Phospho-regulation of the K-Cl cotransporter KCC2

• Applicant: Professor Dr. Hans-Gerd Nothwang
• Subject Area: Molecular Biology and Physiology of Neurons and Glial Cells
• Term: from 2015 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 273749082

The K-Cl cotransporter KCC2 plays a seminal role in fast synaptic inhibition. It is the main Cl-extruder in neurons, thereby generating an inwardly directed Cl-gradient across the plasma membrane. This gradient underlies the rapid hyperpolarizing action of the inhibitory neurotransmitters GABA and glycine. KCC2 is essential and dysfunctions are associated with neurological disorders such as epilepsy, neuropathic pain, and trauma. Phosphorylation has emerged as a key mechanism to regulate KCC2 activity and has been proposed as a pharmacotherapeutic target. Yet, our knowledge of phosphorylation-mediated regulation of KCC2 under physiological and pathophysiological conditions is still very limited. By characterizing native phospho-sites, we recently identified the novel regulatory phospho-site T934/S937. Phosphorylation of these amino acid residues stimulates KCC2 intrinsic transport activity in HEK-293 cells. Furthermore, phosphorylation of this site determines the action of a staurosporine and NEM sensitive but hitherto unknown phospho-site. The project builds on these results by extending the functional characterization of T934/S937. We will characterize its role in neuronal cells and strive for the identification of the molecular mechanisms regulating it. In addition, we will further our analysis on the complex functional crosstalk with the staurosporine and NEM sensitive regulatory phospho-site, as both sites influence each other. These experiments will be performed in cell culture. However, it is essential to address the role of T934/S937 in the entire organism as well. To this end, we will generate essential tools to characterize their in vivo function. We will produce and validate antibodies against the phospho-site. Furthermore, two mouse lines with mutations in T934/S937 will be generated. One mouse line will mimic the dephosphorylated state of the phospho-site, the other will mimic the phosphorylated state of the phospho-site. Both mouse lines will be characterized by us as well as in collaboration with other groups for their role in physiological and pathophysiological processes. Altogether, the project will provide important insight in phospho-regulatory mechanisms of KCC2, which is essential for basic and translational research alike. Furthermore, it will supply essential resources to decode the in vivo roles of KCC2 phosphorylation.

• DFG Programme: Research Grants
• International Connection: Finland
• Cooperation Partner: Professor Claudio Rivera, Ph.D.