西安交通大学学报（医学版）Issue(4)：435-438,443,5.DOI:10.7652/jdyxb201504002
钠通道 SCN5a 突变体 R104W 的构建及验证
Construction and verification of R104W mutant of SCN5a sodium channel
摘要
Abstract
Objective To construct and verify the R104W mutant of SCN5a channel.Methods The SCN5a-R104W mutant was constructed by rapid site-directed mutagenesis,and the expected mutation was confirmed by direct sequencing.The mutant DNA was transfected into HEK293 cells using Lipofectamine TM3000.Function of the SCN5a-R104W mutant was tested by Western blot analysis and whole-cell patch clamp recording.Results Sequencing results showed that the base on 310 was changed from C to T of SCN5a-R104W mutant DNA.Protein expression of SCN5a-R104W mutant was lower than that of wild-type SCN5a (SCN5a-WT)channel.SCN5a-WT channels were expressed on the cell surface and SCN5a-R104W channels were mainly expressed in the cytoplasm. Patch clamping result showed that no sodium current was recorded from the cells expressing SCN5a-R104W mutant channel,and SCN5a-R104W exerted dominant-negative effect on SCN5a-WT channel.Conclusion Trafficking deficient SCN5a-R104W mutant channel was successfully constructed and verified.关键词
钠通道/R104W 突变/Brugada 综合征/定点诱变Key words
sodium channel/R104W mutant/Brugada syndrome/site-directed mutagenesis分类
医药卫生引用本文复制引用
胡培静,杜媛,王娅,王亭忠,王娟娟,陈春燕,马爱群..钠通道 SCN5a 突变体 R104W 的构建及验证[J].西安交通大学学报（医学版）,2015,(4):435-438,443,5.基金项目
国家自然科学基金资助项目(No.81170175) Supported by the National Natural Science Foundation of China (No.81170175) （No.81170175）
