Neuronal chloride homeostasis is determined by KCC2 and NKCC1 transporters responsible for chloride efflux and influx. KCC2 internalization and increased NKCC1 surface expression leads to a pathological state by accumulation of chloride in neurons. Molecules designed to reduce intra-neuronal chloride levels - by stabilizing KCC2 or destabilizing NKCC1 at the membrane - represent promising therapeutic strategies for diseases in which inhibition is impaired. The WNK/SPAK kinase pathway, by regulating the phosphorylation and membrane stability of both NKCC1 and KCC2, constitutes a new target of interest in the pathology. Interestingly, NKCC1 has two SPAK-binding motifs, whereas KCC2b, the main form of KCC2 in mature neurons, does not. Here, we tested the hypothesis that NKCC1 acts as a scaffolding protein for SPAK, allowing phosphorylation and internalization of KCC2. First, we showed that 60% of NKCC1 aggregates colocalize with KCC2 aggregates and, conversely, 40% of KCC2 aggregates colocalize with NKCC1 aggregates. Furthermore, the colocalized aggregates were denser in NKCC1/KCC2 molecules than the isolated ones, suggesting that they share the same molecular scaffold. Then, using molecular modelling, we developed 12 peptides that mimicked the SPAK binding sites on NKCC1 in order to inhibit the SPAK-NKCC1 interaction and test the impact of these peptides on SPAK submembrane recruitment and KCC2 membrane stabilization. One of these peptides significantly enhanced KCC2 membrane clustering, leading to the conclusion that NKCC1-SPAK interaction does regulate KCC2. This peptide will be tested in vivo, which will determine its therapeutic potential.