Functional analysis of CLCN5 mutations in patients with Dent's disease

Abstract

Dent's disease is an X‐linked renal disease characterized by low molecular weight proteinuria, hypercalciuria and nephrolithiasis, which can be due to loss‐of‐function mutations in the CLCN5 gene encoding the ClC‐5 2Cl−/H+ exchanger. Here, we investigated three published CLCN5 mutations: E267A, S270G and S270R (Hoopes et al., Kidney Int., 2004; Igarashi et al., Kidney Int., 1998).We studied wild‐type (WT) and ClC‐5 mutants in X. laevis oocytes using two‐electrode voltage‐clamp for measuring currents and chemiluminescence for evaluating surface expression. Protein expression and subcellular localization was investigated in HEK293 cells using western blots and immunocytochemistry.No significant current was recorded with the S270R mutant. The E267A and S270G mutants produced 20–50% lower currents as compared to WT. We found no difference between these mutants and WT ClC‐5 in terms of subcellular targeting and protein expression levels. They trafficked normally to the cell surface and to the early endosomes and displayed complex N‐glycosylation like WT ClC‐5.Our study shows that these CLCN5 mutations considerably alter electrical activity of ClC‐5.