Journal
CELLULAR SIGNALLING
Volume 20, Issue 10, Pages 1769-1779Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.cellsig.2008.06.011
Keywords
KCC2; chloride transporter; endocytosis; AP-2
Categories
Funding
- Canadian Institute of Health Research (CIHR) [MOP-62822, MOP-49590]
Ask authors/readers for more resources
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, 657LLYXEE662, 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. (C) 2008 Elsevier Inc. All rights reserved.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available