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Thomas J Jentsch - One of the best experts on this subject based on the ideXlab platform.
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leukoencephalopathy upon disruption of the Chloride Channel clc 2
The Journal of Neuroscience, 2007Co-Authors: Judith Blanz, Thomas J Jentsch, Muriel Auberson, Michaela Schweizer, Hannes Maier, Adrian Muenscher, Christian A HubnerAbstract:ClC-2 is a broadly expressed plasma membrane Chloride Channel that is modulated by voltage, cell swelling, and pH. A human mutation leading to a heterozygous loss of ClC-2 has previously been reported to be associated with epilepsy, whereas the disruption of Clcn2 in mice led to testicular and retinal degeneration. We now show that the white matter of the brain and spinal cord of ClC-2 knock-out mice developed widespread vacuolation that progressed with age. Fluid-filled spaces appeared between myelin sheaths of the central but not the peripheral nervous system. Neuronal morphology, in contrast, seemed normal. Except for the previously reported blindness, neurological deficits were mild and included a decreased conduction velocity in neurons of the central auditory pathway. The heterozygous loss of ClC-2 had no detectable functional or morphological consequences. Neither heterozygous nor homozygous ClC-2 knock-out mice had lowered seizure thresholds. Sequencing of a large collection of human DNA and electrophysiological analysis showed that several ClC-2 sequence abnormalities previously found in patients with epilepsy most likely represent innocuous polymorphisms.
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the clc 5 Chloride Channel knock out mouse an animal model for dent s disease
Pflügers Archiv: European Journal of Physiology, 2003Co-Authors: Willy Gunther, Nils Piwon, Thomas J JentschAbstract: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 α-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.
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molecular dissection of gating in the clc 2 Chloride Channel
The EMBO Journal, 1997Co-Authors: Sveneric Jordt, Thomas J JentschAbstract:The ClC-2 Chloride Channel is probably involved in the regulation of cell volume and of neuronal excitability. Site-directed mutagenesis was used to understand ClC-2 activation in response to cell swelling, hyperpolarization and acidic extracellular pH. Similar to equivalent mutations in ClC-0, neutralizing Lys566 at the end of the transmembrane domains results in outward rectification and a shift in voltage dependence, but leaves the basic gating mechanism, including swelling activation, intact. In contrast, mutations in the cytoplasmic loop between transmembrane domains D7 and D8 abolish all three modes of activation by constitutively opening the Channel without changing its pore properties. These effects resemble those observed with deletions of an amino-terminal inactivation domain, and suggest that it may act as its receptor. Such a 'ball-and-chain' type mechanism may act as a final pathway in the activation of ClC-2 elicited by several stimuli.
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idiopathic low molecular weight proteinuria associated with hypercalciuric nephrocalcinosis in japanese children is due to mutations of the renal Chloride Channel clcn5
Journal of Clinical Investigation, 1997Co-Authors: Sarah E Lloyd, Thomas J Jentsch, Takashi Igarashi, Willy Gunther, Simon H S Pearce, H Kawaguchi, Rajesh V ThakkerAbstract:The annual urinary screening of Japanese children above 3 yr of age has identified a progressive proximal renal tubular disorder characterized by low molecular weight proteinuria, hypercalciuria, and nephrocalcinosis. The disorder, which has a familial predisposition and occurs predominantly in males, has similarities to three X-linked proximal renal tubular disorders that are due to mutations in the renal Chloride Channel gene, CLCN5. We have investigated four unrelated Japanese kindreds with this tubulopathy and have identified four different CLCN5 mutations (two nonsense, one missense, and one frameshift). These are predicted to lead to a loss of Chloride Channel function, and heterologous expression of the missense CLCN5 mutation in Xenopus oocytes demonstrated a 70% reduction in Channel activity when compared with the wild-type. In addition, single-stranded conformation polymorphism (SSCP) analysis was found to be a sensitive and specific mutational screening method that detected > 75% of CLCN5 mutations. Thus, the results of our study expand the spectrum of clinical phenotypes associated with CLCN5 mutations to include this proximal renal tubular disorder of Japanese children. In addition, the mutational screening of CLCN5 by SSCP will help to supplement the clinical evaluation of the annual urinary screening program for this disorder.
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two physically distinct pores in the dimeric clc 0 Chloride Channel
Nature, 1996Co-Authors: Uwe Ludewig, Michael Pusch, Thomas J JentschAbstract:The Torpedo Chloride Channel ClC-0 is the prototype of a large family of Chloride Channels that have roles in transepithelial transport and in regulating electrical excitability and cell volume. ClC-0 opens in bursts with two identical conductance levels of approximately 8pS. Hyperpolarization slowly increases the probability of bursts ('slow gating'), and depolarization increases Channel opening within bursts ('fast gating'). Replacing serine 123 by threonine changes rectification, ion selectivity and gating, but retains the typical bursting behaviour with two identical independent albeit reduced, conductance states (approximately 1.5 pS). Coexpression with wild-type ClC-0, either as covalently linked concatamers or as independent proteins, leads to bursting Channels with two different pores. Our experiments strongly suggest that conductance, ion selectivity and 'fast' gating are determined only by the single subunit forming a single pore, independent from the attached pore; in contrast, 'slow' gating is a function of both subunits. Thus ClC-0 is a homodimer with two largely independent pores.
Klaus Steinmeyer - One of the best experts on this subject based on the ideXlab platform.
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cloning and functional expression of rat clc 5 a Chloride Channel related to kidney disease
Journal of Biological Chemistry, 1995Co-Authors: Klaus Steinmeyer, Blanche Schwappach, Marcelle Bens, Alain Vandewalle, Thomas J JentschAbstract:Abstract We have cloned a novel member of the CLC Chloride Channel family from rat brain, rCLC-5. The cDNA predicts a 83-kDa protein belonging to the branch including CLC-3 and CLC-4, with which it shares 80% identity. Expression of rCLC-5 in Xenopus oocytes elicits novel anion currents. They are strongly outwardly rectifying and have a conductivity sequence of NO3 > Cl > Br > I glutamate. Although CLC-5 has consensus sites for phosphorylation by protein kinase A, raising the intracellular cAMP concentration had no effect on these currents. Currents were also unchanged when rCLC-5 was coexpressed with rCLC-3 and rCLC-4, either singly or in combination. rCLC-5 is expressed predominantly in kidney and also in brain, lung, and liver. Along the nephron, rCLC-5 message is detectable in all tubule segments investigated, but expression in the glomerulus and the S2 segment of the proximal tubule is low.
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Mutations in dominant human myotonia congenita drastically alter the voltage dependence of the CIC-1 Chloride Channel
Neuron, 1995Co-Authors: Michael Pusch, Klaus Steinmeyer, Manuela C. Koch, Thomas J JentschAbstract:Abstract Autosomal dominant myotonia congenita (Thomsen's disease) is caused by mutations in the muscle Chloride Channel CIC-1. Several point mutations found in affected families (1290M, R317Q, P480L, and G1552R) dramatically shift gating to positive voltages in mutant/WT heterooligomeric Channels, and, when measurable, even more so in mutant homooligomers. These Channels can no longer contribute to the repolarization of action potentials, fully explaining why they cause dominant myotonia. Most replacements of the isoleucine at position 290 shift gating toward positive voltages. Mutant/WT heterooligomers can be partially activated by repetitive depolarizations, suggesting a role in shortening myotonic runs. Remarkably, a human mutation affecting an adjacent residue (E291 K) is fully recessive. Large shifts in the voltage dependence of gating may be common to many mutations in dominant myotonia congenita.
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Genomic organization of the human muscle Chloride Channel CIC-1 and analysis of novel mutations leading to Becker-type myotonia
Human Molecular Genetics, 1994Co-Authors: C Lorenz, Klaus Steinmeyer, Christof Meyer-kleine, Manuela C. Koch, Thomas J JentschAbstract:The muscle Chloride Channel CIC-1 regulates the electric excitability of the skeletal muscle membrane. Mutations in the gene encoding this Chloride Channel (CLCN1) are responsible for both human purely myotonic disorders, autosomal recessive generalized myotonia (Becker's disease, GM) and autosomal dominant myotonia congenita (Thomsen's disease, MC). We now show that the protein-coding sequence of the CLCN1 gene is organized into 23 exons. The CIC-1 upstream region contains a canonical TATA box, several consensus binding sites for myogenic transcription factors and two other putative regulatory elements. SSCA analysis of a German GM family revealed that affected members are compound heterozygotes having two novel mutations
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Multimeric structure of ClC-1 Chloride Channel revealed by mutations in dominant myotonia congenita (Thomsen).
The EMBO Journal, 1994Co-Authors: Klaus Steinmeyer, Michael Pusch, C Lorenz, M C Koch, Thomas J JentschAbstract:Voltage-gated ClC Chloride Channels play important roles in cell volume regulation, control of muscle excitability, and probably transepithelial transport. ClC Channels can be functionally expressed without other subunits, but it is unknown whether they function as monomers. We now exploit the properties of human mutations in the muscle Chloride Channel, ClC-1, to explore its multimeric structure. This is based on analysis of the dominant negative effects of ClC-1 mutations causing myotonia congenita (MC, Thomsen's disease), including a newly identified mutation (P480L) in Thomsen's own family. In a co-expression assay, Thomsen's mutation dramatically inhibits normal ClC-1 function. A mutation found in Canadian MC families (G230E) has a less pronounced dominant negative effect, which can be explained by functional WT/G230E heterooligomeric Channels with altered kinetics and selectivity. Analysis of both mutants shows independently that ClC-1 functions as a homooligomer with most likely four subunits.
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Nonsense and missense mutations in the muscular Chloride Channel gene Clc- 1 of myotonic mice
Journal of Biological Chemistry, 1994Co-Authors: M. Gronemeier, A. Condie, Jane Prosser, Klaus Steinmeyer, Thomas J Jentsch, Harald JockuschAbstract:In mature vertebrate muscle, the Chloride Channel Clc-1 is necessary for the stabilization of the resting potential. Its functional defect leads to the disease myotonia. The ADR mouse (phenotype ADR, genotype adr/adr) is an animal model for human myotonias. The adr gene is a member of a family of non-complementing recessive autosomal mutations ("alleles" of adr) that cause myotonia in the mouse. The standard allele adr has arisen by the insertion of a retroposon into the Chloride Channel gene Clc-1 (Steinmeyer, K., Klocke, R., Ortland, C., Gronemeier, M., Jockusch, H., Gründer, S., and Jentsch, T. J. (1991) Nature 354, 304-308). In order to study the nature of two other alleles, adrmto and adrK, we have analyzed overlapping Clc-1 cDNA amplification products by the hydroxylamine and osmium tetroxide modification technique and direct sequencing. A comparison between ADR*MTO and C57BL/6 wild type showed six base pair substitutions, one of which resulted in a stop codon in position 47, whereas the five others are either silent or lead to amino acid substitutions in non-conserved regions of the Clc-1 sequence and were already present in the wild type inbred SWR/J strain from which adrmto was derived. The detection of the stop codon in the adrmto allele is further indication of the identity of the Clc-1 Chloride Channel with the adr myotonia gene in the mouse, because a chain termination close to the N terminus would necessarily destroy gene function. For the ethylnitrosourea-induced mutation adrK, an Ile-->Thr exchange in codon 553 was identified. As this affects a conserved residue within a highly conserved region of the Clc-1 gene, a functional significance of this residue is suggested.
Willy Gunther - One of the best experts on this subject based on the ideXlab platform.
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the clc 5 Chloride Channel knock out mouse an animal model for dent s disease
Pflügers Archiv: European Journal of Physiology, 2003Co-Authors: Willy Gunther, Nils Piwon, Thomas J JentschAbstract: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 α-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.
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functional characterization of renal Chloride Channel clcn5 mutations associated with dent sjapan disease
Kidney International, 1998Co-Authors: Takashi Igarashi, Willy Gunther, Takashi Sekine, Jun Inatomi, Hiroshi Shiraga, Shouri Takahashi, Junzou Suzuki, Noboru Tsuru, Toshio Yanagihara, Mitsunobu ShimazuAbstract:Functional characterization of renal Chloride Channel, CLCN5, mutations associated with Dent's Japan 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’s Japan 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.
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idiopathic low molecular weight proteinuria associated with hypercalciuric nephrocalcinosis in japanese children is due to mutations of the renal Chloride Channel clcn5
Journal of Clinical Investigation, 1997Co-Authors: Sarah E Lloyd, Thomas J Jentsch, Takashi Igarashi, Willy Gunther, Simon H S Pearce, H Kawaguchi, Rajesh V ThakkerAbstract:The annual urinary screening of Japanese children above 3 yr of age has identified a progressive proximal renal tubular disorder characterized by low molecular weight proteinuria, hypercalciuria, and nephrocalcinosis. The disorder, which has a familial predisposition and occurs predominantly in males, has similarities to three X-linked proximal renal tubular disorders that are due to mutations in the renal Chloride Channel gene, CLCN5. We have investigated four unrelated Japanese kindreds with this tubulopathy and have identified four different CLCN5 mutations (two nonsense, one missense, and one frameshift). These are predicted to lead to a loss of Chloride Channel function, and heterologous expression of the missense CLCN5 mutation in Xenopus oocytes demonstrated a 70% reduction in Channel activity when compared with the wild-type. In addition, single-stranded conformation polymorphism (SSCP) analysis was found to be a sensitive and specific mutational screening method that detected > 75% of CLCN5 mutations. Thus, the results of our study expand the spectrum of clinical phenotypes associated with CLCN5 mutations to include this proximal renal tubular disorder of Japanese children. In addition, the mutational screening of CLCN5 by SSCP will help to supplement the clinical evaluation of the annual urinary screening program for this disorder.
Takashi Igarashi - One of the best experts on this subject based on the ideXlab platform.
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functional characterization of renal Chloride Channel clcn5 mutations associated with dent sjapan disease
Kidney International, 1998Co-Authors: Takashi Igarashi, Willy Gunther, Takashi Sekine, Jun Inatomi, Hiroshi Shiraga, Shouri Takahashi, Junzou Suzuki, Noboru Tsuru, Toshio Yanagihara, Mitsunobu ShimazuAbstract:Functional characterization of renal Chloride Channel, CLCN5, mutations associated with Dent's Japan 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’s Japan 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.
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idiopathic low molecular weight proteinuria associated with hypercalciuric nephrocalcinosis in japanese children is due to mutations of the renal Chloride Channel clcn5
Journal of Clinical Investigation, 1997Co-Authors: Sarah E Lloyd, Thomas J Jentsch, Takashi Igarashi, Willy Gunther, Simon H S Pearce, H Kawaguchi, Rajesh V ThakkerAbstract:The annual urinary screening of Japanese children above 3 yr of age has identified a progressive proximal renal tubular disorder characterized by low molecular weight proteinuria, hypercalciuria, and nephrocalcinosis. The disorder, which has a familial predisposition and occurs predominantly in males, has similarities to three X-linked proximal renal tubular disorders that are due to mutations in the renal Chloride Channel gene, CLCN5. We have investigated four unrelated Japanese kindreds with this tubulopathy and have identified four different CLCN5 mutations (two nonsense, one missense, and one frameshift). These are predicted to lead to a loss of Chloride Channel function, and heterologous expression of the missense CLCN5 mutation in Xenopus oocytes demonstrated a 70% reduction in Channel activity when compared with the wild-type. In addition, single-stranded conformation polymorphism (SSCP) analysis was found to be a sensitive and specific mutational screening method that detected > 75% of CLCN5 mutations. Thus, the results of our study expand the spectrum of clinical phenotypes associated with CLCN5 mutations to include this proximal renal tubular disorder of Japanese children. In addition, the mutational screening of CLCN5 by SSCP will help to supplement the clinical evaluation of the annual urinary screening program for this disorder.
Mitsunobu Shimazu - One of the best experts on this subject based on the ideXlab platform.
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functional characterization of renal Chloride Channel clcn5 mutations associated with dent sjapan disease
Kidney International, 1998Co-Authors: Takashi Igarashi, Willy Gunther, Takashi Sekine, Jun Inatomi, Hiroshi Shiraga, Shouri Takahashi, Junzou Suzuki, Noboru Tsuru, Toshio Yanagihara, Mitsunobu ShimazuAbstract:Functional characterization of renal Chloride Channel, CLCN5, mutations associated with Dent's Japan 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’s Japan 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.