与HERG钾通道相互作用的FHL2蛋白对该通道功能的调控  

Modulatory effect of FHL2 protein interacted with HERG potassium channel

作　　者：沈秀张[1] 林吉进[1,2] 武君[1] 黄瑞燕[1] 

机构地区：[1]汕头大学医学院第一附属医院心内科,广东汕头515041 [2]广东省人民医院广东省心血管病研究所心内科,广东广州510080

出　　处：《心脏杂志》2009年第2期155-160,共6页Chinese Heart Journal

基　　金：国家自然科学基金项目资助(30600254);广东省自然科学基金项目资助(06301076);中国博士后科学基金项目资助(20060400211)

摘　　要：目的筛选与心脏人类果蝇相关基因(HERG)的编码蛋白钾通道存在相互作用的蛋白质,并进一步研究该相互作用蛋白对HERG钾通道的调控作用。方法①以带有编码人类心脏HERG钾通道的cDNA为模板,通过PCR方法得到编码人类心脏HERG钾通道氨基末端(404个氨基酸)的DNA片段。将该片段克隆入pGBKT7载体,构建"诱饵"质粒pGBKT7-HERG-NT。②应用酵母双杂交技术筛选人类心脏cDNA文库。③以PCR法扩增4个半LIM结构域(FHL2)基因的开放读框片段(ORF),并克隆入pcDNA3.0载体。④以pcDNA3.0-herg转染HEK293细胞,应用G418筛选得到HEK293/HERG细胞株。以pcDNA3.0-FHL2转染HEK293细胞,筛选得到HEK293/FHL2细胞株后,再将pcDNA3.0-herg转染入该细胞株。⑤应用膜片钳技术,研究FHL2对HERG通道功能的影响。结果①用酵母双杂交技术筛选得到37个阳性克隆,其中含有表达FHL2蛋白的克隆。②膜片钳检测发现,FHL2蛋白在增加HERG电流幅度的同时并调节其激活过程。结论FHL2蛋白能与HERG氨基末端相互作用而影响HERG钾通道的功能。AIM To screen the proteins interacted with human ether-a-go-go-related gene （HERG） potassium channel and to study the modulatory effect of the interacting protein on this channel. METHODS （1）The N-terminal fragment of HERG （aa 1-404 ） was PCR-amplified by using the human cDNA that encoded the HERG potassium channel as template, and the fragment was then cloned into the pGBKT7 to construct the bait plasmid pGBKT7-HERG-NT. （2）The yeast two-hybrid technique was used to screen the human heart cDNA library. （3）The open reading frame of four and half LIM domain （FHL2） gene was PCR-amplified and cloned into the pcDNA3.0. （4）The HEK293 cells were transfected with pcDNA3.0- herg, and the cells were then screened with G418 to obtain the HEK293/HERG cells. The HEK293 cells were transfected with pcDNA3.0-FHL2 to obtain the HEK293/FHL2 cells and the HEK293/FHL2 cells were transfected with pcDNA3.0-berg. （5）The patch-clamp technique was used to study the effect of the interacting protein on the HERG channel property. RESULTS （1）Thirty-seven clones were selected by the yeast two-hybrid screen, one of which was FHL2. （2）The patch clamp electrophysiological experiment showed that FHL2 increased the HERG current amplitude and accelerated the activation rate of the HERG potassium channel. CONCLUSION FHL2 interacts with the amino terminus of HERG and modulates the HERG potassium channel function.

关 键 词：酵母双杂交 HERG FHL2 膜片钳技术 

分 类 号：R541.7[医药卫生—心血管疾病]