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

Abstract

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.

Staurosporine and NEM mainly impair WNK-SPAK/OSR1 mediated phosphorylation of KCC2 and NKCC1

“…Inhibition of KCC2 promotes formation of pathological conditions whereas inhibition of NKCC1 seems to prevent or at least alleviate pathological phenotypes [37]. This renders these two transporters a prime pharmacotherapeutic target and fosters interest in understanding posttranslational mechanisms of their regulation [12,[38][39][40]. The main focus is set on phospho-related mechanisms.…”

“…Phosphorylation of Ser 932 , Thr 934 , Ser 937 , or the protein kinase C (PKC) site Ser 940 , all residing in exon 22, increases its transport activity [47,68,77]. The multiplicity of phosphorylation/dephosphorylation sites on KCC2 offers a complex toolbox to gradually fine-regulate its activity and integrate different signaling pathways [4,40,65,68].…”

“…Initially, both staurosporine and NEM were thought to act through a similar mechanism [47], but recent findings revealed that they act differentially on specific KCC2 phospho-sites [40]. Furthermore, staurosporine and NEM mediated effects involve both KCC2 phosphorylation and dephosphorylation [40,48].…”

Impaired KCC2 phosphorylation leads to neuronal network dysfunction and neurodevelopmental pathogenesis

“…However, due to the post-translational regulation of KCC2, there is no simple relationship between the abundance of KCC2 protein and its activity as chloride extruder (Kahle et al, 2013). Phosphorylation of KCC2 at specific residues strongly affects the ion transport activity of the transporter (Lee et al, 2007(Lee et al, , 2010Inoue et al, 2012;Weber et al, 2014;Friedel et al, 2015;Cordshagen et al, 2018).…”

“…Among multiple KCC2 phosphorylation sites (Payne et al, 1996;Lee et al, 2007Lee et al, , 2010Weber et al, 2014;Agez et al, 2017;Cordshagen et al, 2018), the dual (de)phosphorylation of threonine 906 and 1007 (Thr 906 /Thr 1007 ) is a particularly potent regulator of KCC2 activity (Lee et al, 2007(Lee et al, , 2011Rinehart et al, 2009;Inoue et al, 2012;Friedel et al, 2015;Moore et al, 2018). Thr 906 /Thr 1007 becomes progressively dephosphorylated during neuronal development (Rinehart et al, 2009;Friedel et al, 2015).…”