Acute epileptiform activity induced by gabazine involves proteasomal rather than lysosomal degradation of KCa2.2 channels
Neurobiology of Disease(2018)
Abstract
Voltage-independent, Ca2+-activated K+ channels (KCa2.2, previously named SK2) are typically activated during a train of action potentials, and hence, are powerful regulators of cellular excitability by generating an afterhyperpolarizing potential (AHP) following prolonged excitation. In the acute in vitro epilepsy model induced in hippocampal brain slice preparations by exposure to the GABAA receptor blocker gabazine (GZ), the AHP was previously shown to be significantly decreased. Here, we asked the question whether KCa2.2 protein degradation occurs in this model and which pathways are involved. To this end, we applied either gabazine alone or gabazine together with inhibitors of proteasomal and lysosomal protein degradation pathways, Z-Leu-Leu-Leu-CHO (MG132) and chloroquine (CQ), respectively. Western blot analysis showed a significant decrease of total KCa2.2 protein content in GZ-treated slices which could be rescued by concomitant incubation with MG132 and CQ. Using HEK293 cells transfected with a green fluorescent protein-tagged KCa2.2 construct, we demonstrated that proteasomal rather than lysosomal degradation was involved in KCa2.2 reduction. We then recorded epileptiform afterdischarges at hippocampal Schaffer collateral-CA1 synapses and confirmed that the GZ-induced increase was significantly attenuated by both MG132 and CQ, with MG132 being significantly more effective than CQ. Epileptiform afterdischarges were almost prevented by co-application of protein degradation inhibitors. Furthermore, epileptiform afterdischarges could be re-established by using the KCa2.2 blocker UCL 1684 suggesting involvement of KCa2.2. We conclude that in GZ-induced acute epilepsy, KCa2.2 degradation by proteasomal rather than lysosomal pathways plays a major role in the generation of epileptiform afterdischarges.
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Key words
Gabazine,Epilepsy,KCa2.2,UCL1684,PKA
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