Implications of the N-terminal heterogeneity for the neuronal K-Cl cotransporter KCC2 function

•KCC2a isoform is expressed at the neuronal plasma membrane.•KCC2a isoform mediates chloride extrusion in cultured neurons.•SPAK differently regulates kinetic activity of KCC2a and KCC2b isoforms. The neuron-specific K-Cl cotransporter KCC2 maintains the low intracellular chloride concentration required for the fast hyperpolarizing responses of the inhibitory neurotransmitters γ-aminobutyric acid (GABA) and glycine. The two KCC2 isoforms, KCC2a and KCC2b differ by their N-termini as a result of alternative promoter usage. Whereas the role of KCC2b in mediating the chloride transport is unequivocal, the physiological role of KCC2a in neurons has remained obscure. We show that KCC2a isoform can decrease the intracellular chloride concentration in cultured neurons and attenuate calcium responses evoked by application of the GABAA receptor agonist muscimol. While the biotinylation assay detected both KCC2 isoforms at the cell surface of cultured neurons, KCC2a was not detected at the plasma membrane in immunostainings, suggesting that the N-terminal KCC2a epitope is masked. Confirming this hypothesis, KCC2a surface expression was detected by the C-terminal KCC2 pan antibody but not by the N-terminal KCC2a antibody in KCC2b-deficient neurons. One possible cause for the epitope masking is the binding site of Ste20-related proline-alanine-rich kinase (SPAK) in the KCC2a N-terminus. SPAK, a known regulator of K-Cl cotransporters, was co-immunoprecipitated in a complex with KCC2a but not KCC2b isoform. Moreover, SPAK overexpression decreased the transport activity of KCC2a but not that of KCC2b, as revealed by rubidium flux assay in HEK293 cells. Thus, our data indicate that both KCC2 isoforms perform as chloride cotransporters in neuronal cells, while their N-terminal heterogeneity could play an important role in fine-tuning of the K-Cl transport activity.