正常血钾型周期性麻痹SCN4A基因R675Q新突变细胞模型的建立及初步研究  

The construction and preliminary investigation of the cell model of a novel nmtation R675Q in the SCN4A gene identified in a Chinese family with normokalemic periodic paralysis

作　　者：吴磊[1] 吴卫平[2] 颜光涛[2] 王晓辉[2] 刘洁晓[2] 

机构地区：[1]浙江大学医学院附属第二医院神经内科,杭州310006 [2]解放军总医院神经内科

出　　处：《中华医学遗传学杂志》2008年第6期629-632,共4页Chinese Journal of Medical Genetics

基　　金：国家自然科学基金（30370495）

摘　　要：目的建立一个正常血钾型周期性麻痹（normokalemic periodic paralysis，normoKPP）R675Q新突变的细胞模型并对其通道电流进行初步研究。方法用编码野生型人骨骼肌钠离子通道α亚单位基因（skeletal muscle Nachannel type 4 alpha subunitgene，SCN4A）的cDNA为模板，PCR技术定点突变后磷酸钙沉淀法瞬时转染突变质粒至293细胞，逆转录PCR和Western印迹验证目的基因和蛋白的表达；膜片钳技术全细胞记录野生和突变型钠通道电流。结果突变经测序得以证实；转染后24h和48h，突变通道的基因及蛋白表达量显著增高；相同的测试电压下R675Q钠通道相对电流在到达峰值前小于野生型钠通道，到达峰值后大于野生型钠通道；R675Q和野生型钠通道均在0mV测试电压刺激下到达峰值。结论成功建立了正常血钾型周期性麻痹R675Q新突变的细胞模型；R675Q突变同时提高了通道的激活和失活，其所在的S4区域可能和患者发作性力弱的症状密切相关。Objective To construct and investigate the cell model of a novel mutation R675Q in the skeletal muscle Na channel type 4 alpha subunit gene （ SCN4A ） identified from a Chinese family with normokalemic periodic paralysis. Methods cDNA encoding the adult isoform of SCN4A was used as a template for in v/tro site-directed mutagenesis by PCR method. The mutated plasmid was transiently transfected into HEK-293 cells by calcium phosphate pre- cipitation. Twenty four and 48 hours after transfection, the expression level of SCN4A was detected by reverse transerip- tion-polymerase chain reaction （RT-PCR） and Western blot. Whole cell voltage-clamp recording was used to study the current of sodium channels. Results The site-mutagenesis of the plasmid was confirmed by sequencing. The expression ofSC2C4A gene was significantly elevated 24 h and 48 h after transfection. The relative current of R675Q is smaller than that of wide type before reaching peak current under the same test voltage, but larger than that of wild type current after reaching peak current. They both had the largest peak current under 0 mV test pulse. Conclusion A cell model of normokalemic periodic paralysis was successfully constructed. The R675Q mutation of the SCN4A gene enhances the ac- tivation and inactivation of the sodium channel, and the S4 transmembrane setpnent may have intimate relationship with the attack of weakness in normoKPP patients.

关 键 词：正常血钾型周期性麻痹 细胞模型 膜片钳 

分 类 号：R686[医药卫生—骨科学]