Li Yu, Cui Chang-cong, Zhao Yong-hui, Xue Xiao-lin, Zhang Ai-feng, Lian Jiang-fang, Huang Chen
Department of Cardiology, First Hospital of Xi'an Jiaotong University, Xi'an 710061, China.
Zhonghua Xin Xue Guan Bing Za Zhi. 2007 Feb;35(2):143-6.
To investigate the functional expression of HERG mutation A561V detected in a Chinese congenital long QT syndrome family.
The mutation gene A561V was cloned into eukaryotic expressive vector pcDNA3 by quick site-directed mutagenesis PCR and restriction enzymes. The wild-type HERG, heterozygous type HERG and HERG mutation A561V were respectively cotransfected with pRK5-GFP into HEK293 cells by Suprefact transfection regent. The protein expression was measured by immunofluorescence method and Western blot. The electrophysiological characteristics of transfected cells were determined by whole cell patch-clamp technique.
Direct sequence analyses revealed a C to T transition at position 1682. A561V mutation was correctly combined to eukaryotic expressive vector pcDNA3 and expressed in HEK293 cells. The protein expression of mutation and heterozygosis were located in cytoplasm and cellular membrane. 155 kDa and 135 kDa protein bands were detected in wild type HERG channel while only 135 kDa protein band was shown in heterozygous and mutational channels. Significant HERG tail-current was recorded in wild type HERG channel but not in mutation and heterozygosis channels.
This study evidenced a functional dominant-negative current suppression in HEK293 cells transfected with HERG mutation A561V.
研究在中国一个先天性长QT综合征家系中检测到的HERG突变A561V的功能表达。
通过快速定点诱变PCR和限制性内切酶将突变基因A561V克隆到真核表达载体pcDNA3中。野生型HERG、杂合型HERG和HERG突变A561V分别通过Suprefact转染试剂与pRK5-GFP共转染入HEK293细胞。采用免疫荧光法和蛋白质印迹法检测蛋白质表达。通过全细胞膜片钳技术测定转染细胞的电生理特性。
直接序列分析显示第1682位存在C到T的转变。A561V突变正确地连接到真核表达载体pcDNA3并在HEK293细胞中表达。突变型和杂合型的蛋白质表达位于细胞质和细胞膜。在野生型HERG通道中检测到155 kDa和135 kDa的蛋白条带，而在杂合型和突变型通道中仅显示135 kDa的蛋白条带。在野生型HERG通道中记录到明显的HERG尾电流，而在突变型和杂合型通道中未记录到。
本研究证明在转染HERG突变A561V的HEK293细胞中存在功能性显性负电流抑制。
