| Abstract No.: | A-B1069 |
| Country: | Canada |
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| Title: | IDENTIFICATION OF THE MOLECULAR SIGNALS MEDIATING K+-CL- COTRANSPORTER KCC2 ENDOCYTOSIS |
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| Authors/Affiliations: | 1 Beibei Zhao*; 2 Adrian Wong; 1 Derek Bowie; 1 John Presley; 1 Fiona Bedford;
1 McGill University, Montreal, QC, Canada; 2 Ottawa Health Research Institute, Ottawa, ON, Canada
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| Content: | Objectives: The neuron-specific potassium-chloride cotransporter 2 (KCC2) plays a crucial role, by controlling chloride extrusion, in the development and maintenance of inhibitory neurotransmission. Neuronal mechanisms that regulate KCC2 function therefore potentially offer an indirect means by which the fidelity of inhibitory neurotransmission and associated neuronal functions can be controlled. 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, an important pathway controlling KCC2 membrane expression.
Materials and Methods: We used a combination of approaches, including a fluorescence-based single cell endocytosis assay, surface biotinylation, chimeric endocytosis-reporter systems, and site-directed mutagenesis to characterize the cellular mechanism and molecular motif regulating the endocytosis of KCC2 from the plasma membrane.
Results: 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 complex. Using an endocytosis reporter system, we identify the presence of an autonomous endocytosis motif in the carboxyl cytoplasmic terminus of KCC2. Using site-directed mutagenesis we demonstrate that this novel KCC2 endocytic motif is essential for the constitutive internalization of KCC2. 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.
Conclusion: Our results indicate constitutive internalization of KCC2 is clathrin-mediated and dependent on a novel endocytic motif. Furthermore, that this process appears to be an evolutionarily conserved mechanism amongst functionally homologous cotransporters. Given the essential role of KCC2 in synaptic inhibition and also certain neuropathological conditions, our findings are important for the study of these disorders and elucidating novel mechanisms by which neuronal plasticity may be regulated.
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