Brugada综合征SCN5A基因G1712C突变的功能分析  

Functional analysis of a novel SCN5A mutation G1712C identified in Brugada syndrome

作　　者：陈燕玉[1] 刘深荣 谢亮真 朱庭延 陈益臻 邓晓江[1] 孟素荣[1] 彭健[1] 

机构地区：[1]南方医科大学南方医院心内科,广东广州510515

出　　处：《南方医科大学学报》2017年第2期256-260,共5页Journal of Southern Medical University

基　　金：广东省科技计划项目(2013B021800140);广州市科技计划项目(201300000146)

摘　　要：目的探讨Brugada综合征SCN5A基因新突变G1712C的电生理机制。方法采用体外定点诱变法构建携带有基因突变G1712C的p Rc/CMV-h H1的表达载体,lipo3000脂质转染法建立稳定表达p GFP-IRES-hβ1质粒的HEK293细胞系,并用G418进行筛选鉴定。分别做野生型的p Rc/CMV-h H1(h H1)和携带有基因突变G1712C的p Rc/CMV-h H1(mh H1)瞬时转染表达。进行全细胞膜片钳实验记录钠通道电流。实验结果由Patch Master以及IGOR Pro 6.0软件分析。结果 G1712C位于Na+通道蛋白α亚单位的DⅣ区S5与S6之间的P-loop上。在瞬时转染野生型的h H1的细胞系中,指令电位从-60 m V逐渐上升时,钠电流也渐变大,在-20 m V时完全激活;激活电压在-60 m V到-50 m V,反转电位在50 m V左右。在瞬时转染突变型G1712C的细胞系中,没有发现钠电流。结论野生型h H1所表达的钠通道蛋白与正常心肌细胞钠通道电生理特性相似。SCN5A基因G1712C突变导致Nav1.5通道失去功能,可能是该家系Brugada综合征的病因。Objective To elucidate the molecular and electrophysiological mechanisms of Brugada syndrome through functional analysis of a novel SCN5A gene mutation G1712C. Methods A recombinant plasmid pRc/CMV-hH1 containing the mutant human cardiac sodium channel α subunit （hill） cDNA was constructed using in vitro PCR-based site-directed mutagenesis technique. LipofectamineTM 3000 was used to transfect the plasmid DNA into HEK293 cell line to induce stable expression of Na＋ channel β1-subunit, and the positive colonies were selected by screening with G418. The standard liposome method was used to transiently transfect HEK293 cells with either the wild-type or mutant Na ＋ channel subunits （hill and mhH1, respectively）, and the macroscopic Na＋ currents were recorded using whole-cell patch-clamp technique. Data acquisition and analysis, generation of voltage commands and curve fitting were accomplished with EPC-10, PatchMaster and IGOR Pro 6.0. Results An HEK293 cell line that stably expressed Na ＋ channel β1-subunit was successfully established. After transient transfection with the WT subunit, large Na＋ currents were recorded from the stable β1-cell line. Transient transfection with the G1712C subunit, however, did not elicit a Na ＋ current in the cells. Conclusion Compared with normal Na ＋ channel, the wild-type channel exhibits a similar sodium current. The characteristic kinetics of sodium channel of WT-hH1 was identical to that in normal cardiac muscle cell, and the missense mutation （G1712C） in the P-loop region of the domain W may have caused the failure of sodium channel expression.

关 键 词：BRUGADA综合征 SCN5A基因 G1712C 钠电流 

分 类 号：R541.7[医药卫生—心血管疾病]