Coexpression of complementary fragments of ClC-5 and restoration of chloride channel function in a Dent's disease mutation

2004 ◽  
Vol 286 (1) ◽  
pp. C79-C89 ◽  
Author(s):  
L. Mo ◽  
W. Xiong ◽  
T. Qian ◽  
H. Sun ◽  
N. K. Wills
Keyword(s):  
Chloride Channel ◽  
Functional Activity ◽  
Terminal Region ◽  
Channel Activity ◽  
Loss Of Function ◽  
C Protein ◽  
Dent’S Disease ◽  
Protein Fragments ◽  
Disease Mutation ◽  
Dent's Disease

The human hereditary disorder Dent's disease is linked to loss-of-function mutations of the chloride channel ClC-5. Many of these mutations involve insertion of premature stop codons, resulting in truncation of the protein. We determined whether the functional activity of ClC-5 could be restored by coexpression of the truncated protein (containing the NH2-terminal region) with its complementary “missing” COOH-terminal region. Split channel constructs for ClC-5, consisting of complementary N and C protein regions, were created at an arbitrary site in the COOH-terminal region (V655) and at four Dent's disease mutation sites (R347, Y617, R648, and R704). Coexpression of complementary fragments for the split channel at V655 produced currents with anion and pH sensitivity similar to those of wild-type ClC-5. Channel activity was similarly restored when complementary split channel constructs made for Dent's mutation R648 were coexpressed, but no ClC-5 currents were found when split channels for mutations R347, Y617, or R704 were coexpressed. Immunoblot and immunofluorescence studies of COS-7 cells revealed that N or C protein fragments could be transiently expressed and detected in the plasma membrane, even in split channels that failed to show functional activity. The results suggest that ClC-5 channel activity can be restored for specific Dent's mutations by expression of the missing portion of the ClC-5 molecule.

Conformational defects underlie proteasomal degradation of Dent's disease-causing mutants of ClC-5

2013 ◽  
Vol 452 (3) ◽  
pp. 391-400 ◽  
Author(s):  
Christina D’Antonio ◽  
Steven Molinski ◽  
Saumel Ahmadi ◽  
Ling-Jun Huan ◽  
Leigh Wellhauser ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Chloride Channel ◽  
Proteasomal Degradation ◽  
Terminal Region ◽  
Intramolecular Interactions ◽  
Structural Basis ◽  
Membrane Domain ◽  
Dent’S Disease ◽  
Terminal Domain ◽  
Dent's Disease

Mutations in the CLCN5 (chloride channel, voltage-sensitive 5) gene cause Dent's disease because they reduce the functional expression of the ClC-5 chloride/proton transporter in the recycling endosomes of proximal tubule epithelial cells. The majority (60%) of these disease-causing mutations in ClC-5 are misprocessed and retained in the ER (endoplasmic reticulum). Importantly, the structural basis for misprocessing and the cellular destiny of such ClC-5 mutants have yet to be defined. A ClC-5 monomer comprises a short N-terminal region, an extensive membrane domain and a large C-terminal domain. The recent crystal structure of a eukaryotic ClC (chloride channel) transporter revealed the intimate interaction between the membrane domain and the C-terminal region. Therefore we hypothesized that intramolecular interactions may be perturbed in certain mutants. In the present study we examined two misprocessed mutants: C221R located in the membrane domain and R718X, which truncates the C-terminal domain. Both mutants exhibited enhanced protease susceptibility relative to the normal protein in limited proteolysis studies, providing direct evidence that they are misfolded. Interestingly, the membrane-localized mutation C221R led to enhanced protease susceptibility of the cytosolic N-terminal region, and the C-terminal truncation mutation R718X led to enhanced protease susceptibility of both the cytosolic C-terminal and the membrane domain. Together, these studies support the idea that certain misprocessing mutations alter intramolecular interactions within the full-length ClC-5 protein. Further, we found that these misfolded mutants are polyubiquitinated and targeted for proteasomal degradation in the OK (opossum kidney) renal epithelial cells, thereby ensuring that they do not elicit the unfolded protein response.

scholarly journals Mutations in CLCN5 chloride channel in Japanese patients with low molecular weight proteinuria.

1998 ◽  
Vol 9 (5) ◽  
pp. 811-818
Author(s):  
T Morimoto ◽  
S Uchida ◽  
H Sakamoto ◽  
Y Kondo ◽  
H Hanamizu ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Chloride Channel ◽  
Expression System ◽  
Hypophosphatemic Rickets ◽  
Low Molecular Weight ◽  
Loss Of Function ◽  
Dent’S Disease ◽  
Clcn5 Gene ◽  
Low Molecular Weight Proteinuria ◽  
Dent's Disease

Mutations in the CLCN5 gene have been demonstrated in three disorders of hypercalciuric nephrolithiasis, i.e., Dent's disease, X-linked recessive nephrolithiasis, and X-linked recessive hypophosphatemic rickets. Recently, a number of Japanese children with low molecular weight proteinuria (LMWP) showing symptoms similar to those shown by patients with Dent's disease in British families have also been reported to have mutations in the CLCN5 gene. The present study examines five unrelated Japanese families with LMWP, two of which lacked any signs other than LMWP, and three of which had several signs other than LMWP, i.e., hypercalciuria, aminoaciduria, hypophosphatemia, and rickets. One nonsense (E118X) and one missense (W22G) mutation were found in three patients in the two families having only LMWP. One genomic deletion including exons 5 to 8 in the CLCN5 gene was found in a patient with hypophosphatemic rickets, and a nonsense mutation (R347X) was found in one patient with LMWP and slight hypercalciuria. No mutations of the exons and exon-intron boundaries in the CLCN5 gene were found in one patient with LMWP, aminoaciduria, and hypokalemia. In addition to the predicted loss of chloride channel function in these nonsense and deletion mutations, the loss of function in the missense mutation W22G was confirmed in the Xenopus oocyte expression system. These results clarified four novel mutations in the CLCN5 genes, and additionally suggested that the loss-of-function mutation of the CLCN5 does not necessarily lead to hypercalciuria and nephrocalcinosis in the early stage of the disease, and that LMWP is an early and essential manifestation of disorders of the CLC-5 chloride channel.

scholarly journals Novel truncating mutations in the ClC-5 chloride channel gene in patients with Dent's disease

2003 ◽  
Vol 18 (4) ◽  
pp. 717-723 ◽  
Author(s):  
I. Carballo-Trujillo ◽  
V. Garcia-Nieto ◽  
F. J. Moya-Angeler ◽  
M. Anton-Gamero ◽  
C. Loris ◽  
...  
Keyword(s):  
Chloride Channel ◽  
Dent’S Disease ◽  
Channel Gene ◽  
Dent's Disease

scholarly journals Renal Chloride Channel, CLCN5, Mutations in Dent's Disease

1999 ◽  
Vol 14 (9) ◽  
pp. 1536-1542 ◽  
Author(s):  
Jeremy P. D. Cox ◽  
Katsusuke Yamamoto ◽  
Paul T. Christie ◽  
Carol Wooding ◽  
Terry Feest ◽  
...  
Keyword(s):  
Chloride Channel ◽  
Dent’S Disease ◽  
Dent's Disease ◽  
Clcn5 Mutations

scholarly journals Characterization of Renal Chloride Channel (CLCN5) Mutations in Dent's Disease

2000 ◽  
Vol 11 (8) ◽  
pp. 1460-1468 ◽  
Author(s):  
KATSUSUKE YAMAMOTO ◽  
JEREMY P. D. T. COX ◽  
THOMAS FRIEDRICH ◽  
PAUL T. CHRISTIE ◽  
MARTIN BALD ◽  
...  
Keyword(s):  
Splice Site ◽  
Chloride Channel ◽  
Nonsense Mutation ◽  
Donor Splice Site ◽  
Dent’S Disease ◽  
Donor Splice ◽  
Dent's Disease ◽  
Renal Tubular Disorder ◽  
Oligonucleotide Hybridization ◽  
Clcn5 Mutations

Abstract. Dent's disease is an X-linked renal tubular disorder characterized by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis, and renal failure. The disease is caused by mutations in a renal chloride channel gene, CLCN5, which encodes a 746 amino acid protein (CLC-5), with 12 to 13 transmembrane domains. In this study, an additional six unrelated patients with Dent's disease were identified and investigated for CLCN5 mutations by DNA sequence analysis of the 11 coding exons of CLCN5. This revealed six mutations: four frameshift deletions involving codons 392, 394, 658, and 728, one nonsense mutation (Tyr617Stop), and anAtoTtransversion at codon 601 that would result in either a missense mutation (Asp601Val) or creation of a novel donor splice site. These mutations were confirmed by restriction endonuclease or sequence-specific oligonucleotide hybridization analysis and were not common polymorphisms. The frameshift deletions and nonsense mutation predict truncated and inactivated CLC-5. The effects of the putative missense Asp601Val mutant CLC-5 were assessed by heterologous expression inXenopusoocytes, and this revealed a chloride conductance that was similar to that observed for wild-type CLC-5. However, an analysis of the mutant CLCN5 transcripts revealed utilization of the novel donor splice site, resulting in a truncated CLC-5. Thus, all of the six mutations are likely to result in truncated CLC-5 and a loss of function, and these findings expand the spectrum of CLCN5 mutations associated with Dent's disease.

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