Multi-site effects of staurosporine and NEM on phospho-specific residues in KCC2 and NKCC1 - Oldenburg university

invited by Igor Medina

abstract: The plasma membrane K+-Cl- outward cotransporter KCC2 and the Na+, K+, Cl- inward cotransporter NKCC1 belong to the Cation-Chloride-Cotransporter (CCC; SLC12) family. In neurons, KCC2 and NKCC1 are involved in the development and maintenance of inhibitory neurotransmission. For instance, KCC2 generates a low intracellular [Cl-]i concentration required for fast inhibitory neurotransmission. Binding of γ-aminobutyric acid (GABA) or glycine to their receptors, that are ligand-gated Cl- channels, leads to a Cl- influx and therefore hyperpolarization. By contrast, NKCC1 is more prevalent in immature inhibitory neurons. There, its action results in a high [Cl-]i causing GABA or glycine to elicit a depolarizing action that opens L-type voltage-gated Ca2+ channels necessary for proper synapse formation. Their opposite transport direction and their presence in the same neurons require regulatory mechanisms to coordinate their activity. One potent mechanism to regulate their activity is phospho-regulation. Previously analyses showed, that treatment with the broad kinase inhibitor staurosporine or N-ethylmaleimide (NEM), that was also suggested to modulate the activity of kinases and phosphatases, reciprocally regulate the activity of KCC2 (activation) and NKCC1 (inactivation).
Here, I will present data on the impact of both reagents on KCC2 and NKCC1 in stably transfected KCC2 HEK293 cells and immature hippocampal cultures, using large-scale phosphoproteomics studies and phospho-specific antibodies. The most prominent way to reciprocally regulate the function of KCC2 and NKCC1 is based on the WNK/SPAK/OSR1 mediated phosphorylation pathway. Both reagents reduce the phosphorylation and therefore function of the WNK/SPAK/OSR1 mediated phosphorylation pathway that results in a dephosphorylation of Thr906 and Thr1007 in KCC2 (activation) and Thr203, Thr207 and Thr212 in NKCC1 (inactivation). Finally, the impact of NEM on PKC mediated phosphorylation of Ser940 is brain region specific (enhanced phosphorylation in immature cortical neurons vs. dephosphorylation in immature hippocampal neurons). In conclusion, further in-depth analyses are required to elucidate the tissue specific regulation of single and combined phospho-sites prior to any therapeutically targeting of CCC phosphor-sites.


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