Dravet综合征相关NaV1．1（F1237fsX1269）截短突变对钠通道膜蛋白表达和功能的影响  

Influence of truncated mutation of Dravet syndrome associated NaV1.1 （F1237fsX1269） in membrane protein expressions

作　　者：徐海清[1] 蔡秀曲 余璐[1] 汪洁[1] 廖卫平[1] 石奕武[1] 高玫梅[1] 汤斌[1] 

机构地区：[1]广州医科大学附属第二医院神经致病基因与离子通道病省部共建教育部重点实验室,广州510260

出　　处：《中华神经医学杂志》2015年第12期1266-1270,共5页Chinese Journal of Neuromedicine

基　　金：广东省教育厅育苗工程项目（2013LYM_0071）

摘　　要：目的研究Dravet综合征相关Ⅰ型电压依赖性钠通道（NaV1．1）截短突变体F1237fsXl269蛋白在HEK293T细胞中总蛋白及膜蛋白的表达水平变化，探讨NaY1．1截短突变的可能致病机制。方法以质粒pCMV—SCN1A-WT为模板，构建带有增强型黄色荧光蛋白（EYFP）融合表达的野生型与截短突变质粒（分别标记为pCMV-EYFP-SCN1A-WT、pCMV-EYFP-3710），并转染至HEK293T细胞，48h后运用生物素标记方法提取总蛋白与膜蛋白，应用Westernblotting检测野生型蛋白与截短突变蛋白的总蛋白及膜蛋白表达水平。应用全细胞膜片钳技术记录野生型蛋白与截短突变蛋白所产生的钠电流。结果Westernblotting检测结果显示：pCMV-EYFP-3710质粒转染HEK293T细胞后可检测到相应截短突变蛋白，相对分子质量约为191000，且均可在细胞胞浆及胞膜上表达。截短突变蛋白的总蛋白表达水平与野生型蛋白相比明显减少，膜蛋白表达水平只有野生型蛋白的43％，差异有统计学意义（P〈0．05）。电生理检测结果显示：野生型蛋白可检测到正常钠离子电流[电流密度为（-149．0±7．7）pA／pF]，而截短突变蛋白未检测到钠离子电流。结论Dravet综合征相关NaV1．1截短突变体F1237fsXl269产生的截短突变蛋白的膜蛋白表达水平降低，并丧失钠离子通道功能，其机制可能与野生型蛋白竞争β1、β2亚基而产生显性负效应有关。Objective To investigate the total protein and surface protein expression levels of Dravet syndrome associated NaV1.1 truncated mutant F1237fsX1269 protein in HEK293T cells, and explore the potential pathogenic mechanism of NaV1.1 truncated mutation. Methods The wide-type plasmids （pCMV- EYFP-SCN1A-WT） and trancated mutation plasmids （pCMV-EYFP-3710） expressed EYFP were constructed on the templates of pCMV-SCN1A-WT; and then, these plasmids were transfected into the HEK293T cells; 48 h after that, the input and surface proteins were extracted. Western blotting was employed to detect the surface protein expression; whole cell patch-clamp technique was used to record the sodium current created by pCMV- EYFP-SCN1A-WT and pCMV-EYFP-3710 proteins. Results Western blotting indicated that the truncated mutation proteins could be noted in the pCMV-EYFP-3710NaV1.1 （F1237fsX1269） HEK293T cells, with the relative molecular mass reaching to 191 000; they could express in the cytoplasm and membrane of pCMV-EYFP-3710NaV1.1 （F1237fsX1269） HEK293T cells. Total protein expression in the pCMV-EYFP-3710NaVl.1 （F1237fsX1269） HEK293T cells was obviously deceased as compared with that in the pCMV- EYFP-SCN1A-WT HEK293T cells （P〈0.05）, and the level of surface protein expression in the pCMV-EYFP-3710NaVl.1 （F1237fsX1269） HEK293T cells only enjoyed 43 % of that in the pCMV- EYFP-SCN1A-WT HEK293T cells （P〈0.05）. Normal sodium current created by pCMV- EYFP-SCN1A-WT protein （electric current density： [-149.0±7.7] pA/pF） was noted, while no in the pCMV-EYFP-3710 proteins Conclusion The reduction of surface expression of F1237fsX1269 truncated protein indicates impaired trafficking, but the residual surface expression may have potential pathogenicity by dominant negative effect.

关 键 词：DRAVET综合征 SCN1A基因 Ⅰ型电压依赖性钠通道 截短突变 膜蛋白 

分 类 号：R742.1[医药卫生—神经病学与精神病学]