桥尾蛋白磷酸化参与大鼠螺旋神经节神经元GABA_(A)受体内化  

Phosphorylation of Gephyrin Is Involved in GABA_(A) Receptors Internalization in Rat Spiral Ganglion Neurons

作　　者：林晓宇 韦芳玉 覃江圆[1] 叶莞丽 易健启 陈慧英[1] 刘金兰[1] 覃继新[2] 苏纪平[1] Lin Xiaoyu;Wei Fangyu;Qin Jiangyuan;Ye Wanli;Yi Jianqi;Chen Huiying;Liu Jinlan;Qin Jixin;Su Jiping(Department of Otolaryngology-Head and Neck Surgery,the First Affiliated Hospital of Guangxi Medical University,Nanning,530021,China;不详)

机构地区：[1]广西医科大学第一附属医院耳鼻咽喉头颈外科,南宁530021 [2]广西右江民族医学院附属医院耳鼻咽喉头颈外科

出　　处：《听力学及言语疾病杂志》2022年第2期192-196,共5页Journal of Audiology and Speech Pathology

基　　金：国家自然科学基金项目(81560174,81360157);广西自然科学基金(2014GXNSFAA118137)资助。

摘　　要：目的探讨桥尾蛋白(gephyrin)丝氨酸S270位点磷酸化在水杨酸钠(sodium salicylate,SS)介导的GABA_(A)受体(GABA type A receptors,GABA_(A)Rs)内化的作用。方法将12只SD大鼠分成4组,分别为SS处理组、LiCl处理组、SS+LiCl处理组和对照组,每组3只,急性分离各组耳蜗螺旋神经节神经元(spiral ganglion neurons,SGNs),SGNs原代培养48 h后分别用5 mM SS、1 mM LiCl、5 mM SS联合1 mM LiCl处理1 h,对照组不予处理;采用实时荧光定量PCR(RT-qPCR)检测GABA_(A)Rsα2亚基、桥尾蛋白基因表达的改变;采用Western blot技术检测各组GABA_(A)Rsα2膜蛋白和总蛋白表达的改变、桥尾蛋白及其S270位点磷酸化的改变。结果RT-qPCR结果显示,各组GABA_(A)Rsα2亚基mRNA、桥尾蛋白mRNA与对照组比较均无明显变化(P>0.05),Western blot结果显示,SS处理组GABA_(A)Rsα2亚基膜蛋白(0.08±0.02)较对照组(0.18±0.04)表达显著下调(P<0.01),LiCl处理组(0.14±0.03)有效逆转SS诱导的GABA_(A)Rsα2亚基膜蛋白下调(SS组为0.08±0.02)(P<0.05),LiCL组膜蛋白、各组总蛋白与对照组比较无明显变化(P>0.05);SS处理组桥尾蛋白S270磷酸化(0.84±0.02)较对照组(0.35±0.14)显著增强(P<0.001)。LiCl处理组(0.66±0.10)有效逆转SS诱导的桥尾蛋白S270磷酸化增强(SS组为0.84±0.02,P<0.05),LiCl处理组桥尾蛋白磷酸化、各处理组桥尾蛋白与对照组比较无明显变化。结论桥尾蛋白S270位点磷酸化可增强参与SS诱导的SGNs GABA_(A)Rs内化。Objective To investigate the role of gephyrin serine 270 phosphorylation in the internalization of GABA type A receptors(GABA_(A)Rs)mediated by sodium salicylate(SS).Methods A total of 12 neonatal rats were randomly divided into four groups with 3 in each group.Spiral ganglion neurons(SGNs)were isolated and treated separately with 5 mM SS,1 mM LiCl,and 5 mM SS+1 mM LiCl for 1 h after primary culture for 48 h,while the control group was not treated.The gene expression changes of GABA_(A)Rsα2 subunit and gephyrin were detected by real-time fluorescence quantitative PCR.The changes of GABA_(A)Rsα2 membrane protein and total protein expression and the changes of gephyrin protein and its phosphorylation at the S270 were detected by Western blot.Results There were no significant changes in GABA_(A)Rsα2 subunit and gephyrin gene expression in all treatment groups compared with the control group(P>0.05).Western blot results showed that GABA_(A)Rsα2 membrane protein expression was decreased significantly compared with the control group(0.08±0.02 vs 0.18±0.04,P<0.01).LiCl pretreatment effectively reversed the downregulation of GABA_(A)Rsα2 subunit membrane protein(0.14±0.03 in the SS+LiCl group vs 0.08±0.02 in the SS group,P<0.05).Compared with the control group,there were no significant expression changes in membrane protein in the LiCl group and total protein in all treatment groups(P>0.05).Phosphorylation of gephyrin S270 in the SS group was significantly increased compared with the control group(0.84±0.02 vs 0.35±0.14,P<0.001).LiCl pretreatment effectively reversed the enhanced gephyrin S270 phosphorylation(0.66±0.10 in SS+LiCl group vs 0.84±0.02 in SS group,P<0.05).The phosphorylation of gephyrin S270 in the LiCl group and the expression of gephyrin protein in all treatment groups were not significantly different from that in the control group(P>0.05).Conclusion The enhanced phosphorylation at S270 of gephyrin is involved in GABA_(A)Rs internalization induced by SS on SGNs.

关 键 词：桥尾蛋白 磷酸化 GABA_(A)受体 内化 螺旋神经节神经元 

分 类 号：R764.3[医药卫生—耳鼻咽喉科]