Coordinated downregulation of KCC2 and GABA_A receptor contributes to inhibitory dysfunction during seizure induction

The GABA_A receptor (GABA_AR) is the main inhibitory receptor in the adult mammalian brain. GABA_AR function is dependent on its expression, distribution, and the chloride (Cl^-) transmembrane gradient, which is determined by the potassium-chloride cotransporter 2 (KCC2) in the adult brain. KCC2 and GABA_AR are downregulated in an activity-dependent manner during seizure induction. Functionally, KCC2 and GABA_AR are closely related membrane proteins which modulate GABAergic inhibition. However, it remains unclear how their downregulation during seizure induction is coordinated. This study aimed to assess this interaction. Our results revealed that KCC2 and GABA_AR were simultaneously downregulated in both in vivo and in vitro seizure models induced by the convulsant cyclothazide (CTZ), which was at least partly due to structural coupling in hippocampal neuronal membranes. Immunohistochemistry revealed colocalization of gephyrin with KCC2 and co-immunoprecipitation exhibited a direct coupling between GABA_AR α1-subunit and KCC2 protein in hippocampal cell membranes. KCC2 specific short hairpin RNA (KCC2-shRNA) was employed to specifically reduce the expression of KCC2 in cultured hippocampal neurons. This resulted in a significant reduction in KCC2-independent GABAergic miniature inhibitory post-synaptic current (mIPSC) amplitude in shKCC2-transfected neurons. Further, pre-treatment with furosemide, a KCC2 inhibitor, during CTZ stimulation followed by washout significantly prevented convulsant stimulation-induced membrane KCC2 downregulation and significantly attenuated GABA_AR downregulation concomitant with recovery of suppressed KCC2-independent GABAergic mIPSC amplitude. Our results suggest that the coordinated downregulation of KCC2 and GABA_AR during seizure induction exerts a strong functional impact on GABA_AR, highlighting an important regulatory mechanism in epilepsy.