Construction and verification of R104W mutant of SCN5a sodium channel

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.