An upregulation of the Na+-K+-2Cl− cotransporter NKCC1, the main chloride importer in mature neurons, can lead to depolarizing/excitatory responses mediated by GABA type A receptors (GABAARs) and, thus, to hyperactivity. Understanding the regulatory mechanisms of NKCC1 would help prevent intra-neuronal chloride accumulation that occurs in pathologies with defective inhibition. The cell mechanisms regulating NKCC1 are poorly understood. Here, we report in mature hippocampal neurons that GABAergic activity controls the membrane diffusion and clustering of NKCC1 via the chloride-sensitive WNK lysine deficient protein kinase 1 (WNK1) and the downstream Ste20 Pro-line Asparagine Rich Kinase (SPAK) kinase that directly phosphorylates NKCC1 on key threonine residues. At rest, this signaling pathway has little effect on intracellular Cl− concentration, but it participates in the elevation of intraneuronal Cl− concentration in hyperactivity conditions associated with an up-regulation of NKCC1. The fact that the main chloride exporter, the K+-Cl− cotransporter KCC2, is also regulated in mature neurons by the WNK1 pathway indicates that this pathway will be a target of choice in the pathology.

An upregulation of the Na+-K+-2Cl- co-transporter NKCC1, the main chloride importer in mature neurons, can lead to depolarizing/excitatory responses mediated by GABAA receptors and thus to hyperactivity. Understanding the regulatory mechanisms of NKCC1 would help prevent intra-neuronal chloride accumulation that occurs in pathologies with defective inhibition. The cellular and molecular regulatory mechanisms of NKCC1 are poorly understood. Here, we report in mature hippocampal neurons that GABAergic activity controls the membrane diffusion and clustering of NKCC1 via the chloride-sensitive WNK1 kinase and the downstream SPAK kinase that directly phosphorylates NKCC1 on key threonine residues. At rest, this signaling pathway has little effect on intracellular Cl- concentration but it participates to the elevation of intraneuronal Cl- concentration in hyperactivity condition associated with an up-regulation of NKCC1. The fact that the chloride exporter KCC2 is also regulated in mature neurons by the WNK1 pathway indicates that this pathway will be a target of choice in the pathology.