Journal
PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY
Volume 468, Issue 7, Pages 1183-1196Publisher
SPRINGER HEIDELBERG
DOI: 10.1007/s00424-016-1808-7
Keywords
CLC-5; Gating glutamate; Dent's disease; V-ATPase; Endocytosis; Endosomal acidification
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Funding
- Ministry of Education, Culture, Sports, Science and Technology of Japan
- Grants-in-Aid for Scientific Research [15K19441, 25461241, 16K09640] Funding Source: KAKEN
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Dent's disease is characterized by defective endocytosis in renal proximal tubules (PTs) and caused by mutations in the 2Cl(-)/H+ exchanger, CLC-5. However, the pathological role of endosomal acidification in endocytosis has 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 endosomal acidification. 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 endosomal acidification 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-ATPase and 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 disease in humans. In addition, defective endosomal acidification as a result of insufficient V-ATPase activation may still be important in the pathogenesis of Dent's disease.
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