Role of ClC-5 in renal endocytosis is unique among ClC exchangers and does not require PY-motif-dependent ubiquitylation

J Biol Chem. 2010 Jun 4;285(23):17595-603. doi: 10.1074/jbc.M110.115600. Epub 2010 Mar 29.

Abstract

Inactivation of the mainly endosomal 2Cl(-)/H(+)-exchanger ClC-5 severely impairs endocytosis in renal proximal tubules and underlies the human kidney stone disorder Dent's disease. In heterologous expression systems, interaction of the E3 ubiquitin ligases WWP2 and Nedd4-2 with a "PY-motif" in the cytoplasmic C terminus of ClC-5 stimulates its internalization from the plasma membrane and may influence receptor-mediated endocytosis. We asked whether this interaction is relevant in vivo and generated mice in which the PY-motif was destroyed by a point mutation. Unlike ClC-5 knock-out mice, these knock-in mice displayed neither low molecular weight proteinuria nor hyperphosphaturia, and both receptor-mediated and fluid-phase endocytosis were normal. The abundances and localizations of the endocytic receptor megalin and of the Na(+)-coupled phosphate transporter NaPi-2a (Npt2) were not changed, either. To explore whether the discrepancy in results from heterologous expression studies might be due to heteromerization of ClC-5 with ClC-3 or ClC-4 in vivo, we studied knock-in mice additionally deleted for those related transporters. Disruption of neither ClC-3 nor ClC-4 led to proteinuria or impaired proximal tubular endocytosis by itself, nor in combination with the PY-mutant of ClC-5. Endocytosis of cells lacking ClC-5 was not impaired further when ClC-3 or ClC-4 was additionally deleted. We conclude that ClC-5 is unique among CLC proteins in being crucial for proximal tubular endocytosis and that PY-motif-dependent ubiquitylation of ClC-5 is dispensable for this role.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Animals
  • Chloride Channels / chemistry*
  • Chloride Channels / metabolism
  • Cytoplasm / metabolism
  • Endocytosis
  • Female
  • Gene Expression Regulation*
  • Kidney Tubules / metabolism
  • Low Density Lipoprotein Receptor-Related Protein-2 / chemistry
  • Male
  • Mice
  • Mice, Knockout
  • Models, Biological
  • Protein Structure, Tertiary
  • Ubiquitin / chemistry*

Substances

  • CLC5 protein, mouse
  • Chloride Channels
  • Low Density Lipoprotein Receptor-Related Protein-2
  • Ubiquitin