Chloride neuronal homeostasis, which is determined by the KCC2 and NKCC1 transporters, responsible for Cl- efflux and influx respectively, regulates the efficiency and polarity of GABAergic transmission in the brain. The dysfunction of these transporters leads to a pathological state by a build-up of chloride in neurons. Drugs aimed at reducing neuronal chloride levels therefore represent promising therapeutic strategies. We have shown in mature neurons that the regulation of the membrane trafficking/activity of KCC2 and NKCC1 involves the With-No-lysine-(K)-serine-threonine kinase WNK1 and its effector, Ste20-Proline-Asparagine-Rich-Kinase (SPAK). Once activated, SPAK is recruited below the plasma membrane allowing a local control of its activity. NKCC1 but not KCC2 directly binds SPAK. We propose that NKCC1 regulates KCC2 by recruiting SPAK near inhibitory synapses. We developed several peptides that prevent SPAK binding to NKCC1 and showed that they regulate KCC2 as a proof of concept. We want now to validate the SPAK scaffold hypothesis and identif new small compounds, through interdisciplinary approaches (chemistry/molecular modeling/cell biology/imaging/animal behavior), able to interact with the NKCC1/SPAK complex and inhibit SPAK activity and thus prevent KCC2 internalization and chloride build-up in neurons. This project will shed light on the virtually unknown role of a signaling pathway in the nervous system and may lead to the discovery of a non-canonical role for NKCC1 in neurons. It may also lead to the discovery of new therapeutic strategies in diseases with inhibition defects.