长QT综合征KCNH2基因突变L413P和L559H的致病机制  

The mechanistic role of KCNH2 gene IA13P and L559H mutations in long QT syndrome

作　　者：李翠兰[1] 胡大一[1] 刘文玲[1] 李蕾[1] 王洪涛[2] 齐书英[3] 龚秋明 周正锋 

机构地区：[1]北京大学人民医院心内科,100044 [2]首都医科大学附属北京同仁医院心脏中心 [3]石家庄国际和平医院心内科 [4]美国俄勒冈卫生科学大学心血管医学系

出　　处：《中华内科杂志》2007年第10期838-841,共4页Chinese Journal of Internal Medicine

基　　金：国家自然科学基金(30170381和30540038);国家985工程(985-2-034-24);北京市科技新星计划(2004-BG-01)

摘　　要：目的研究长 QT 综合征致病基因之一 KCNH2基因 L413P 和 L559H 突变的致病机制。方法 DNA 定点突变技术构建 L413P 和 L559H 表达载体,脂质体介导法转染人胚胎肾细胞表达。用膜片钳技术记录 KCNH2通道电流变化情况,观察 HERG 蛋白表达和细胞内定位情况。结果电生理学显示 L413P 和 L559H 突变后完全无电流。在转染 L413P 或 L559H 的细胞中只观察到未成熟 HERG 蛋白,而转染 WT 的细胞可观察到成熟和未成熟两种 HERG 蛋白形式。L413P 和 L559H 突变蛋白主要分布在细胞核周围的局部区域,提示突变蛋白滞留于内质网中而 WT 蛋白均匀分布在细胞膜核细胞质中。L413P 或 L559H 与等量野生型质粒共转染后电流相比无显著变化。低温与 E-4031不能纠正 HERG 突变的异常。结论 L413P 和 L559H 突变可引起突变蛋白转运障碍,但基因表达和电生理学结果显示突变对野生型无负显性作用,突变的致病机制可能是单倍体不足。Objective To investigate the molecular pathogenesis for two novel mutations L413P and L559H of KCNH2 found in Chinese patients with long QT syndrome. Methods L413P and L559H mutant constructs were generated by site-directed mutagenesis using human wild-type （WT） pcDNA3-HERG cDNA as a template. WT and mutant constructs were transiently transfected into human embryonic kidney 293 cells using lipofectamine method. After transfection, the recording of HERG current was performed using patch clamp technique. The expression and cellular localization of HERG protein were studied with Western blot and immunofluorescence methods. Results Electrophysiological recordings showed that L413P and L559H mutations did not express HERG current. Western blot analysis revealed that only 135 000 immature HERG protein was expressed in L413P and L559H-transfected cells, whereas both mature and immature forms of HERG protein were observed in WT-transfected cells. Immunofluorescence study showed that L413P and L559H mutant proteins were predominantly localized around the nucleus, suggesting that the mutant channels are retained in the endoplasmic reticulum. When L413P or L559H was co-transfected with equal amount of WT plasmids, both 135 000 and 155 000 forms of HERG protein were observed, and the HERG current was not significantly changed as compared with that of WT transfection alone. Low temperature and E-4031could not rescue these two mutant channels. Conclusions The L413P and L559H mutations resulted in protein trafficking defects with failure of mutant proteins to reach the plasma membrane. However, both biochemical and electrophysiological results showed that the mutations did not have a dominant-negative effect on WT, indicating that the mechanism of the L413P and L559H mutations might be haploinsufficiency.

关 键 词：QT延长综合征 基因 离子通道 

分 类 号：R541.7[医药卫生—心血管疾病]