Diazepam reduces synaptic GABA type areceptor availability via multiple trafficking mechanisms  

作　　者：Joshua M LORENZ-GUERTIN Sabyasachi DAS Tija C JACOB 

机构地区：[1]Department of Pharmacology & Chemical Biology,University of Pittsburgh

出　　处：《中国药理学与毒理学杂志》2017年第10期952-952,共1页Chinese Journal of Pharmacology and Toxicology

基　　金：supported by National Institute of Health(T32GM008424);Whitehall Foundation;William C DEGROAT Neuropharmacology Departmental Fellowship;Pharmacology and Chemical Biology Startup Funds

摘　　要：OBJECTIVE Investigate the effects of diazepam(DZP) on γ2 subunit containing GABA type A receptor(GABA A R) trafficking.METHODS Immunofluorescence microscopy measured surface GABA A Rs and gephyrin in rat cortical neurons after 24 h exposure of 1.0 μmol·L^(-1) DZP.Biochemical studies of mice injected with 10 mg·kg^(-1) DZP vs vehicle were assessed for γ2 subunit and total gephyrin cortical levels 12 h post-injection.Ubiquitination of the γ2 subunit was studied by immunoprecipitation after 12 h of 1.0 μmol·L^(-1) DZP exposure.A γ2 subunit encoding an N terminal fluorogen-activating peptide and pH-sensitive green fluorescent protein(γ2 pH FAP) measured lysosomal targeting of γ2 containing GABA A Rs.RFP-gephyrin and γ2 pH FAP synaptic diffusion rates were examined using fluorescence recovery after photobleaching(FRAP).RESULTS Extrasynaptic levels of γ2 GABA A Rs decreased by 12.2%,while synaptic gephyrin S270 phosphorylation increased by 18.3% in DZP-treated neurons after 24 h compared to control(P<0.05).Dendritic levels of gephyrin were also reduced to 74.1% of control,while S270 phosphorylation was elevated by 25.2%(P<0.05;P<0.01).Mice 12 h post-DZP injection demonstrated a 12.7% and 26.1% decrease in total γ2 and gephyrin levels,respectively(P<0.05;P<0.01).12 h DZP treatment enhanced γ2 subunit ubiquitination 1.13-fold relative to control(P<0.05).Internalized γ2 pH FAP GABA A Rs associated with lysosomes was 8.0% higher in neurons treated with 12-16 h DZP compared to control.Pilot FRAP experiments suggest gephyrin and γ2 have increased mobility and turnover at synapses following DZP.CONCLUSION DZP treatment decreases γ2 GABA A R levels and gephyrin scaffolding function after one day of exposure,which may contribute to the formation of DZP tolerance.OBJECTIVE Investigate the effects of diazepam（DZP） on γ2 subunit containing GABA type A receptor（GABA A R） trafficking.METHODS Immunofluorescence microscopy measured surface GABA A Rs and gephyrin in rat cortical neurons after 24 h exposure of 1.0 μmol·L^（-1） DZP.Biochemical studies of mice injected with 10 mg·kg^（-1） DZP vs vehicle were assessed for γ2 subunit and total gephyrin cortical levels 12 h post-injection.Ubiquitination of the γ2 subunit was studied by immunoprecipitation after 12 h of 1.0 μmol·L^（-1） DZP exposure.A γ2 subunit encoding an N terminal fluorogen-activating peptide and pH-sensitive green fluorescent protein（γ2 pH FAP） measured lysosomal targeting of γ2 containing GABA A Rs.RFP-gephyrin and γ2 pH FAP synaptic diffusion rates were examined using fluorescence recovery after photobleaching（FRAP）.RESULTS Extrasynaptic levels of γ2 GABA A Rs decreased by 12.2%,while synaptic gephyrin S270 phosphorylation increased by 18.3% in DZP-treated neurons after 24 h compared to control（P〈0.05）.Dendritic levels of gephyrin were also reduced to 74.1% of control,while S270 phosphorylation was elevated by 25.2%（P〈0.05;P〈0.01）.Mice 12 h post-DZP injection demonstrated a 12.7% and 26.1% decrease in total γ2 and gephyrin levels,respectively（P〈0.05;P〈0.01）.12 h DZP treatment enhanced γ2 subunit ubiquitination 1.13-fold relative to control（P〈0.05）.Internalized γ2 pH FAP GABA A Rs associated with lysosomes was 8.0% higher in neurons treated with 12-16 h DZP compared to control.Pilot FRAP experiments suggest gephyrin and γ2 have increased mobility and turnover at synapses following DZP.CONCLUSION DZP treatment decreases γ2 GABA A R levels and gephyrin scaffolding function after one day of exposure,which may contribute to the formation of DZP tolerance.

关 键 词：GABA type A receptor GEPHYRIN TRAFFICKING DIAZEPAM 

分 类 号：R96[医药卫生—药理学]