A pure chloride channel mutant of CLC-5 causes Dent’s disease via insufficient V-ATPase activation

Dent’s diseaseis characterized by defective endocytosisin renal proximal tubules(PTs) and caused by mutations in the 2Cl−/H+ exchanger, CLC-5. However, the pathological role of endosomalacidification in endocytosishas recently come into question. To clarify the mechanism of pathogenesis for Dent’s disease, we examined the effects of a novel gating glutamate mutation, E211Q, on CLC-5 functions and endosomalacidification. In Xenopus oocytes, wild-type (WT) CLC-5 showed outward-rectifying currents that were inhibited by extracellular acidosis, but E211Q and an artificial pure Cl− channel mutant, E211A, showed linear currents that were insensitive to extracellular acidosis. Moreover, depolarizing pulse trains induced a robust reduction in the surface pH of oocytes expressing WT CLC-5 but not E211Q or E211A, indicating that the E211Q mutant functions as a pure Cl− channel similar to E211A. In HEK293 cells, E211A and E211Q stimulated endosomalacidification and hypotonicity-inducible vacuolar-type H+-ATPase ( V-ATPase) activation at the plasma membrane. However, the stimulatory effects of these mutants were reduced compared with WT CLC-5. Furthermore, gene silencing experiments confirmed the functional coupling between V-ATPaseand CLC-5 at the plasma membrane of isolated mouse PTs. These results reveal for the first time that the conversion of CLC-5 from a 2Cl−/H+ exchanger into a Cl− channel induces Dent’s diseasein humans. In addition, defective endosomalacidification as a result of insufficient V-ATPaseactivation may still be important in the pathogenesis of Dent’s disease.