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.%目的 探讨Brugada综合征SCN5A基因G1712C新突变的电生理机制.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pRe/CMV-hHl (hHl)和携带有基因突变G1712C的pRc/CMV-hHl (mhHl)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.
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