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PubMed1998

Partial restoration of cAMP-stimulated CFTR chloride channel activity in DeltaF508 cells by deoxyspergualin.

Deletion of the codon encoding phenylalanine 508 (DeltaF508) is the most common mutation in cystic fibrosis (CF) and results in a trafficking defect. Mutant DeltaF508-CF transmembrane conductance regulator (CFTR) protein retains functional activity, but the nascent protein is recognized as abnormal and, in consequence, is retained in the endoplasmic reticulum (ER) and degraded. It has been proposed that this retention in the ER is mediated, at least in part, by the cellular chaperones heat shock protein (HSP) 70 and calnexin. We have investigated the ability of deoxyspergualin (DSG), a compound known to compete effectively for binding with HSP70 and HSP90, to promote trafficking of DeltaF508-CFTR to the cell membrane. We show that DSG treatment of immortalized human CF epithelial cells (DeltaF508) and cells expressing recombinant DeltaF508-CFTR partially restored cAMP-stimulated CFTR Cl- channel activity at the plasma membrane. Although there are several possible explanations for these results, one simple interpretation is that DSG may have altered the interaction between DeltaF508-CFTR and its associated chaperones. If this is correct, agents capable of altering the normal functioning of cellular chaperones may provide yet another means of restoring CFTR Cl- channel activity to CF subjects harboring this class of mutations.

1-Methyl-3-isobutylxanthine↗
PubMed1996

Glibenclamide blockade of CFTR chloride channels.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a protein kinase A- and ATP-regulated Cl- channel located in the apical membranes of epithelial cells. Previously Sheppard and Welsh (J. Gen. Physiol. 100: 573-591, 1992) showed that glibenclamide, a compound which binds to the sulfonylurea receptor and thus blocks nucleotide-dependent K+ channels, reduced CFTR whole cell current. The aim of this study was to identify the mechanism underlying this inhibition in cell-free membrane patches containing CFTR Cl- channels. Exposure to gliben-clamide caused a reversible reduction in current carried by CFTR which was paralleled by a decrease in channel open probability (Po). The decrease in Po was concentration dependent, and half-maximum inhibition (ki) occurred at 30 microM. Fluctuation analysis indicated a flickery-type block of open CFTR channels. Event duration analysis supported this notion by showing that the glibenclamide-induced decrease in Po was accompanied by interruptions of open bursts [i.e., an apparent reduction in the burst duration (Tburst)] with only a slight reduction in closed time (Tc). The plot of the corresponding open-to-closed (Tburst-1) and closed-to-open (Tc-1) rates as a function of glibenclamide concentration were consistent with a pseudo-first-order open-blocked mechanism and provided estimates of the on rate (kon = 1.17 microM-1S-1), the off rate (koff = 16 s-1), and the dissociation constant (Kd = 14 microM). The difference between the Ki (30 microM) and the Kd (14 microM) is the result expected for a closed-open-blocked model with an initial Po of 0.47. Since the initial Po was 0.50 +/- 0.02 (n = 12), we can conclude that glibenclamide blocks CFTR by a closed-open-blocked mechanism.

Animals↗
PubMed1999

Chloride channels in the eel intestine

Patches of freshly isolated epithelial cells from eel Anguilla anguilla intestine bathed by the same solution on both sides in the cell-attached configuration had conductances of 57.0+/-1.8 pS (for positive voltages) and 13.3+/-0.7 pS (for negative voltages) (means +/- s.e.m., N=25). Electrical activity was spontaneous in the cell-attached configuration, but was frequently lost after excision. In inside-out patches, channel activity was restored by strong hyperpolarization (-150 mV for 5 s) or depolarization (+150 mV for 5 s). Channel activity was inhibited by the Cl- transport blocker DIDS (1 mmol l-1). The membrane potential measured using the nystatin slow whole-cell technique in primary cultured eel intestine epithelial cells was -35.4+/-1.0 mV (N=14), similar to the expected equilibrium potential for Cl- (-38.2 mV). Removal of Cl- from the bath or application of DIDS caused 16 mV and 6-7 mV depolarizing shifts in reversal potential, respectively. In one experiment, DIDS also induced a reduction in cell conductance from 0. 011+/-0.014 to 0.002+/-0.005 nS. The addition of 0.5 mmol l-1 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (a membrane-permeable analogue of cyclic AMP) to the bath caused an increase in conductance without affecting the reversal potential.

Journal Article↗
PubMed2006

Atrial natriuretic peptide modulates cystic fibrosis transmembrane conductance regulator chloride channel expression in rat proximal colon and human intestinal epithelial cells.

The cystic fibrosis transmembrane conductance regulator (CFTR) is one of the most intensively investigated Cl- channels. Different mutations in the CFTR gene cause the disease cystic fibrosis (CF). CFTR is expressed in the apical membrane of various epithelial cells including the intestine. The major organ affected in CF patients is the lung, but it also causes an important dysfunction of intestinal ion transport. The modulation of CFTR mRNA expression by atrial natriuretic peptide (ANP) was investigated in rat proximal colon and in human intestinal CaCo-2 cells by RNase protection assay and semi-quantitative reverse transcriptase PCR techniques. Groups of rats subjected to volume expansion or intravenous infusion of synthetic ANP showed respective increases of 60 and 50% of CFTR mRNA expression in proximal colon. CFTR mRNA was also increased in cells treated with ANP, reaching a maximum effect at 10(-9) M ANP, probably via cGMP. ANP at 10(-9) M was also able to stimulate both the CFTR promoter region (by luciferase assay) and protein expression in CaCo-2 cells (by Western blot and immunoprecipitation/phosphorylation). These results suggested the involvement of ANP, a hormone involved with extracellular volume, in the expression of CFTR in rat proximal colon and CaCo-2 intestinal cells.

Animals↗
PubMed2001

Thiocyanate as a probe of the cystic fibrosis transmembrane conductance regulator chloride channel pore.

Immediately following exposure to thiocyanate (SCN-)-containing solutions, the cystic fibrosis conductance regulator Cl- channel exhibits high unitary SCN conductance and anomalous mole fraction behaviour, suggesting the presence of multiple anion binding sites within the channel pore. However, under steady-state conditions SCN-conductance is very low. Here I show, using patch clamp recording from CFTR-transfected mammalian cell lines, that under steady-state conditions neither SCN- conductance nor SCN- permeability show anomalous mole fraction behaviour. Instead, SCN conductance, permeability, and block of Cl- permeation can all be reproduced by a rate theory model that assumes only a single intrapore anion binding site. These results suggest that under steady-state conditions the interaction between SCN- and the CFTR channel pore can be understood by a simple model whereby SCN- ions enter the pore more easily than Cl-, and bind within the pore more tightly than Cl-. The implications of these findings for investigating and understanding the mechanism of anion permeation are discussed.

Animals↗
PubMed1988

Ethanol potentiation of GABAergic transmission in cultured spinal cord neurons involves gamma-aminobutyric acidA-gated chloride channels.

The interaction of ethanol with gamma-aminobutyric acid (GABA)-mediated 36-Cl-influx and its modulation by various drugs was investigated in C57 mice spinal cord cultured neurons. Ethanol (5-100 mM) potentiated the effect of GABA on 36Cl-influx; whereas at concentrations greater than or equal to 50 mM ethanol activated Cl- channels directly. The effect of ethanol was specific for GABAA receptor-gated Cl- channels, as ethanol did not potentiate glycine-induced 36Cl-influx in the same neurons. Both the enhancing and direct effects of ethanol on 36Cl-influx were blocked by GABA antagonists like bicuculline, picrotoxinin and inverse agonists of the benzodiazepine site like the imidazodiazepine R015-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5 alpha], [1,4]benzodiazepine-3-carboxylate) and N-methyl-beta-carboline-3-carboxamide (FG-7142). Ethanol potentiating effect of GABA-induced 36Cl-influx was also reversed by methyl-6,7-dimethyl-4-ethyl-beta-carboline-3-carboxylate. The effects of the inverse agonists were blocked by the benzodiazepine receptor antagonist R015-1788. Both R015-4513 and FG-7142 reversed direct and GABA potentiating effects of ethanol effect at concentrations lower than those that exhibit inverse agonistic activity in the 36Cl-influx assay in cultured neurons. These results suggest that ethanol facilitation of GABAAergic transmission involves GABA receptor-gated Cl- channels and that this interaction may be responsible for some of the pharmacological effects of ethanol.

Animals↗
PubMed1993

The GABAA receptor channel mediated chloride ion translocation through the plasma membrane: new insights from 36Cl- ion flux measurements.

GABAA receptors in plasma membranes of neurons are integral oligomers which form chloride channels. The binding of GABA molecules at recognition sites for channel opening triggers a transient increase in transmembrane chloride ion flux. The multiplicity and drug specificity of GABAA receptor, kinetics of channel opening, and desensitization of GABAA receptor and its short- and long-term regulation have been investigated by the use of tracer amounts of the radioactive chloride isotope, 36Cl- ion. Results and new insights from 36Cl- ion flux measurements have been reviewed.

Animals↗
PubMed2000

Burst properties of a supergated double-barrelled chloride ion channel.

The chloride selective channel from Torpedo electroplax, ClC-0, is the prototype of a large gene family of chloride channels that behave as functional dimers, with channel currents exhibiting two non-zero conductance levels. Each pore has the same conductance and is controlled by a subgate, and these have seemingly identical fast gating kinetics. However, in addition to the two subgates there is a single slower 'supergate' which simultaneously affects both channels. In the present paper, we consider a six state Markov model that is compatible with these observations and develop approximations as well as exact results for relevant properties of groupings of openings, known as bursts. Calculations with kinetic parameter values typical of ClC-0 suggest that even simple approximations can be quite accurate. Small deviations from the assumption of independence within the model lead to marked changes in certain predicted burst properties. This suggests that analysis of these properties may be helpful in assessing independence/non-independence of gating in this type of channel. Based on simulations of models of both independent and non-independent gating, tests using binomial distributions can lead to false conclusions in each situation. This is made more problematic by the difficulty of selecting an appropriate critical time in defining a burst empirically.

Animals↗
PubMed2000

Retinoic acid-induced apoptotic pathway in T-cell lymphoma: Identification of four groups of genes with differential biological functions.

Retinoic acid (RA) has been used to induce the regression of refractory T-cell lymphoma. In vitro and in vivo studies have shown that RA exerts this effect through the induction of apoptosis. This study was designed to investigate the molecular pathway of RA-induced apoptosis in T-lymphoma cell lines.RA-induced apoptosis was verified by morphology, flow cytometry, and DNA ladder analysis. Differential display method using a combination of 12 poly(A)-anchored primers and 20 arbitrary primers was adopted for gene cloning. Total RNAs were extracted from H9 cell line at 0, 6, 12, and 24 hours after All-trans RA (ATRA) treatment and the serial expression patterns of the candidate fragments were recognized. The cloned gene fragments were then analyzed and confirmed by Northern blot analysis on H9 and SR786 cell lines.ATRA-induced apoptosis of T-cell lymphoma was protein synthesis-dependent. The execution or irreversible phase of apoptosis appeared to occur at 6-12 hours of RA treatment. Among the 60,000 arbitrarily displayed bands, 25 of 250 candidate fragments were selected for further cloning and sequencing. A total of 14 clones could be matched to known genes and were categorized into four groups: A) transcription factors: prothymosin, CA150, p78 serine/threonine kinase, IL-1beta-stimulating gene, glucocorticoid receptor, MLN64/CAB1, gastrin-binding protein, and polypeptide from glioblastoma; B) chaperone: 90 kDa heat shock protein; C) ion channel: chloride channel protein 3; and D) cytoskeleton: cytovillin2/ezrin and vimentin. Another two clones of genes were of unrecognized functions. The remaining 11 clones belonged to unmatched or novel genes. The expression of these genes varied, either upregulated or downregulated, in response to ATRA treatment.RA-induced apoptosis may involve a cascade of genes that are related to transcription regulation, stress response, housekeeping, and the execution of apoptosis. The clarification of the RA-induced apoptotic pathway will help us to understand the molecular mechanism of cancer differentiation agents.

Apoptosis↗
PubMed1999

Signal transduction involving cyclic AMP-dependent and cyclic AMP-independent mechanisms in the control of steroidogenesis.

The control of steroidogenesis via signal transduction mechanisms involving cAMP-dependent and cAMP-independent mechanisms is reviewed. Several structurally unrelated factors that are potent stimulators of steroidogenesis whose actions do not require cAMP and/or synthesis of proteins have been identified. These include various interleukins, a lipophilic factor from macrophages, a steroidogenic inducing protein from follicular fluid and an imidazole compound, calmidazolium. All of these factors are capable of inducing maximum steroidogenesis. Calcium is required for steroidogenesis in all steroidogenic cells. With the exception of the effects of angiotensin II, there is little evidence for a role of IP3 in the stimulation of the release of calcium from intracellular stores in steroidogenic cells under physiological conditions. There may however, be a cAMP-mediated activation of a plasma membrane calcium channel. Chloride channels that can be regulated by cAMP-dependent and -independent mechanisms, are present in steroidogenic cells. Chloride ions exert a negative effect on steroidogenesis because exclusion of chloride from the extracellular medium markedly enhances cAMP-stimulated steroidogenesis. Arachidonic acid and its lipoxygenase products are involved in the control of steroidogenesis via cAMP mediated processes. An arachidonic acid related thioesterase has been isolated that is activated by ACTH and which may be involved in the release of arachidonic acid. It is concluded that while cAMP is a second messenger for LH/ACTH in the control of steroidogenesis, other signalling systems exist which are potentially equally effective in controlling steroidogenesis. In addition, the action of cAMP requires other signalling pathways involving calcium and chloride ions, as well as arachidonic acid and its lipoxygenase products.

Animals↗
PubMed2001

PDGF-induced coupling of function with metabolism in microvascular pericytes of the retina.

PURPOSE: The aim of this study was to test the hypothesis that platelet-derived growth factor (PDGF)-BB regulates the physiology of retinal pericytes, which are contractile cells located on the abluminal surface of capillaries. The expression of PDGF-BB and its cognate receptor in retinal vessels suggests a vasoactive function. However, although endothelium-derived PDGF-BB appears vital for the development of pericyte-containing microvessels, its role in the mature vasculature remains uncertain. METHODS: Based on the premise that ion channels mediate the responses of pericytes to vasoactive signals, the perforated-patch configuration of the patch-clamp technique was used to determine the effect of PDGF-BB on the ionic currents and membrane potential of pericytes located on microvessels freshly isolated from the adult rat retina. Changes in pericyte calcium levels were monitored with the calcium indicator fluo-4. Differential interference contrast optics and image analysis software aided in assessing the effects of PDGF-BB on the lumens of isolated pericyte-containing microvessels. In some experiments, blockers of adenosine triphosphate (ATP) synthesis created chemical ischemia. RESULTS: Electrophysiological recordings from pericytes showed that PDGF-BB can activate nonspecific cation channels, chloride channels, and ATP-sensitive potassium channels. The metabolic status of an isolated capillary determined which of these ion channels were activated by PDGF-BB and thereby whether the membrane potential decreased or increased, the cell calcium rose or fell, and the vessel lumen constricted or dilated. CONCLUSIONS: The ability of PDGF-BB to be a vasoconstrictor when energy supplies are ample and to be a vasodilator under ischemic conditions may provide an efficient mechanism to link capillary function to local metabolic needs.

Aniline Compounds↗
PubMedDate not supplied

Bicarbonate-dependent lipid ordering and protein aggregation are part of the nongenomic action of progesterone on capacitated spermatozoa.

The nongenomic action of progesterone (P) on capacitated sperm in mediating acrosomal exocytosis operates through transmembrane signal transduction involving increased intracellular calcium ions and modulation of protein kinases and phospholipid metabolism through a second messenger pathway. Conflicting views exist regarding the nature of the nongenomic receptor of P. It is thought to be a cell-surface receptor having the properties of a calcium channel, chloride channel, bicarbonate/chloride exchanger, and gamma aminobutyric acid type A (GABA(A)). In this study, we tried to understand the role of bicarbonate and/or P in inducing membrane perturbations in capacitated and acrosome-reacting spermatozoa. We also attempted to characterize the membrane responses in P-stimulated and bicuculline-poisoned sperm. The presence of a high level of bicarbonate in the medium favored high rotational mobility of lipids and proteins in capacitated sperm. However, the capacitated sperm exhibited low lipid ordering and unaltered protein ordering in response to the high bicarbonate concentration. P and bicuculline appeared to bind to the same or similar binding sites, probably a GABA(A) receptor, eliciting similar motional perturbations in sperm membranes. It appears that both P and bicuculline can induce receptor aggregation and lipid ordering in sperm membranes, which alter the capability of sperm to bind zona pellucida, and that the P action on sperm takes effect through a bicarbonate-dependent signal transduction mechanism.

Acrosome Reaction↗
PubMed1988

Properties of single chloride selective channel from sarcoplasmic reticulum.

The behavior of single chloride channels in sarcoplasmic reticulum of rabbit and trout skeletal muscle was examined by fusing isolated vesicle fractions into planar lipid bilayers. The channel exhibited a full open state with a unit conductance of 65 pS (in 100 mM Cl-) and several subconductance states with reversal potentials which were dependent on the chloride gradient across the bilayer. Open probability was 0.6-0.95 for membrane potentials ranging from -60 to +60 mV. The kinetic behaviour could be described by assuming one time constant for the fully conducting channel, and at least two time constants for the non-conducting channel. In the presence of methane sulfonate, sulfate and phosphate anions, a decrease in the unit current amplitude but not open time argued in favor of a competition between these anions and Cl- at the transport site of the channel. Chloride channel activity was not affected by variations of Ca2+ concentration in both chambers or by the presence of Mg2+. Similarly, neither millimolar ATP nor the presence of the drugs taurine (up to 10 mM), lidocaine (2-40 microM) or the calmodulin antagonist W7 (5-150 microM), modified channel behavior. Finally, pH variations between 6.8 to 8 were without effect.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1997

Molecular cloning and expression of a chloride ion channel of cell nuclei.

Ion channels are known to be present on the plasma membrane of virtually all cells and have been found on the membranes of various intracellular organelles. However, until recently they were believed not to occur at the nuclear membrane. In this study we describe the molecular cloning and characterization of a nuclear ion channel protein, designated nuclear chloride channel-27 (NCC27), from the human myelomonocytic cell line, U937. NCC27 is a novel chloride ion channel protein that was found to localize principally to the cell nucleus. Its only known homologue is a bovine chloride ion channel protein (p64) believed to localize to internal organelles. NCC27 therefore represents the first human member of a new class of organellar chloride ion channel proteins.

Amino Acid Sequence↗
PubMed1998

X-linked recessive nephrolithiasis: presentation and diagnosis in children.

We report a new X-linked recessive nephrolithiasis kindred. X-linked recessive nephrolithiasis is a recently described disease characterized by recurrent nephrolithiasis, nephrocalcinosis, and progressive renal failure, associated with mutations in a renal chloride channel gene, chloride channel number 5. Screening individuals at risk with renal ultrasonography and measurement of urinary excretion of low molecular weight proteins and calcium will exclude boys without X-linked recessive nephrolithiasis kindred and identify boys likely to have the disease.

Calcium↗
PubMed1997

Nonmuscle myosin IIA copurifies with chloride channel-enriched membranes from epithelia.

Hydrophobic affinity chromatography can be used to isolate, from bovine tracheal epithelial cells, an intracellular membrane vesicle fraction enriched in a single type of chloride channel. The intracellular origin of these vesicles and the function of this chloride channel has not been established. As a first step in this direction, we have purified a 200 kDa protein that copurifies with these membranes. Partial amino acid sequencing and immunoblotting with specific antibodies identify this protein as nonmuscle myosin IIA. Since the vesicle chloride channel is present in plasma membrane fractions as well, we hypothesize that these vesicles represent an intracellular storage or recycling compartment for epithelial chloride channels, which translocate to the plasma membrane during periods of peak demand for epithelial fluid secretion.

Amino Acid Sequence↗