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

• Published in: Cellular signalling 2008-10, Vol.20 (10), p.1769-1779
• Main Authors: Zhao, Beibei, Wong, Adrian Y.C., Murshid, Ayesha, Bowie, Derek, Presley, John F., Bedford, Fiona Kay
• Format: Article
• Language: English
• Subjects: Adaptor Protein Complex 2 - metabolism; Amino Acid Motifs; Amino Acid Sequence; Animals; AP-2; Cell Line; Chloride transporter; Clathrin - metabolism; Endocytosis; Endosomes - metabolism; Hemagglutinins - metabolism; Humans; K Cl- Cotransporters; KCC2; Leucine - metabolism; Mice; Molecular Sequence Data; Neurons - metabolism; Protein Binding; Recombinant Fusion Proteins - metabolism; Structure-Activity Relationship; Symporters - chemistry; Symporters - metabolism
• ISSN: 0898-6568; 1873-3913
• DOI: 10.1016/j.cellsig.2008.06.011

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, 657LLXXEE 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.