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An internalization signal in ClC-5, an endosomal Cl-channel mutated in dent's disease
M Schwake1, T Friedrich, T J Jentsch
1Zentrum für Molekulare Neurobiologie Hamburg, Hamburg University, Falkenried 94, D-20246 Hamburg, Germany.
Abstract:
The ClC-5 chloride channel resides mainly in vesicles of the endocytotic pathway and contributes to their acidification. Its disruption in mice entails a broad defect in renal endocytosis and causes secondary changes in calciotropic hormone levels. Inactivating mutations in Dent's disease lead to proteinuria and kidney stones. Possibly by recycling, a small fraction of ClC-5 also reaches the plasma membrane. Here we identify a carboxyl-terminal internalization motif in ClC-5. It resembles the PY motif, which is crucial for the endocytosis and degradation of epithelial Na(+) channels. Mutating this motif increases surface expression and currents about 2-fold. This is probably because of interactions with WW domains, because dominant negative mutants of the ubiquitin-protein ligase WWP2 increased surface expression and currents of ClC-5 only when its PY motif was intact. Stimulating endocytosis by expressing rab5 or its GTPase-deficient Q79L mutant decreased WT ClC-5 currents but did not affect channels with mutated motifs. Similarly, decreasing endocytosis by expressing the inactive S34N mutant of rab5 increased ClC-5 currents only if its PY-like motif was intact. Thus, the endocytosis of ClC-5, which itself is crucial for the endocytosis of other proteins, depends on the interaction of a carboxyl-terminal internalization signal with ubiquitin-protein ligases containing WW domains.
Insights
The ClC-5 chloride channel
Area of Science:
- Molecular Biology
- Cell Biology
- Nephrology
Background:
- The ClC-5 chloride channel is vital for endosomal acidification and renal endocytosis.
- Disruption of ClC-5 leads to kidney dysfunction, including proteinuria and kidney stones in Dent's disease.
- A small fraction of ClC-5 is found at the plasma membrane, suggesting surface expression regulation.
Purpose of the Study:
- To identify the mechanism regulating ClC-5 surface expression and endocytosis.
- To investigate the role of a specific internalization motif in ClC-5 trafficking.
- To elucidate the interaction of ClC-5 with other proteins involved in endocytosis.
Main Methods:
- Site-directed mutagenesis of the ClC-5 carboxyl-terminal motif.
- Analysis of ClC-5 surface expression and channel currents using electrophysiology.
- Manipulation of endocytic pathway components (rab5, WWP2) to assess ClC-5 trafficking.
Main Results:
- A carboxyl-terminal PY-like motif was identified in ClC-5, crucial for its endocytosis.
- Mutating this motif increased ClC-5 surface expression and currents twofold.
- ClC-5 endocytosis depends on interactions with WW domain-containing ubiquitin ligases like WWP2 and is regulated by rab5.
Conclusions:
- ClC-5 endocytosis is regulated by its PY-like motif and WW domain-containing ubiquitin ligases.
- This process is essential for maintaining normal ClC-5 function and its role in renal endocytosis.
- Understanding ClC-5 trafficking provides insights into Dent's disease pathogenesis and potential therapeutic targets.