[1]陈燕玉,刘深荣,谢亮真,等.Brugada综合征SCN5A基因G1712C突变的功能分析[J].南方医科大学学报,2017,(02):256.
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Brugada综合征SCN5A基因G1712C突变的功能分析()
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《南方医科大学学报》[ISSN:/CN:]

卷:
期数:
2017年02期
页码:
256
栏目:
出版日期:
2017-01-22

文章信息/Info

Title:
Functional analysis of a novel SCN5A mutation G1712C identified in Brugada syndrome
作者:
陈燕玉刘深荣谢亮真朱庭延陈益臻邓晓江孟素荣彭健
关键词:
Brugada综合征SCN5A基因G1712C钠电流
Keywords:
Brugada syndrome SCN5A G1712C sodium current
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pRc/CMV-hH1的表达载体,lipo3000脂质转染法建立稳定表达pGFP-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pRc/CMV-hH1(hH1)和携带有基因突变G1712C的pRc/CMV-hH1(mhH1)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PatchMaster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hH1的细胞系中,指令电位从-60 mV逐渐上升时,钠电流也 渐变大,在-20 mV时完全激活;激活电压在-60 mV到-50 mV,反转电位在50 mV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hH1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。
Abstract:
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 (hH1) 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 (hH1 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 IV may have caused the failure of sodium channel expression.

相似文献/References:

[1]彭健,崔英凯,阮发晖,等.发热与Brugada综合征(21例报告)[J].南方医科大学学报,2005,(04):432.
 PENG Jian,CUI Ying-kai,YUAN Fa-hui,et al.Fever and Brugada syndrome: report of 21 cases[J].,2005,(02):432.

更新日期/Last Update: 1900-01-01