INTRODUCTION: The neuron-specific K-Cl cotransporter KCC2 maintains low intracellular chloride levels, which are crucial for fast GABAergic and glycinergic neurotransmission. KCC2 also plays a pivotal role in the development of excitatory glutamatergic neurotransmission by promoting dendritic spine maturation. The cytoplasmic C-terminal domain (KCC2-CTD) plays a critical regulatory role in the molecular mechanisms controlling the cotransporter activity through dimerization, phosphorylation, and protein interaction. METHODS: To identify novel CTD regulatory motifs, we used the Mu transposon-based mutagenesis system to generate a library of KCC2 mutants with 5 amino acid insertions randomly distributed within the KCC2-CTD. We determined the insertion positions in 288 mutants by restriction analysis and selected clones with a single insertion site outside known KCC2 regulatory motifs. We analyzed the subcellular distribution of KCC2-CTD mutants in cultured cortical neurons using immunocytochemistry and selected ten mutants with ectopic expression patterns for detailed characterization. RESULTS: A fluorescent Cl(-)-transport assay in HEK293 cells revealed mutants with both reduced and enhanced Cl(-)-extrusion activity, which overall correlated with their glycosylation patterns. Live-cell immunostaining analysis of plasma membrane expression of KCC2-CTD mutants in cultured cortical neurons corroborated the glycosylation data. Furthermore, the somatodendritic chloride gradient in neurons transfected with the KCC2-CTD mutants correlated with their Cl(-)-extrusion activity in HEK293 cells. Gain- and loss-of-function mutant positions were analyzed using available KCC2 cryo-EM structures. DISCUSSION: Two groups of mutants were identified based on 3D structural analysis. The first group, located near the interface of transmembrane and cytoplasmic domains, may affect interactions with the N-terminal inhibitory peptide regulating KCC2 activity. The second group, situated on the external surface of the cytoplasmic domain, may disrupt interactions with regulatory proteins. Analyzing CTD mutations that modulate KCC2 activity enhances our understanding of its function and is essential for developing novel anti-seizure therapies.