Abstract
Dent's disease is associated with impaired renal endocytosis and endosomal acidification. It is linked to mutations in the membrane chloride/proton exchanger ClC-5; however, a direct link between localization in the protein and functional phenotype of the mutants has not been established until now. Here, two Dent's disease mutations, G212A and E267A, were investigated using heterologous expression in HEK293T cells, patch-clamp measurements and confocal imaging. WT and mutant ClC-5 exhibited mixed cell membrane and vesicular distribution. Reduced ion currents were measured for both mutants and both exhibited reduced capability to support endosomal acidification. Functionally, mutation G212A was capable of mediating anion/proton antiport but dramatically shifted the activation of ClC-5 toward more depolarized potentials. The shift can be explained by impeded movements of the neighboring gating glutamate Gluext, a residue that confers major part of the voltage dependence of ClC-5 and serves as a gate at the extracellular entrance of the anion transport pathway. Cell surface abundance of E267A was reduced by ~50% but also dramatically increased gating currents were detected for this mutant and accordingly reduced probability to undergoing cycles associated with electrogenic ion transport. Structurally, the gating alternations correlate to the proximity of E267A to the proton glutamate Gluin that serves as intracellular gate in the proton transport pathway and regulates the open probability of ClC-5. Remarkably, two other mammalian isoforms, ClC-3 and ClC-4, also differ from ClC-5 in gating characteristics affected by the here investigated disease-causing mutations. This evolutionary specialization, together with the functional defects arising from mutations G212A and E267A, demonstrate that the complex gating behavior exhibited by most of the mammalian CLC transporters is an important determinant of their cellular function.
Highlights
Dent’s disease (Dent and Friedman, 1964) is a X-linked hereditary disease coupled to impaired function of the kidney
The pH of vesicles containing either ClC-5 WT or the mutants G212A and E267A was measured by automatic vesicle identification (Figures 2F–H) and ratiometric fluorescence measurements
The analysis revealed that expression of WT ClC-5 leads to significantly stronger endosomal acidification in HEK293T cells
Summary
Dent’s disease (Dent and Friedman, 1964) is a X-linked hereditary disease coupled to impaired function of the kidney. The pathophysiology of the disease has been connected to mutations in two different genes – CLCN5 and OCRL1 (Lloyd et al, 1996; Hoopes et al, 2005). OCRL1 encodes a Golgi-localized PI(4,5)P2 5-phosphatase that interacts with clathrin and regulates. The second gene, CLCN5, encodes for the membrane transporter ClC-5 that resides in endocytotic vesicles (Günther et al, 1998; Sakamoto et al, 1999) and mediates secondary active chloride/proton exchange (Picollo and Pusch, 2005; Scheel et al, 2005). Knockout of Clcn in mice is associated with impaired renal endocytosis and significantly slowed rates of endosomal acidification (Piwon et al, 2000; Günther et al, 2003). Impaired endocytosis and endosomal ion homeostasis seem to represent the major mechanisms leading to Dent’s disease
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