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|本期目录/Table of Contents|

与HERG钾通道相互作用的FHL2蛋白对该通道功能的调控

《心脏杂志》[ISSN:1009-7236/CN:61-1268/R]

期数:
2009年第2期
页码:
155
栏目:
基础研究
出版日期:
2009-03-30

文章信息/Info

Title:
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
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
Author(s):
SHEN Xiu-zhang1;  LIN Ji-jin1; 2;  WU Jun1;  HUANG Rui-yan1
1.Department of Cardiology, First Affiliated Hospital of Medical College, Shantou University, Shantou 515041, Guangdong, China; 2.Department of Cardiology, Guangdong Provincial Cardiovascular Institute, Guangdong Provincial People’s Hospital, Guangzhou 510080, Guangdong, China
关键词:
酵母双杂交; HERG; FHL2; 膜片钳技术
Keywords:
yeast two-hybrid;  HERG;  FHL2;  patch clamp technique
分类号:
R541.7
DOI:
-
文献标识码:
A
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。
Abstract:
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 ①The 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. ②The yeast two-hybrid technique was used to screen the human heart cDNA library. ③The open reading frame of four and half LIM domain(FHL2) gene was PCR-amplified and cloned into the pcDNA3.0. ④The 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-herg. ⑤The patch-clamp technique was used to study the effect of the interacting protein on the HERG channel property. RESULTS ①Thirty-seven clones were selected by the yeast two-hybrid screen, one of which was FHL2. ②The 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.

参考文献/References

[1］ Sanguinetti MC, Tristani-Firouzi M. hERG potassium channels and cardiac arrhythmia［J］. Nature, 2006, 440(7083):463-469.

［2］ Curran ME， Splawski I, Timothy KW, et al. A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome［J］. Cell, 1995, 80(5):795-803.

［3］ Modell SM, Lehmann MH. The long QT syndrome family of cardiac ion channelopathies: a HuGE review［J］. Genet Med, 2006, 8(3):143-155.

［4］ Thomas D, Gut B, Wendt-Nordahl G, et al. The antidepressant drug fluoxetine is an inhibitor of human ether-a-go-go-related gene (HERG) potassium channels［J］. J Pharmacol Exp Ther, 2002, 300(2):543-548.

［5］ Yao X, McIntyre MS, Lang DG, et al. Propranolol inhibits the human ether-a-go-go-related gene potassium channels［J］. Eur J Pharmacol, 2005, 519(3):208-211.

［6］ Lin J, Friesen MT, Bocangel P, et al. Characterization of mesenchyme homeobox 2 (MEOX2) transcription factor binding to RING finger protein 10［J］. Mol Cell Biochem, 2005, 275(1-2):75-84.

［7］ Johannessen M, Moller S, Hansen T, et al. The multifunctional roles of the four-and-a-half-LIM only protein FHL2［J］. Cell Mol Life Sci, 2006, 63(3):268-284.

［8］ Kupershmidt S, Yang IC, Sutherland M, et al. Cardiac-enriched LIM domain protein fhl2 is required to generate Iks in a heterologous system［J］. Cardiovasc Res, 2002, 56(1):93-103.

备注/Memo

备注/Memo:
收稿日期：2008-1-17.基金项目：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） 通讯作者：林吉进，副主任医师，主要从事心血管疾病临床及基础研究Email:linjijin@yahoo.com.cn 作者简介：沈秀张,医师, 硕士生Email: shenxz8212@yahoo.com.cn
更新日期/Last Update: 2009-04-16