中华心血管病杂志
中華心血管病雜誌
중화심혈관병잡지
Chinese Journal of Cardiology
2011年
3期
238-241
,共4页
张竞涛%黄健%滕思勇%王蓉蓉%张银辉%浦介麟%惠汝太%张澍
張競濤%黃健%滕思勇%王蓉蓉%張銀輝%浦介麟%惠汝太%張澍
장경도%황건%등사용%왕용용%장은휘%포개린%혜여태%장주
钠通道%真核细胞%RNA干扰%突变
鈉通道%真覈細胞%RNA榦擾%突變
납통도%진핵세포%RNA간우%돌변
Sodium channels%Eukaryotic cells%RNA interference%Mutation
目的 研究探讨真核释放因子eRF3a的RNA干扰如何影响心脏钠通道无义突变的表达和功能.方法 构建心脏钠通道无义突变和干扰eRF3a RNA的真核表达载体,独立或共同转染HEK293细胞,全细胞膜片钳记录钠通道的表达电流和动力学,同时应用蛋白印迹和免疫荧光证明钠通道的表达和定位.结果 独立转染W822X无义突变的HEK293细胞没有检测到全长钠通道蛋白,表达的钠电流水平很低,仅相当野生型钠电流3%.共同转染HEK293细胞可以检测到全长钠通道蛋白,表达的钠电流水平明显增加,相当于野生型钠电流30%.免疫荧光也显示,独立转染W822X无义突变的HEK293细胞没有出现荧光细胞,而共同转染的HEK293细胞出现较多的荧光细胞.结论 真核释放因子eRF3a的RNA干扰促进心脏钠通道无义突变的翻译,部分恢复钠通道全长表达和功能.
目的 研究探討真覈釋放因子eRF3a的RNA榦擾如何影響心髒鈉通道無義突變的錶達和功能.方法 構建心髒鈉通道無義突變和榦擾eRF3a RNA的真覈錶達載體,獨立或共同轉染HEK293細胞,全細胞膜片鉗記錄鈉通道的錶達電流和動力學,同時應用蛋白印跡和免疫熒光證明鈉通道的錶達和定位.結果 獨立轉染W822X無義突變的HEK293細胞沒有檢測到全長鈉通道蛋白,錶達的鈉電流水平很低,僅相噹野生型鈉電流3%.共同轉染HEK293細胞可以檢測到全長鈉通道蛋白,錶達的鈉電流水平明顯增加,相噹于野生型鈉電流30%.免疫熒光也顯示,獨立轉染W822X無義突變的HEK293細胞沒有齣現熒光細胞,而共同轉染的HEK293細胞齣現較多的熒光細胞.結論 真覈釋放因子eRF3a的RNA榦擾促進心髒鈉通道無義突變的翻譯,部分恢複鈉通道全長錶達和功能.
목적 연구탐토진핵석방인자eRF3a적RNA간우여하영향심장납통도무의돌변적표체화공능.방법 구건심장납통도무의돌변화간우eRF3a RNA적진핵표체재체,독립혹공동전염HEK293세포,전세포막편겸기록납통도적표체전류화동역학,동시응용단백인적화면역형광증명납통도적표체화정위.결과 독립전염W822X무의돌변적HEK293세포몰유검측도전장납통도단백,표체적납전류수평흔저,부상당야생형납전류3%.공동전염HEK293세포가이검측도전장납통도단백,표체적납전류수평명현증가,상당우야생형납전류30%.면역형광야현시,독립전염W822X무의돌변적HEK293세포몰유출현형광세포,이공동전염적HEK293세포출현교다적형광세포.결론 진핵석방인자eRF3a적RNA간우촉진심장납통도무의돌변적번역,부분회복납통도전장표체화공능.
Objective In this study we investigated the functional restoration of nonsense mutations in the SCN5A gene. Methods The readthrough-enhancing reagents were introduced to HEK293 cells to suppress one nonsense mutation W822X in the SCN5A gene. Patch-clamp was used to record the whole-cell current and dynamics. Western blot and immunofluorescence staining were used to certify the expression and the location of the sodium channel. Results In transfected HEK293 cells, the nonsense mutation in SCN5A inhibited the expression level of full-length protein, and the sodium currents from the mutant channels were less than 3% of the wild-type level. Readthrough enhancement by decreasing translation termination efficiency with a siRNA targeting eukaryotic release factor eRF3a ( a GTPase that binds eRF1 ), the sodium current from the mutant cDNAs was restored to as much as 30% of the wild-type. After the treatment by the readthrough-enhancing reagents, the channels from cDNA carrying W822X remained the features of wild-type phenotype, and Western blot and immunochemical staining also showed the expression of full-length channel proteins. Conclusion Readthrough-enhancing reagents could effectively suppress nonsense mutations in SCN5A and partially restore the function of sodium channel and the expression of full-length channels.
