KCC2 phosphorylation dynamics relate to oligomerization during development and after spinal cord injury

Effective synaptic inhibition relies on KCC2, a K+-Cl- cotransporter that forms oligomers to extrude chloride. However, how phosphorylation regulates oligomerization remains unclear. To address this, we studied the phosphorylation of KCC2 in the lumbar spinal cord of neonatal rats by subcellular fractionation and phospho-specific western blotting across three paradigms with distinct expression profiles: developmental upregulation, spinal cord injury (SCI)-induced downregulation, and pharmacological rescue with the 5-HT2A/2C agonist DOI. KCC2 moved from cytoplasm to membrane during development, exhibiting increased intracellular phosphorylation at serine, threonine, and tyrosine residues and decreased membrane tyrosine phosphorylation as a result of the oligomer formation. SCI caused a shift in this ratio in favor of lower oligomers, higher membrane tyrosine phosphorylation and lower intracellular threonine phosphorylation. DOI partially reversed these deficits, reinforcing oligomerization and adapting phosphorylation to a developmental profile. In sum, this study identifies tyrosine dephosphorylation at the membrane as a reliable correlate of oligomerization and offers a unified quantitative model for the regulation of KCC2 in development, after injury, and upon pharmacological rescue.