Characterization of Dent's disease mutations of CLC-5 reveals a correlation between functional and cell biological consequences and protein structure

Abstract

Mutations of the human CLCN5 gene, which encodes the CLC-5 Cl−/H+ exchanger, lead to Dent's disease. Mutations result in functional defects that range from moderate reductions to complete loss of whole cell currents, although the severity of the functional defect rarely correlates with the severity of the disease. To further elucidate the basis of CLC-5 mutations causing Dent's disease, we examined the functional and cell biological consequences of seven previously reported missense mutants, utilizing electrophysiological and cell biological techniques. This revealed three classes of Dent's disease-causing CLC-5 mutations. Class 1 mutations lead to endoplasmic reticulum retention and degradation of CLC-5. Class 2 mutations appear to have little effect on subcellular distribution of CLC-5 but cause defective function resulting in severe defects in endosomal acidification. Class 3 mutations lead to alterations in the endosomal distribution of CLC-5 but are otherwise able to support endosomal acidification. Molecular modeling demonstrates a structural basis that may underlie the nature of the defect resulting from each mutation with each class occupying discrete regions of the protein quaternary structure. Thus these results demonstrate that the cell biological consequences of CLC-5 mutations are heterogeneous and can be classified into three major groups and that a correlation between the nature of the defect and the location of the mutation in the structure may be drawn. This model may prove to be useful as a tool to aid in the diagnosis and future therapeutic intervention of the disease.

Receptor-mediated endocytosis and endosomal acidification is impaired in proximal tubule epithelial cells of Dent disease patients

“…CLC-5 is postulated to have a role in acidification of the endosome by providing a parallel chloride conductance to the V-ATPase (5). Previous studies using heterologous expression systems have shown that CLC-5 mutations have different effects on endosomal acidification (17). Expression of wild-type CLC-5 and CLC-5 mutants 30:insH and R637X in HEK293 cells revealed that proton transport by the CLC-5 mutant R637X was significantly decreased compared with wild-type CLC-5, whereas that by the CLC-5 mutant 30:insH was similar to wild-type CLC-5 (Fig.…”

“…1A, Fig. S1, and Table 1) (17). Thus, some Dent disease causing CLC-5 mutations may not impair endosomal acidification, while resulting in defective proximal tubular endocytosis, suggesting endocytosis and endosomal trafficking may not be coupled to endosomal acidification.…”

“…Thus, in vitro studies have demonstrated that Dent disease causing CLC-5 mutants markedly reduce or abolish Cl − conductance and that some, but not all, CLC-5 mutants may also impair endosomal acidification (17). Thus, the in vitro expression studies of Dent disease causing CLC-5 mutants in HEK cells have shown that: CLC-5 mutants S270R, located in the H-I loop, G513E and R516W (in helix O), I524K and E527D (in helix P), and R637X [in cystathionine-β-synthase (CBS) domain-1 in the C terminus] resulted in a lack of Cl − currents, largely due to retention and degradation of the mutant CLC-5s in the endoplasmic reticulum (ER); whereas CLC-5 mutants consisting of a His insertion at codon 30 (30:insH), G57V, and R280P (in helix A, B, and I, respectively), had markedly reduced Cl − currents but did not impair endosomal acidification (Fig.…”

A Patient with Nephrotic-Range Proteinuria and Focal Global Glomerulosclerosis

“…Some patients with Dent disease have proximal tubule cells with inverted H 1 -ATPase polarity and redistribution to the basolateral regions, further evidence that ClC-5 plays an important role in intracellular traffic (40). A recent study in a mammalian expression system suggests that endoplastic reticulum retention and degradation of ClC-5, defective endosomal acidification, or altered endosomal distribution of ClC-5 without defective endosomal acidification may play a role in the disease (51). As a result, LMW proteins that are freely filtered by the glomerulus cannot be reabsorbed by the proximal tubular cells.…”

Clinical utility gene card for: Dent disease (Dent-1 and Dent-2)

“…The sensitivity of sequence analysis of PCR-amplified products approaches 100%. A lot of variants have been tested functionally, [13][14][15][16] and the pathogenicity of most mutants has been predicted by publically available algorithms. Nonetheless, errors may occur due to allele dropout and mutations outside the coding region in the promoter, polyA-site, enhancers or intronic variants may be missed.…”