Identification of a novel di-leucine motif mediating K(+)/Cl(-) cotransporter KCC2 constitutive endocytosis

Cell Signal. 2008 Oct;20(10):1769-79. doi: 10.1016/j.cellsig.2008.06.011. Epub 2008 Jun 24.

Abstract

The neuron-specific potassium-chloride cotransporter 2 (KCC2) plays a crucial role, by controlling chloride extrusion, in the development and maintenance of inhibitory neurotransmission. Although it is now well established that activity-dependent mechanisms can down regulate KCC2 gene expression, the role of post-translational mechanisms in controlling KCC2 expression, specifically at the cell-surface, are poorly understood. We therefore set out to identify the mechanisms and motifs regulating KCC2 endocytosis, one important pathway that may control KCC2 membrane expression. Using a fluorescence-based assay, we show KCC2 when expressed in HEK293 cells is constitutively internalized via a dynamin- and clathrin-dependent pathway. Consistent with this, we demonstrate KCC2 from adult mouse brain associates in vivo with the clathrin-binding adaptor protein-2 (AP-2) complex. Using an endocytosis reporter system, we identify the presence of an autonomous endocytosis motif in the carboxyl cytoplasmic terminus of KCC2. By site-directed mutagenesis we define this novel KCC2 endocytic motif as a non-canonical di-leucine motif, (657)LLXXEE(662). Finally by mutating this motif in the context of full-length KCC2 we demonstrate that this novel KCC2 endocytic motif is essential for the constitutive internalization of KCC2 and for binding to the AP-2 complex. Subsequent sequence analysis reveals this motif is highly conserved between the closely related K(+)/Cl(-) family members that mediate chloride efflux, but absent from the more distant related cotransporters controlling chloride influx. In conclusion, our results indicate constitutive internalization of KCC2 is clathrin-mediated and dependent on the binding of AP-2 to this novel endocytic motif. Furthermore, that this process appears to be an evolutionarily conserved mechanism amongst functionally homologous cotransporters.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Protein Complex 2 / metabolism
  • Amino Acid Motifs
  • Amino Acid Sequence
  • Animals
  • Cell Line
  • Clathrin / metabolism
  • Endocytosis*
  • Endosomes / metabolism
  • Hemagglutinins / metabolism
  • Humans
  • Leucine / metabolism*
  • Mice
  • Molecular Sequence Data
  • Neurons / metabolism
  • Protein Binding
  • Recombinant Fusion Proteins / metabolism
  • Structure-Activity Relationship
  • Symporters / chemistry*
  • Symporters / metabolism*

Substances

  • Adaptor Protein Complex 2
  • Clathrin
  • Hemagglutinins
  • Recombinant Fusion Proteins
  • Symporters
  • potassium-chloride symporters
  • Leucine