Project Summary The level of activity in the nervous system is tightly controlled by the interplay between excitatory glutamatergic neurons and inhibitory GABA (γ–aminobutyric acid) neurons. When inhibition is compromised, the resultant hyperexcitability is linked to epilepsy, neurodevelopmental disease and psychiatric disorders. The efficacy of GABAergic inhibition is maintained by continuous extrusion of Cl– by the potassium cotransporter KCC2, which permits GABAA receptors to inwardly conduct those ions, resulting in neuron hyperpolarization. KCC2 also exhibits a structural role independent of its transporter function: it affects the maturation of dendritic spine morphology and glutamatergic signaling. KCC2 is upregulated during the first two postnatal weeks in mice, which is roughly equivalent to the final stages of preterm human development. In keeping with this, rare de novo mutations have recently been identified in the kcc2 gene that cause EIMFS, a form of epilepsy in infancy. In surviving patients, the epileptic phenotype persists into adulthood. We have evidence that the point mutations L288H, W318S, and ∆S748 impair the activity of KCC2. Mechanistically, each mutation decreased total protein expression; however, each differently affected the amount of the protein on the cell surface. While these and other experiments demonstrate that the mutations each confer a different pattern of changes to KCC2 that may account for their decreased function, they are limited in that they rely on overexpression of the gene in immortalized cell lines, which are fundamentally different from the neurons in which KCC2 normally functions. By developing a platform where novel mutations in the kcc2 gene can be rapidly characterized, we can guide the development of mouse lines which recapitulate the human disease state and screen for tailored therapeutics to each clinical population. We propose to generate neuron cultures where the normal KCC2 protein is removed and replaced by one of three above mutations found in patients with epilepsy. In addition to a characterization of these mutations in a neuronal context, our approach provides an opportunity to translate how altered KCC2 function as a result of these mutations may predispose to hyperexcitability and epilepsy. In the first aim we will determine how mutations affect the distribution and biochemistry of the protein, and if they disturb the neurons' anatomical maturation. We will also show that each mutation diminishes the capability of KCC2 to maintain hyperpolarizing inhibition. Importantly, KCC2 function and membrane trafficking is affected by phosphorylation at KCC2-T1007. This site is phosphorylated by SPAK kinase, which is phosphorylated and activated by WNK kinase. We have a recently developed WNK kinase inhibitor and our preliminary data indicate that it potentiates KCC2 activity by reducing KCC2-T1007 phosphorylation. In the second aim, we will show how mutations in KCC2 affect seizure activity a...