全面性癫痫伴热性惊厥附加症SCN1A基因G302D突变的膜片钳电生理研究

Functional Analysis of a Novel SCN1A Mutation G302D Identified in GEFS+

目的:探讨全面性癫痫伴热性惊厥附加症(GEFS+)SCN1A基因G302D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G302D的pCMV-SCN1A的表达载体,lipo2000脂质体转染法共转染pCMV-SCN1A-G302D质粒和pCD8-IRES-SCN1B质粒到HEK-293细胞系,进行全细胞膜片钳电生理实验记录Navl.1通道电流及动力学参数,由pClamp10.0以及OriginPro8.0软件分析。结果:SCN1A-G302D突变体与野生型相比,电流密度降低,激活速度减慢,失活后恢复时间延长。失活参数两者相比没有显著性统计学差异。结论:SCN1A基因G302D突变导致Navl.1通道功能部分丧失,可能是导致GEFS+的病因。

Objective: To elucidate the molecular and electrophysiological mechanisms of generalized epilepsy with febrile seizures plus through functional analysis of a novel SCN1A gene mutation G302D. Methods: A recombinant plasmid pCMV-SCN1A containing the mutant G302 D was constructed by the PCR-based site-directed mutagenesis technique. Lipofectamine 2000 was used to co-transfect the plasmid pCMV-SCN1A-G302D and pCD8-IRES-SCN1B into HEK-293 cells line to induce stable expression of α1 and β1 subunit of Na+channel. During patch clamp detection, the standard stimulus was given in the whole-cell voltage clamp mode. Data acquisition and analysis were accomplished with PatchMaster10.0 and OriginPro8.0. Results: SCN1A-G302D mutants displayed a decreased current density, a slower activation and a significantly delayed recovery from inactivation. There were no significantly differences in the steady-state inactivation between two groups. Conclusions: These data indicates that SCN1A-G302D mutants result in partial loss of function, which may be the cause of GEFS +.