Phosphoregulation of the intracellular termini of K-Cl cotransporter 2 (KCC2) enables flexible control of its activity.

The pivotal role of K-Cl cotransporter 2 (KCC2) in inhibitory neurotransmission and severe human diseases fosters interest in understanding posttranslational regulatory mechanisms such as (de)phosphorylation. Here, the regulatory role of the five phosphosites Ser, Thr, Ser, Thr, and Thr was investigated by the use of alanine and aspartate mutants. Tl-based flux analyses in HEK-293 cells demonstrated increased transport activity for S932D (mimicking phosphorylation) and T1008A (mimicking dephosphorylation), albeit to a different extent. Increased activity was due to changes in intrinsic activity, as it was not caused by increased cell-surface abundance. Substitutions of Ser, Thr, or Thr had no effect. Additionally, we show that the indirect actions of the known KCC2 activators staurosporine and -ethylmaleimide (NEM) involved multiple phosphosites. S31D, T34A, S932A/D, T999A, or T1008A/D abrogated staurosporine mediated stimulation, and S31A, T34D, or S932D abolished NEM-mediated stimulation. This demonstrates for the first time differential effects of staurosporine and NEM on KCC2. In addition, the staurosporine-mediated effects involved both KCC2 phosphorylation and dephosphorylation with Ser and Thr being target sites. In summary, our data reveal a complex phosphoregulation of KCC2 that provides the transporter with a toolbox for graded activity and integration of different signaling pathways.