TY - JOUR
T1 - Coordinated downregulation of KCC2 and GABAA receptor contributes to inhibitory dysfunction during seizure induction
AU - Wan, Li
AU - Chen, Lulan
AU - Yu, Jiangning
AU - Wang, Guoxiang
AU - Wu, Zheng
AU - Qian, Binbin
AU - Liu, Xu
AU - Wang, Yun
N1 - Funding Information:
This work was funded by a postdoctoral grant 2019M653075 from the China Postdoctoral Science Foundation to LW and NSFC grants to XL ( 81301108 , 81971204 ) and YW ( 31471027 , 31771188 ). It was also partially supported by Shanghai Municipal Science and Technology Major Project (No. 2018SHZDZX01 ) and ZJLab, and a grant from the Sanming Project of Medicine in Shenzhen (No. SZSM201512011 ).
Publisher Copyright:
© 2020 Elsevier Inc.
PY - 2020/11/12
Y1 - 2020/11/12
N2 - The GABAA receptor (GABAAR) is the main inhibitory receptor in the adult mammalian brain. GABAAR 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 GABAAR are downregulated in an activity-dependent manner during seizure induction. Functionally, KCC2 and GABAAR 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 GABAAR 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 GABAAR 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 GABAAR downregulation concomitant with recovery of suppressed KCC2-independent GABAergic mIPSC amplitude. Our results suggest that the coordinated downregulation of KCC2 and GABAAR during seizure induction exerts a strong functional impact on GABAAR, highlighting an important regulatory mechanism in epilepsy.
AB - The GABAA receptor (GABAAR) is the main inhibitory receptor in the adult mammalian brain. GABAAR 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 GABAAR are downregulated in an activity-dependent manner during seizure induction. Functionally, KCC2 and GABAAR 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 GABAAR 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 GABAAR 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 GABAAR downregulation concomitant with recovery of suppressed KCC2-independent GABAergic mIPSC amplitude. Our results suggest that the coordinated downregulation of KCC2 and GABAAR during seizure induction exerts a strong functional impact on GABAAR, highlighting an important regulatory mechanism in epilepsy.
UR - https://www.scopus.com/pages/publications/85090229323
UR - https://www.scopus.com/pages/publications/85090229323#tab=citedBy
U2 - 10.1016/j.bbrc.2020.08.082
DO - 10.1016/j.bbrc.2020.08.082
M3 - Article
C2 - 32892950
AN - SCOPUS:85090229323
SN - 0006-291X
VL - 532
SP - 489
EP - 495
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 3
ER -