OBJECTIVEIn this study we investigated the functional restoration of nonsense mutations in the SCN5A gene.

METHODSThe 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.

RESULTSIn 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.

CONCLUSIONReadthrough-enhancing reagents could effectively suppress nonsense mutations in SCN5A and partially restore the function of sodium channel and the expression of full-length channels.