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PubMed1998

Functional characterization of renal chloride channel, CLCN5, mutations associated with Dent'sJapan disease.

BACKGROUND: The annual urinary screening of Japanese children above three years of age has identified a progressive renal tubular disorder characterized by low molecular weight proteinuria, hypercalciuria and nephrocalcinosis, and this represents a variant of Dent's disease. Hitherto, 12 mutations of the X-linked renal specific chloride channel, CLCN5, have been reported in the Dent'sJapan variant. To further identify such CLCN5 mutations and to define the structure-function relationships of this channel, we have investigated five unrelated, non-consanguinous Japanese families with this disorder. METHODS: Leukocyte DNA from probands was used with CLCN5 primers for PCR amplification of the coding region, and the DNA sequences of the products determined. Functional studies were performed by expressing the mutants in Xenopus oocytes. RESULTS: Five CLCN5 mutations consisting of two nonsense (R648X and R704X), two missense (S270R and L278F) and one acceptor splice site mutation (ag-->cg) in intron 4 were identified. The missense and splice site mutations represent novel abnormalities. Heterologous expression in Xenopus oocytes of wild-type and the missense mutants demonstrated that the mutations, which were translated, either abolished or markedly reduced chloride conductance. CONCLUSIONS: These results expand the spectrum of CLCN5 mutations associated with this renal disorder and provide insight into possible structure-function relationships. For example, both the missense mutations are located within a short putative loop between two transmembrane domains, and our results suggest that this region may have an important functional role in the regulation of channel activity.

Adolescent↗
PubMed1987

Characterization of human sweat duct chloride conductance by chloride channel blockers.

To characterize the chloride conductance of human sweat duct the effect of various analogues of diphenylamine-2-carboxylate was investigated on the transepithelial potential difference (PDT) and resistance (RT) of isolated microperfused sweat ducts. Although the most powerful analogues which block Cl- channels in various secretory and absorptive epithelia were ineffective, a number of analogues (in particular Cl substituted ones) were found which at high concentrations significantly and reversibly increased PDT and RT. The data suggest that the main chloride conductance pathway of sweat duct epithelium resides in the cell membranes rather than in the tight junctions. In addition the different blocking spectra of the chloride conductances of sweat duct and tracheal epithelium (Welsh MJ, Science 232:1648, 1986) suggest that the combined impairment of both conductances in cystic fibrosis does not result from a molecular defect in the Cl- channels.

Chlorides↗
PubMed2003

Chloride channel ClCN7 mutations are responsible for severe recessive, dominant, and intermediate osteopetrosis.

UNLABELLED: Among 94 osteopetrotic patients presenting with a severe clinical picture and diagnosed early in life, 12 bore mutations in the ClCN7 gene, but only 7 of them had the expected two recessive mutations. The remaining five patients seem to be heterozygous for a ClCN7 mutation, and significant variations were observed in the clinical manifestations of their disease, even within the same family. INTRODUCTION: Human osteopetroses are a heterogeneous group of diseases that include both infantile severe, autosomal recessive (ARO) and adult autosomal dominant (ADO) forms. Two genes, Atp6a3 (TCIRG1) and ClCN7, have been shown to be associated with human ARO, the latter of which is also thought to be responsible for ADO-II. However, patients with an intermediate phenotype have been described: the genetic basis of these observances is unknown. MATERIALS AND METHODS: In this study, we report the clinical and molecular analysis of 94 patients in which a diagnosis of severe osteopetrosis was made within the first 2 years of age. Both TCIRG1 and CLCN7 genes were sequenced in all patients and the molecular findings were correlated to clinical parameters. RESULTS AND CONCLUSIONS: In 56 of 94 patients with a classical picture of ARO, TCIRG1-dependent recessive mutations were found. In contrast, ClCN7 mutations were found in 12 cases (13%) of severe osteopetrosis, but only 7 of them had two recessive mutations identified: in 6 of these 7 cases, central nervous system manifestations were noted, and these patients had a poor prognosis. The remaining five cases were heterozygous for a ClCN7 mutation, including two brothers from a large family with a history of ADO-II in which the presence of a second ClCN7 mutation was formally excluded. Despite an early and severe clinical presentation, these five patients all reached adulthood, suggesting that the degree of dominant interference with chloride channel function can vary widely. Our findings suggest that recessive ClCN7-dependent ARO may be associated with CNS involvement and have a very poor prognosis, whereas heterozygous ClCN7 mutations cause a wide range of phenotypes even in the same family, ranging from early severe to nearly asymptomatic forms. These findings have prognostic implications, might complicate prenatal diagnosis of human osteopetroses, and could be relevant to the management of these patients.

Adolescent↗
PubMed1998

A novel frameshift mutation 840delA and a novel polymorphism D203A in the steroidogenic acute regulatory protein gene in a Japanese patient with congenital lipoid adrenal hyperplasia. Mutations in brief no. 117. Online.

Congenital lipoid adrenal hyperplasia (CLAH) is an autosomalrecessive disorder characterized by impaired production of all steroids including glucocorticoids, mineralocorticoids and sexsteriods. It has recently been reported that mutations in the steriodogenic acute regulatory protein (StAR) gene cause CLAH. We analyzed the StAR gene in a Japanese patient with CLAH. The patient was revealed to be a compound heterozygote bearing a nonsense mutation Q258X, changing codon 258 (CAG) encoding Gln to the stop codon TAG, and a novel framshift mutation 840delA resulting from deletion of one of the three adenosines normally present in codon 238 (AAA), thus leading to a frameshift after codon 237 (Thr) in the StAR gene. The patient was also revealed to be homozygous for a novel missense point mutation D203A, changing codon 203 (GAC) encoding Asp to GCC encoding Ala in the StAR gene. To elucidate the significance of the D203A mutation, we analyzed the StAR gene sequence in twenty normal subjects, and found that all of them were homozygous for the D203A mutation, indicating that the D203A mutation is an innocent polymorphism. In conclusion, we have identified a novel frameshift mutation 840delA which seems to cause 840delA and the first polymorphism D203A in the human StAR gene.

Adrenal Hyperplasia, Congenital↗
PubMed2003

Functional analysis of CFTR chloride channel activity in cells with elevated MDR1 expression.

Using the patch-clamp method, we investigated a relationship between MDR1 expression and its effects on the CFTR channel function. Incubation of CaCo-2 cells with increasing concentrations of doxorubicin resulted in a reduction of CFTR chloride channel activity in a dose-dependent manner. This reduction was associated with a decrease of CFTR mRNA and simultaneous up-regulation of MDR1 mRNA in the presence of doxorubicin. Similar alteration of the CFTR function was observed in CaCo-2 cells transiently overexpressing MDR1. No alterations of the cAMP-dependent chloride currents were observed in COS-1 cells transiently co-expressing CFTR and MDR1 from strong CMV promoters. This indicated that repression of CFTR by MDR1 induction requires the presence of the native CFTR promoter.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Completely functional double-barreled chloride channel expressed from a single Torpedo cDNA.

We have performed an electrophysiological analysis of the recently cloned Torpedo marmorata Cl- channel. Functional expression of Cl- channels in oocytes of Xenopus laevis previously injected with cRNA yielded an outward-rectifying current activated by hyperpolarization. Replacement of Cl- with other anions significantly reduced or inhibited the current. Single-channel recordings from cell-attached patches exhibited burst-like Cl- channel activity with rapid fluctuations between three equally spaced substates (0 pS, 9 pS, and 18 pS). The properties of the cloned Cl- channel were almost identical to those of the reconstituted native T. californica Cl- channel and were in full agreement with the predictions of the double-barreled channel model [Hanke, W. & Miller, C. (1983) J. Gen. Physiol. 82, 25-45]. Our results imply that the cloned cDNA codes for the completely functional Torpedo electroplax Cl- channel.

Animals↗
PubMed2000

Inhibition of cystic fibrosis transmembrane conductance regulator chloride channel currents by arachidonic acid.

Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is inhibited by a number of different classes of organic anions which are able to enter and block the channel pore from its cytoplasmic end. Here I show, using patch clamp recording from CFTR-transfected baby hamster kidney cell lines, that the cis-unsaturated fatty acid arachidonic acid also inhibits CFTR Cl- currents when applied to the cytoplasmic face of excised membrane patches. This inhibition was of a relatively high affinity compared with other known CFTR inhibitors, with an apparent Kd of 6.5 +/- 0.9 microM. However, in contrast with known CFTR pore blockers, inhibition by arachidonic acid was only very weakly voltage dependent, and was insensitive to the extracellular Cl- concentration. Arachidonic acid-mediated inhibition of CFTR Cl- currents was not abrogated by inhibitors of lipoxygenases, cyclooxygenases or cytochrome P450, suggesting that arachidonic acid itself, rather than some metabolite, directly affects CFTR. Similar inhibition of CFTR Cl- currents was seen with other fatty acids, with the rank order of potency linoleic > or = arachidonic > or = oleic > elaidic > or = palmitic > or = myristic. These results identify fatty acids as novel high affinity modulators of the CFTR Cl- channel.

Animals↗
PubMed1992

Actions of ethanol on gamma-aminobutyric acid-activated chloride channels.

Ethanol appears to exert its intoxicating effects, at least partly, by potentiating gamma-aminobutyric acid (GABA) mediated inhibitory synaptic transmission in the brain. Work over the past decade using mouse lines selected for different ethanol sensitivities has implicated GABA-activated chloride channels as a potential target of ethanol. More recently, molecular biological techniques have identified a subunit (gamma 2L) of the GABA channel which appears to be required for the effects of ethanol on GABA channels. The presence of this subunit, however, cannot account for the full degree of potentiation of GABA responses observed in native GABA channels. Ongoing research is directed at identifying other factors which might be involved in the effects of ethanol on GABA channels and in understanding ethanol's molecular mechanism of action. Such information could be vital for designing drugs to combat ethanol intoxication.

Animals↗
PubMed1996

Regulation of the serum-activated Ca(2+)-dependent chloride channel in Xenopus oocytes.

Fetal bovine serum evoked Ca(2+)-dependent chloride currents with two components in Xenopus oocytes. The evoked currents were inhibited by GDP beta S, but not by pertussis toxin (PTX). An inositol 1,4,5-triphosphate (IP3) receptor antagonist, heparin completely inhibited the currents, although a phospholipase C inhibitor, neomycin had no effect. The serum-activated currents were enhanced to 171% by a selective protein kinase C (PKC) inhibitor, GF109203X. By contrast, a potent PKC activator, TPA, abolished the initial component of the currents and arachidonic acid enhanced this effect. The effects of TPA and/or arachidonic acid on the currents inhibited by GF109203X. These results indicate that the receptor for serum is linked to a PTX-insensitive G-protein involving cytosolic Ca2+ release through IP3 and PKC activation by a mechanism independent of a phospholipase C-mediated phospholipid signaling. Furthermore, the evoked currents are regulated by PKC and arachidonic acid appears to potentiate its effect.

Animals↗
PubMed2004

The cystic fibrosis mutation G1349D within the signature motif LSHGH of NBD2 abolishes the activation of CFTR chloride channels by genistein.

Cystic fibrosis (CF) is a common lethal genetic disease caused by autosomal recessive mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel that belongs to the ATP-Binding Cassette (ABC) family of transporters. The class III CF mutations G551D and G1349D are located within the "signature" sequence LSGGQ and LSHGH of NBD1 and NBD2, respectively. We have constructed by site-directed mutagenesis vectors encoding green fluorescent protein (GFP)-tagged wild-type (wt) CFTR or CFTR containing delF508, G551D, G1349D and G551D/G1349D to study their pharmacology after transient expression in COS-7 cells. We show that IBMX and the benzo[c]quinolizinium derivative MPB-91 stimulates the activity of G1349D-, G551D- and G551D/G1349D-CFTR only in the presence of cAMP-promoting agents like forskolin or cpt-cAMP. Similar half-maximal effective concentrations (EC(50)) of MPB-91 (22-36microM) have been determined for wt-, G551D-, G1349D- and G551D/G1349D-CFTR. The isoflavone genistein stimulates wild-type (wt)- and delF508-CFTR channel activity in a non-Michaelis-Menten manner. By contrast, the response of G1349D- and G551D-CFTR to genistein is dramatically altered. First, genistein is not able to stimulate G1349D- and G551D/G1349D-CFTR. Second, genistein stimulates G551D-CFTR without any inhibition at high concentration. We conclude from these results that whereas G551 in NBD1 is an important molecular site for inhibition of CFTR by genistein, the symmetrical G1349 in NBD2 is also one major site but for the activation of CFTR by genistein. Because both mutations alter specifically the mechanism of CFTR channel activation by genistein, we believe that the signature sequences of CFTR act as molecular switches that upon interaction with genistein turn on and off the channel.

Amino Acid Motifs↗
PubMed1992

Isolation and identification from Salvia officinalis of two diterpenes which inhibit t-butylbicyclophosphoro[35S]thionate binding to chloride channel of rat cerebrocortical membranes in vitro.

Ethanolic extracts from dried leaves of sage (Salvia officinalis) showed inhibition of [35S]tertiary-butylbicyclophosphorothionate ([35S]TBPS) binding to rat brain membranes in vitro. This ligand is considered to bind to the chloride channel of the GABA/benzodiazepine receptor complex in brain tissue. Substances having inhibitory activity were purified and their chemical structure identified as the diterpenes carnosic acid and carnosol (IC50 values of 33 +/- 3 microM and 57 +/- 4 microM, respectively). The two compounds did not affect binding of the ligands [3H]muscimol and [3H]diazepam to the GABA/benzodiazepine complex in vitro. Saturation experiments of [35S]TBPS binding indicated that carnosic acid decreases the binding affinity.

Abietanes↗
PubMed2000

Deletion of phenylalanine 508 causes attenuated phosphorylation-dependent activation of CFTR chloride channels.

In cell-attached patches stimulated with cAMP agonists, the single-channel open probability (Po) of the phenylalanine 508-deleted cystic fibrosis transmembrane conductance regulator (DeltaF508-CFTR) channel, the most common disease-associated mutation in cystic fibrosis, was abnormally low (a functional defect). To investigate the mechanism for the poor response of DeltaF508-CFTR to cAMP stimulation, we examined, in excised inside-out patches, protein kinase A (PKA)-dependent phosphorylation activation and ATP-dependent gating of wild-type (WT) and DeltaF508-CFTR channels expressed in NIH3T3 mouse fibroblasts. For WT-CFTR, the activation time course of CFTR channel current upon addition of PKA and ATP followed a sigmoidal function with time constants that decreased as [PKA] was increased. The curvilinear relationship between [PKA] and the apparent activation rate suggests an incremental phosphorylation-dependent activation of CFTR at multiple phosphorylation sites. The time course of PKA-dependent activation of DeltaF508-CFTR channel current also followed a sigmoidal function, but the rate of activation was at least 7-fold slower than that with WT channels. This result suggests that deletion of phenylalanine 508 causes attenuated PKA-dependent phosphorylation of the CFTR chloride channel. Once DeltaF508-CFTR channels were maximally activated with PKA, the mutant channel and WT channel had indistinguishable steady-state Po values, ATP dose-response relationships and single-channel kinetics, indicating that DeltaF508-CFTR is not defective in ATP-dependent gating. By measuring whole-cell current density, we compared the number of functional channels in WT- and DeltaF508-CFTR cell membrane. Our data showed that the estimated channel density for DeltaF508-CFTR was approximately 10-fold lower than that for WT-CFTR, but the cAMP-dependent whole-cell current density differed by approximately 200-fold. We thus conclude that the functional defect (a decrease in Po) of DeltaF508-CFTR is as important as the trafficking defect (a decrease in the number of functional channels in the plasma membrane) in cystic fibrosis pathogenesis.

3T3 Cells↗
PubMed1999

Anion permeability and conduction of adenine nucleotides through a chloride channel in cardiac sarcoplasmic reticulum.

Cardiac sarcoplasmic reticulum (SR) membrane contains several chloride (Cl-) channels. We have characterized a 116-pS Cl- channel (500 mM cis, 50 mM trans Cl-) in cardiac SR that is activated by protein kinase A-dependent phosphorylation. To understand its function further, we examined the permeation of various anions and adenine nucleotides using the planar lipid bilayer-vesicle fusion technique. This Cl- channel showed a high selectivity to anions and its permeability sequence was Br- > Cl- > I- > NO3- > F-. When all anions were replaced with ATP in the cis solution, channel activity persisted. The conductance was 78 pS with 200 mM ATP and 68 pS with 100 mM ATP. The reversal potentials were +63 mV and +41 mV in 200 mM ATP and in 100 mM ATP, respectively. With 100 mM ADP or AMP in the cis solution, channel activities were also observed. The conductances were 87 pS with 100 mM ADP and 115 pS with 100 mM AMP. The apparent adenine selectivity of this channel was ATP > ADP > AMP, assuming that they exist as divalent anions. These results suggest that the SR Cl- channel in cardiac cells may serve as a transporter for the movement of adenine nucleotides between cytosol and SR lumen.

Adenine Nucleotides↗