A pure chloride channel mutant of CLC-5 causes Dent’s disease via insufficient V-ATPase activation
A pure chloride channel mutant of CLC-5 causes Dent’s disease via insufficient V-ATPase activation
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DOI：
10.1007/s00424-016-1808-7
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发表时间：
2016-04
期刊：
影响因子：
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通讯作者：
N. Satoh;Hideomi Yamada;O. Yamazaki;Masashi Suzuki;M. Nakamura;A. Suzuki;A. Ashida;D. Yamamoto;Y. Kaku;T. Sekine;G. Seki;S. Horita
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文献类型：
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作者：
N. Satoh;Hideomi Yamada;O. Yamazaki;Masashi Suzuki;M. Nakamura;A. Suzuki;A. Ashida;D. Yamamoto;Y. Kaku;T. Sekine;G. Seki;S. Horita
登特氏病的特征是肾近端小管(PTS)有缺陷的内吞作用，由2CL−/H+交换因子ClC-5突变引起。然而，内体酸化在内吞作用中的病理作用最近受到了质疑。为了阐明Dent病的发病机制，我们研究了一种新的门控谷氨酸突变E211Q对ClC-5功能和内体酸化的影响。在Xenopus卵母细胞中，野生型(WT)ClC-5显示的外向整流电流可被细胞外酸抑制，而E211Q和人工纯Cl-−通道突变体E211A则显示对胞外酸中毒不敏感的线性电流。此外，去极化脉冲串诱导表达WT ClC-5但不表达E211Q或E211A的卵母细胞表面pH显著降低，表明E211Q突变体的功能类似于E211A的纯氯−通道。在HEK293细胞中，E211A和E211Q可刺激胞浆酸化和低渗诱导的质膜空泡型H+-ATPase(V-ATPase)激活。然而，与WT ClC-5相比，这些突变体的刺激作用有所减弱。此外，基因沉默实验证实了V-ATPase和ClC-5在分离的小鼠PTS质膜上的功能偶联。这些结果首次揭示了ClC-5从2Cl−/H+交换到Cl2−通道的转换导致了人类的登特氏病。此外，由于V-ATPase激活不足而导致的缺陷性内膜酸化可能在Dent病的发病机制中仍然很重要。
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. InXenopusoocytes, 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.