WNK1-regulated inhibitory phosphorylation of the KCC2 cotransporter maintains the depolarizing action of GABA in immature neurons

Activation of Cl--permeable gamma-aminobutyric acid type A (GABA(A)) receptors elicits synaptic inhibition in mature neurons but excitation in immature neurons. This developmental switch in the GABA function depends on a postnatal decrease in intraneuronal Cl- concentration mediated by KCC2, a Cl--extruding K+-Cl- cotransporter. We showed that the serine-threonine kinase WNK1 [with no lysine (K)] forms a physical complex with KCC2 in the developing mouse brain. Dominant-negative mutation, genetic depletion, or chemical inhibition of WNK1 in immature neurons triggered a hyperpolarizing shift in GABA activity by enhancing KCC2-mediated Cl- extrusion. This increase in KCC2 activity resulted from reduced inhibitory phosphorylation of KCC2 at two C-terminal threonines, Thr(906) and Thr(1007). Phosphorylation of both Thr(906) and Thr(1007) was increased in immature versus mature neurons. Together, these data provide insight into the mechanism regulating Cl- homeostasis in immature neurons, and suggest that WNK1-regulated changes in KCC2 phosphorylation contribute to the developmental excitatory-to-inhibitory GABA sequence.