title: A pure chloride channel mutant of CLC-5 causes Dent's disease via insufficient V-ATPase activation.
abstract: 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.
authors.affiliation: Department of Internal Medicine, Faculty of Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Satoh
authors.affiliation: Department of Internal Medicine, Faculty of Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
authors.forename: Hideomi
authors.initials: H
authors.lastname: Yamada
authors.affiliation: Apheresis and Dialysis Center, General Medicine, School of Medicine, Keio University, Tokyo, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Yamazaki
authors.affiliation: Department of Internal Medicine, Faculty of Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Suzuki
authors.affiliation: Department of Internal Medicine, Faculty of Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
authors.forename: Motonobu
authors.initials: M
authors.lastname: Nakamura
authors.affiliation: Department of Internal Medicine, Faculty of Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Suzuki
authors.affiliation: Department of Pediatrics, Osaka Medical College, Takatsuki, Osaka, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Ashida
authors.affiliation: Biomedical Computation Center, Osaka Medical College, Takatsuki, Osaka, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Yamamoto
authors.affiliation: Department of Nephrology, Fukuoka Children's Hospital, Fukuoka, Japan.
authors.forename: Yoshitsugu
authors.initials: Y
authors.lastname: Kaku
authors.affiliation: Department of Pediatrics, Ohashi Medical Center, Toho University, Meguro-ku, Tokyo, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Sekine
authors.affiliation: Yaizu City Hospital, Yaizu, Japan.
authors.forename: George
authors.initials: G
authors.lastname: Seki
authors.affiliation: Department of Internal Medicine, Faculty of Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan. shorita-tky@umin.ac.jp.
authors.forename: Shoko
authors.identifier: http://orcid.org/0000-0003-2103-3605
authors.initials: S
authors.lastname: Horita
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002648:Child; D018118:Chloride Channels / Q000235:genetics* / Q000378:metabolism*; D057973:Dent Disease / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology; D005260:Female; D057809:HEK293 Cells; D006706:Homeostasis / Q000502:physiology; D006801:Humans; D017136:Ion Transport / Q000502:physiology; D007687:Kidney Tubules, Proximal / Q000378:metabolism; D008297:Male; D009154:Mutation / Q000235:genetics*; D009865:Oocytes / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism*; D014982:Xenopus laevis / Q000378:metabolism
chemical_list: C097503:CLC-5 chloride channel; D018118:Chloride Channels; D025262:Vacuolar Proton-Translocating ATPases
keywords: CLC-5; Dent’s disease; Endocytosis; Endosomal acidification; Gating glutamate; V-ATPase