Serine 937 phosphorylation enhances KCC2 activity and strengthens synaptic inhibition

The potassium chloride cotransporter KCC2 is crucial for Cl^- extrusion from mature neurons and thus key to hyperpolarizing inhibition. Auditory brainstem circuits contain well-understood inhibitory projections and provide a potent model to study the regulation of synaptic inhibition. Two peculiarities of the auditory brainstem are (i) posttranslational activation of KCC2 during development and (ii) extremely negative reversal potentials in specific circuits. To investigate the role of the potent phospho-site serine 937 therein, we generated a KCC2 Thr^934Ala/Ser^937Asp double mutation, in which Ser⁹³⁷ is replaced by aspartate mimicking the phosphorylated state, and the neighbouring Thr⁹³⁴ arrested in the dephosphorylated state. This double mutant showed a twofold increased transport activity in HEK293 cells, raising the hypothesis that auditory brainstem neurons show lower [Cl^-]_i. and increased glycinergic inhibition. This was tested in a mouse model carrying the same KCC2 Thr^934Ala/Ser^937Asp mutation by the use of the CRISPR/Cas9 technology. Homozygous KCC2 Thr^934Ala/Ser^937Asp mice showed an earlier developmental onset of hyperpolarisation in the auditory brainstem. Mature neurons displayed stronger glycinergic inhibition due to hyperpolarized E_Cl-. These data demonstrate that phospho-regulation of KCC2 Ser⁹³⁷ is a potent way to interfere with the excitation-inhibition balance in neural circuits.