中国人Brugada综合征SCN5A基因突变K317N的初步功能分析

Functional expression of a novel SCN5A mutation K317N identified in a Chinese Brugada syndrome family

目的继在一个中国人Brugada综合征家系发现了SCN5A基因突变K317N并进行了载体构建之后,为进一步研究Brugada综合征发病的离子、细胞电生理机制,对新的SCN5A基因突变(K317N)进行初步的功能表达鉴定与分析。方法用脂质体转染法建立稳定表达pGFP-IRES-hβ1质粒的293细胞系,并用G418进行筛选鉴定。然后,分别做pRc/CMV-hH1(hH1)和携带有基因突变K317N的pRc/CMV-hH1(mhH1)的瞬时转染表达。采用全细胞膜片钳技术记录钠通道电流。实验结果由pClamp软件分析。结果通过G418的抗生素筛选和荧光鉴定,成功地培养出稳定表达hβ1的293细胞系。在未转染质粒的空白细胞对照中,没有发现钠电流。在瞬时转染野生型的hH1的细胞系中,记录到了明显的钠电流,在瞬时转染突变型K317N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。而突变型K317N的表达则由于突变所造成的氨基酸的改变对心脏电压依赖钠通道蛋白α亚基的关键结构P环产生了影响,钠通道未能正常表达,或者导致细胞上正常钠通道数量的大量减少,这可能是导致在突变型未能记录出钠电流的原因。

Objective After finding a case of novel SCN5A mutation associated with Brugada syndrome in a Chinese family,the primary functional analysis of novel mutation had been made in order to find out the molecular and cellular electrophysiological mechanism of Brugada syndrome.Methods Mammalian cell transfection and expression was carried out in No.293 cell line that stably expresses Na＋channel β1-subunit,and it had been established by using LipofectamineTM2 000.Positive colonies were then selected by antibiotics screening with G418.Then,standard liposome method was used to transiently transfect HEK293 cells with Na＋ channel subunit hH1 or mhH1.Macroscopic Na＋ currents were recorded by using patch-clamp technique in whole cell mode.Data acquisition and analysis,generation of voltage commands and curve fitting were accomplished with pClamp 8.0 software（Axon Instruments）.Results No.293 cell line that stably expresses Na＋channel β1-subunit was established and positive colonies were selected with G418.Cells expressing the construct appeared green under UV epifluorescence.After transient transfection with WT subunit,large Na＋current from stable β1-cell line was recorded.No detectable current was observed in the absence of subunit transfection.However,after transient transfection with K317N subunit,and Na＋current had not been recorded anymore from stable β1-cell line.Conclusion Compared with normal Na＋ channel,the wild-type channel exhibited a similar sodium current.The characteristic kinetics of sodium channel of WT-hH1 is same as normal cardiac muscle cell.The missense mutaion（K317N） in P-Loop region of domain I may be the cause responsible for failure of expression of sodium channel.