定点突变法显示在高龄SHR大鼠心脏组织中KCNE2第98位氨基酸可能存在磷酸化修饰  

KCNE2 protein S98 phosphorylation in heart of old SHR rats detected by point mutagenesis

作　　者：王慧莲[1,2] 吕社民[1] 宁启兰[1] 杨旭东[1] 韩燕[1] 钟波[1] Gea-Ny TSENG 

机构地区：[1]西安交通大学医学院遗传与分子生物学系 [2]佛吉尼亚联邦大学生理学系,美国Richmond VA23298

出　　处：《浙江大学学报（医学版）》2007年第4期364-370,共7页Journal of Zhejiang University(Medical Sciences)

基　　金：西安交通大学暨医学院留学人员回国科研启动基金资助

摘　　要：目的:探究在高龄SHR大鼠心室膜蛋白中,KCNE2蛋白是否发生了磷酸化修饰。方法:用碱性磷酸酶处理高龄SHR大鼠心室膜蛋白,以抗KCNE2的抗体(Ab2)为一抗进行免疫印迹;将在蛋白离体系统中翻译出的含c-myc的KCNE2融合蛋白经碱性磷酸酶处理后,分别以Ab2和抗c-myc的抗体为一抗的免疫印迹;以及一系列的c-myc-hKCNE2的单点(KCNE2Y96F和KCNE2S98A)和双点突变(KCNE2Y96F/S98A)实验。结果:高龄SHR大鼠心室膜蛋白样品经碱性磷酸酶处理后,极大地减弱了Ab2对KCNE2蛋白的识别能力;另外,蛋白离体系统中翻译出的含c-myc的KCNE2融合蛋白经碱性磷酸酶处理后,虽然极大地减弱了Ab2对其融合蛋白的识别能力,却没有影响抗c-myc抗体对KCNE2融合蛋白的识别能力。定点突变的结果显示,虽然以抗c-myc抗体为一抗的免疫印迹结果显示,蛋白质离体翻译系统成功地翻译出了单突变子KCNE2S96A及双突变子KCNE2Y96F/S98A蛋白,但不论样品被碱性磷酸酶处理与否,Ab2在这些样品中均检测出很弱的KCNE2蛋白免疫条带。结论:在高龄SHR大鼠心室组织中,KCNE2第98位丝氨酸可能发生了磷酸化修饰。然而该位点的磷酸化修饰是否会通过影响心脏的电生理,而在SHR大鼠心脏病变过程中起着一定的作用,有待于从电生理的水平上进一步研究。Objective： To investigate the phosphorylation of KCNE2 protein SHR rats. Methods： The membrane proteins from ventricular myocardium extracted, treated with or without alkaline phosphatase and tested binding w of ith in heart of old old SHR were Ab2 （an anti- KCNE2 polyclonal antibody） by Western blot. A KCNE2 fusion protein with c-myc was obtained from in vitro translation system and treated with or without alkaline phosphatase. A series of mono- and double-point mutated fusion KCNE2 proteins with c-myc were obtained from an in vitro translation system, and Western blots with Ab2 or anti-myc antibody were performed. Results： After alkaline phosphatase treatment, Ab2 significantly attenuated its binding with KCNE2. In vitro translation system confirmed that after alkaline phosphatase treatment, Ab2 weakened binding ability to KCNE2 while binding to c-myc was not changed. Point mutation experiments showed that serine residue in position 98 of KCNE2 proteins might be phosphorylated. Conclusion： KCNE2 protein in heart of old SHR rats is phosphorylated and this phosphorylation takes place in serine residue of position 98.

关 键 词：磷酸化 点突变 心脏 Q—T延长综合征 钾通道 KCNE2蛋白 大鼠 近交SHR 

分 类 号：Q78[生物学—分子生物学]