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 bona fide phosphosites Ser³¹, Thr³⁴, Ser⁹³², Thr⁹⁹⁹, and Thr¹⁰⁰⁸ 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 Ser³¹, Thr³⁴, or Thr⁹⁹⁹ had no effect. Additionally, we show that the indirect actions of the known KCC2 activators staurosporine and N-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 Ser⁹³² and Thr¹⁰⁰⁸ being bona fide 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.

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