Calcineurin Inhibitor Cyclosporine Inhibits the Neuronal K‐Cl Co‐transporter KCC2

Abstract

Calcineurin inhibitors (CNI) cyclosporine and tacrolimus are widely used for immunosuppression, but may cause serious neurologic side effects such as seizures suggesting that these drugs may induce hyperexcitability of neocortical neurons. Calcineurin was implicated in the regulation of neuronal excitability and cellular chloride homeostasis. In this context, we tested the hypothesis that CNI interfere with the function of major neuronal cation-chloride cotransporter KCC2. Calcineurin and KCC2 were co-localized in rat neocortical neurons by immuno­fluorescence and interacted by co-immunoprecipitation. Acute administration of cyclosporine to rats (25 mg/kg, 1-4h) increased the level of KCC2 phosphorylation at the activating S940 site (+240%, p<0.05). Evaluation of immunoprecipitated KCC2 using a phospho-tyrosine antibody also suggested its increased phosphorylation and function upon cyclosporine. In contrast, intracellular recordings of chloride homeostasis after iontophoretic Cl- loading revealed strong cyclosporine-induced prolongation of the Cl- extrusion time (+3.4s, p<0.05) which was compatible with KCC2 blockade. To resolve this discrepancy we have evaluated the KCC2-inhibiting WNK/SPAK kinase pathway and found substantially increased expression of SPAK after cyclosporine application (+59%, p<0.05). In sum, our data suggest that short term inhibition of calcineurin by cyclosporine attenuates KCC2 function probably due to stimulation of the WNK/SPAK-signaling with downstream effects on the transporter. Regarding the broad clinical use of CNI our data have clinical implications.