were found to inhibit both conductive Cl- flux in membrane vesicles and single Cl- channels in patch-clamp experiments. In the latter case a type of flicker block was observed with a reduction of channel open probability. Stepwise dilution of the cytosol consistently reduced the inhibitory potency. Since the inhibition was preserved after boiling the cytosol for 10 min, we conclude that the inhibitor is a heat-stable substance. Whether it is identical with the postulated intracellular regulator that couples the defective function of the cystic fibrosis gene product to Cl- channel inhibition cannot be decided at present.

Animals↗
PubMed2001

ClC chloride channels in epithelia: recent progress and remaining puzzles.

ClC chloride channels are widely expressed in epithelia. Recent insights into the roles of specific ClC channels have emerged from molecular and immunolocalization studies, mouse knockout models, and the linkage of mutations of these channels to the human hereditary diseases Bartter's syndrome and Dent's disease.

Animals↗
PubMed2001

Identification of a novel chloride channel expressed in the endoplasmic reticulum, golgi apparatus, and nucleus.

MID-1 is a Saccharomyces cerevisiae gene encoding a stretch-activated channel. Using MID-1 as a molecular probe, we isolated rat cDNA encoding a protein with four putative transmembrane domains. This gene encoded a protein of 541 amino acids. We also cloned the human homologue, which encoded 551 amino acids. Messenger RNA for this gene was expressed abundantly in the testis and moderately in the spleen, liver, kidney, heart, brain, and lung. In the testis, immunoreactivity of the gene product was detected both in the cytoplasm and the nucleus. When expressed in Chinese hamster ovary cells, the gene product was located in intracellular compartments including endoplasmic reticulum and the Golgi apparatus. When microsome fraction obtained from the transfected cells, but not from mock-transfected cells, was incorporated into the lipid bilayer, an anion channel activity was detected. Unitary conductance was 70 picosiemens in symmetric 150 mm KCl solution. We designated this gene Mid-1-related chloride channel (MCLC). MCLC encodes a new class of chloride channel expressed in intracellular compartments.

Amino Acid Sequence↗
PubMed2002

Conformation-dependent regulation of inward rectifier chloride channel gating by extracellular protons.

We have investigated the gating properties of the inward rectifier chloride channel (Cl(ir)) from mouse parotid acinar cells by external protons (H(+)(o)) using the whole-cell patch-clamp technique. Increasing the pH(o) from 7.4 to 8.0 decreased the magnitude of Cl(ir) current by shifting the open probability to more negative membrane potentials with little modification of the activation kinetics. The action of elevated pH was independent of the conformational state of the channel. The effects of low pH on Cl(ir) channels were dependent upon the conformational state of the channel. That is, application of pH 5.5 to closed channels essentially prevented channel opening. In contrast, application of pH 5.5 to open channels actually increased the current. These results are consistent with the existence of two independent protonatable sites: (1) a site with a pK near 7.3, the titration of which shifts the voltage dependence of channel gating; and (2) a site with pK = 6.0. External H(+) binds to this latter site (with a stoichiometry of two) only when the channels are closed and prevent channel opening. Finally, block of channels by Zn(2+) and Cd(2+) was inhibited by low pH media. We propose that mouse parotid Cl(ir) current has a bimodal dependence on the extracellular proton concentration with maximum activity near pH 6.5: high pH decreases channel current by shifting the open probability to more negative membrane potentials and low pH also decreases the current but through a proton-dependent stabilization of the channel closed state.

Algorithms↗
PubMed1995

Antisense oligonucleotides suppress cell-volume-induced activation of chloride channels.

Cell volume regulation is an essential feature of most cells. After swelling in hypotonic media, the simultaneous activation of potassium and chloride channels is believed to be the initial, time-determining step in cell volume regulation. The activation of both pathways is functionally linked and enables the cells to lose ions and water, subsequently leading to cell shrinkage and readjustment of the initial volume. NIH 3T3 fibroblasts efficiently regulate their volume after swelling and bear chloride channels that are activated by decreasing extracellular osmolarity. The chloride current elicited in these cells after swelling is reminiscent of the current found in oocytes expressing an outwardly rectifying chloride current termed ICln. Introduction of antisense oligodeoxynucleotides complementary to the first 30 nucleotides of the coding region of the ICln channel into NIH 3T3 fibroblasts suppresses the activation of the swelling-induced chloride current. The experiments directly demonstrate an unambiguous link between a volume-activated chloride current and a cloned protein involved in chloride transport.

Animals↗
PubMed2002

Cloning and characterisation of amphibian ClC-3 and ClC-5 chloride channels.

Amphibians have provided important model systems to study transepithelial transport, acid-base balance and cell volume regulation. Several families of chloride channels and transporters are involved in these functions. The purpose of this review is to report briefly on some of the characteristics of the chloride channels so far reported in amphibian epithelia, and to focus on recently cloned members of the ClC family and their possible physiological roles. The electrophysiological characterisation, distribution, localisation and possible functions are reviewed and compared to their mammalian orthologs.

Animals↗
PubMed2002

The ClC-5 chloride channel knock-out mouse - an animal model for Dent's disease.

Mutations in the gene CLCN5 encoding the vesicular chloride channel ClC-5 lead to Dent's disease, an X-linked renal disorder. Dent's disease is characterised by proteinuria, hyperphosphaturia and hypercalciuria, which eventually lead to kidney stones and nephrocalcinosis. As it was unclear how mutations in a chloride channel might cause these symptoms, we and others have generated genetic mouse models to elucidate the underlying pathophysiological mechanisms. We review results obtained from these three mouse models and present new data on endosomal acidification and vitamin D metabolism in ClC-5 knock-out (KO) mice. ClC-5 is expressed in apical endosomes of proximal tubular cells where it co-localizes with endocytosed proteins and the proton ATPase. ClC-5 may provide an electric shunt for the efficient operation of the electrogenic H(+)-ATPase. We confirmed this hypothesis by showing that endosomes from CLCN5 KO mice are acidified at a significantly lower rate than wild-type endosomes. This probably results in the drastic impairment of endocytosis observed in ClC-5 KO mice. Parathyroid hormone (PTH) is filtered into the lumen of the nephron, where it is endocytosed and degraded by proximal tubular cells. The defective endocytosis in ClC-5 KO mice entails an increased luminal concentration of PTH, subsequent stimulation of apical PTH receptors which causes an increased endocytosis of the phosphate transporter NaPi and phosphaturia. We now show that it also results in up-regulation of proximal tubular alpha-hydroxylase that generates the active form of vitamin D from its precursor. We discuss how the primary defect in endocytosis leads via secondary changes in calciotropic hormones to the tertiary symptoms hyperphosphaturia, hypercalciuria and kidney stones.

Animals↗
PubMed1994

GABA receptor-linked chloride channels and the behavioral effects of naltrexone in rats.

The present study was conducted to determine whether the effects of naltrexone on schedule-controlled behavior in rats were mediated, at least in part, by the GABAergic system. Because the enhanced sensitivity that has been shown to occur following naltrexone treatment might alter the effects of the treatment compounds, a variety of compounds interacting with the GABA system were tested in both sensitized and nonsensitized animals. Of all the compounds tested in this manner, only the dose-effect function for the GABA agonist muscimol was altered by the naltrexone treatment, with the higher doses of muscimol producing response-rate decreasing effects only in naltrexone-sensitized rats. In the naltrexone-treated animals, these same GABA agonists and antagonists were used as pretreatments prior to the determination of the naltrexone dose-effect function. Although shifts in the naltrexone dose-effect function were observed, the effects were not consistent either within or across receptor class. In contrast, the chloride-channel antagonist picrotoxin clearly shifted the naltrexone dose-effect function in sensitized animals to the left, while the chloride-channel facilitator pentobarbital shifted the function to the right. These results indicate that the effects of naltrexone are at least partially mediated by an action at the GABA-linked chloride channel, rather than directly at the GABA receptor.

Animals↗
PubMed1993

Small and intermediate conductance chloride channels in HT29 cells.

Recently, it has been shown that intermediate conductance outwardly rectifying chloride channels (ICOR) are blocked by cytosolic inhibitor (C. I.) found in the cytosol of human placenta and epithelial cells. C. I. also reduced the baseline current in excised membrane patches of HT29 cells. In the present study, this effect of C. I. was characterized further. Heat treated human placental cytosol was extracted in organic solvents and dissolved in different electrolyte solutions. It is shown that the reduction of baseline conductance (g(o)) is caused by inhibition of small non-resolvable channels, which are impermeable to Na+ and SO4(2-), but permeable to Cl-. The regulation of these small Cl(-)-conducting channels (g(o)) and of ICOR was examined further. First, no activating effects of protein kinase A (PKA) on the open probability (Po) of the ICOR or on the g(o)) were observed. The Po of the ICOR was reduced by 22% in a Ca(2+)-free solution. g(o) was insensitive to changes in the Ca2+ activity. The effects of C. I. from a cystic fibrosis (CF) placenta and the CF pancreatic duct cell line CFPAC-1 were compared with the effects of corresponding control cytosols, and no significant differences between CF and control cytosols were found. We conclude that the excised patches of HT29 cells contain ICOR and small non-resolvable Cl(-)-conducting channels which are similarly inhibited by C. I.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1987

Neurochemical actions of anesthetic drugs on the gamma-aminobutyric acid receptor-chloride channel complex.

Interaction of intoxicant-anesthetic drugs with the gamma-aminobutyric acid (GABA) receptor-chloride channel complex of mouse brain was studied using the binding of [35S]t-butylbicyclophosphorothionate (TBPS) to isolated membranes and the GABA-stimulated uptake of 36Cl- by membrane vesicles. Anesthetic drugs, including barbiturates, chloroform, diethylether and ethanol, inhibited the binding of TBPS and enhanced the GABA-dependent influx of chloride. In the presence of bicuculline, barbiturates increased the binding of TBPS, but this action was not shared by other anesthetic agents. Inhibition of TBPS binding was found with drug concentrations that produce anesthesia in vivo, whereas augmentation of GABA action occurred at subanesthetic concentrations. Effects of a series of n-alcohols (methanol to decanol) were studied on TBPS binding and membrane fluidity (using 1,6-diphenyl-1,3,5-hexatriene as a fluorescent probe). The potencies of the alcohols for inhibiting TBPS binding and fluidizing synaptic membranes were similar to their anesthetic potencies, but there were differences in the relative potencies of the drugs for inhibition of TBPS binding and membrane fluidization. These results, together with effects of assay temperature, suggest that effects of anesthetics on the GABA receptor-chloride channel complex were not due to changes in bulk membrane fluidity. Correlation of anesthetic potencies of chemically diverse agents with both inhibition of TBPS binding and augmentation of GABA-dependent chloride flux suggests a role for the GABA complex in anesthesia.

Alcohols↗
PubMed1996

Mutations in the human skeletal muscle chloride channel gene (CLCN1) associated with dominant and recessive myotonia congenita.

Myotonia, defined as delayed relaxation of muscle after contraction, is seen in a group of genetic disorders that includes autosomal dominant myotonia congenita (Thomsen's disease) and autosomal recessive myotonia congenita (Becker's disease). Both disorders are characterized electrophysiologically by increased excitability of muscle fibers, reflected in clinical myotonia. These diseases are similar except that transient weakness is seen in patients with Becker's, but not Thomsen's disease. Becker's and Thomsen's diseases are caused by mutations in the skeletal muscle voltage-gated chloride channel gene (CLCN1). Genetic screening of a panel of 18 consecutive myotonia congenita (MC) probands for mutation in CLCN1 revealed that a novel Gln-68-Stop nonsense mutation predicts premature truncation of the chloride channel protein. Four previously reported mutations, Arg-894-stop, Arg-338-Gln, Gly-230-Glu, and del 1437-1450, were also noted in our sample set. The Arg-338-Gln and Gly-230-Glu mutations were found in patients with different phenotypes from those of previous reports. Further study of the Arg-338-Gln and Gln-230-Glu alleles may shed light on variable modes of transmission (dominant versus recessive) in different families. Physiologic study of these mutations may lead to better understanding of the pathophysiology of myotonia in these patients and of voltage-gated chloride channel structure/function relationships in skeletal muscles.

Chloride Channels↗
PubMed1998

Golgi localization and functionally important domains in the NH2 and COOH terminus of the yeast CLC putative chloride channel Gef1p.

GEF1 encodes the single CLC putative chloride channel in yeast. Its disruption leads to a defect in iron metabolism (Greene, J. R., Brown, N. H., DiDomenico, B. J., Kaplan, J., and Eide, D. (1993) Mol. Gen. Genet. 241, 542-553). Since disruption of GEF2, a subunit of the vacuolar H+-ATPase, leads to a similar phenotype, it was previously suggested that the chloride conductance provided by Gef1p is necessary for vacuolar acidification. We now show that gef1 cells indeed grow less well at less acidic pH. However, no defect in vacuolar acidification is apparent from quinacrine staining, and Gef1p co-localizes with Mnt1p in the medial Golgi. Thus, Gef1p may be important in determining Golgi pH. Systematic alanine scanning of the amino and the carboxyl terminus revealed several regions essential for Gef1p localization and function. One sequence (FVTID) in the amino terminus conforms to a class of sorting signals containing aromatic amino acids. This was further supported by point mutations. Alanine scanning of the carboxyl terminus identified a stretch of roughly 25 amino acids which coincides with the second CBS domain, a conserved protein motif recently identified. Mutations in the first CBS domain also destroyed proper function and localization. The second CBS domain can be transplanted to the amino terminus without loss of function, but could not be replaced by the corresponding domain of the homologous mammalian channel ClC-2.

Amino Acid Sequence↗