Functional analysis of a novel pathogenic mutation affecting the gating glutamate of the 2Cl−/H+ exchanger ClC‐5 in patients with Dent's disease type 1

Abstract

Dent's disease is an X‐linked recessive renal tubular disorder characterized by low‐molecular‐weight proteinuria, hypercalciuria, nephrolithiasis, nephrocalcinosis, and progressive renal failure, inconstantly associated with other proximal tubular dysfunctions. Inactivating mutations of CLCN5, the gene encoding the 2Cl−/H+ exchanger ClC‐5, are associated with Dent's disease type 1. In the kidney, ClC‐5 is predominantly expressed at the apical membrane of proximal tubule cells and on early endosomes. Impaired endosomal acidification consecutive to ClC‐5 loss‐of‐function might explain the defective endocytosis in proximal tubule cells observed in patients with Dent's disease.Here, we have investigated a novel ClC‐5 pathogenic missense mutation (E211G) affecting the gating glutamate that is responsible for coupling the Cl‐flux to the H+ counter‐transport in ClC 2Cl−/H+ exchangers. The courants were characterized using two‐electrode voltage‐clamp recordings in X. laevis oocytes. ClC‐5 subcellular localization was investigated in X. laevis oocytes using luminescence assays and in HEK293T cells using immunofluorescence. Protein expression was characterized in HEK293T cells using western‐blot.The E211G mutant displayed a nearly linear current/voltage relationship. In contrast to those of wild‐type ClC‐5, currents of the mutant were not sensitive to extracellular acidification. We found no difference between this mutant and wild‐type ClC‐5 in terms of protein expression and subcellular targeting. The mutant trafficked normally to the cell surface and to the early endosomes and displayed complex glycosylation.The present study demonstrates that the E211G mutation considerably affects electrical activity of ClC‐5 in proximal tubule cells.Support or Funding InformationThis work was supported in part by grant “Prix Jeune Chercheur” from “La Fondation du Rein”.