Readthrough of nonsense mutation W822X in the SCN5A gene can effectively restore expression of cardiac Na+ channels W822X
10.3760/cma.j.issn.0253-3758.2011.03.015
- VernacularTitle:心脏钠通道无义突变的分子拯救部分恢复钠通道的表达和功能
- Author:
Jing-Tao ZHANG
1
;
Jian HUANG
;
Si-Yong TENG
;
Rong-Rong WANG
;
Yin-Hui ZHANG
;
Jie-Lin PU
;
Ru-Tai HUI
;
Shu ZHANG
Author Information
1. 中国医学科学院,北京协和医学院,阜外心血管病医院
- Keywords:
Sodium channels;
Eukaryotic cells;
RNA interference;
Mutation
- From:
Chinese Journal of Cardiology
2011;39(3):238-241
- CountryChina
- Language:Chinese
-
Abstract:
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.
