Readthrough of nonsense mutation W822X in the SCN5A gene can effectively restore expression of cardiac Na+ channels W822X / 中华心血管病杂志
Chinese Journal of Cardiology
; (12): 238-241, 2011.
Article
in Zh
| WPRIM
| ID: wpr-272270
Responsible library:
WPRO
ABSTRACT
<p><b>OBJECTIVE</b>In this study we investigated the functional restoration of nonsense mutations in the SCN5A gene.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>
Full text:
1
Database:
WPRIM
Main subject:
Plasmids
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Transfection
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Sodium Channels
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Patch-Clamp Techniques
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Codon, Nonsense
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RNA, Small Interfering
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HEK293 Cells
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Genetics
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Metabolism
Limits:
Humans
Language:
Zh
Journal:
Chinese Journal of Cardiology
Year:
2011
Document type:
Article