Influence of truncated mutation of Dravet syndrome associated NaV1.1 (F1237fsX1269) in membrane protein expressions

Abstract

Objective To investigate the total protein and surface protein expression levels of Dravet syndrome associated NaV1.1 truncated mutant F1237fsX1269 protein in HEK293T cells, and explore the potential pathogenic mechanism of NaV1.1 truncated mutation. Methods The wide-type plasmids (pCMV-EYFP-SCN1A-WT) and truncated mutation plasmids (pCMV-EYFP-3710) expressed EYFP were constructed on the templates of pCMV-SCN1A-WT; and then, these plasmids were transfected into the HEK293T cells; 48 h after that, the input and surface proteins were extracted. Western blotting was employed to detect the surface protein expression; whole cell patch-clamp technique was used to record the sodium current created by pCMV-EYFP-SCN1A-WT and pCMV-EYFP-3710 proteins. Results Western blotting indicated that the truncated mutation proteins could be noted in the pCMV-EYFP-3710NaV1.1 (F1237fsX1269) HEK293T cells, with the relative molecular mass reaching to 191 000; they could express in the cytoplasm and membrane of pCMV-EYFP-3710NaV1.1 (F1237fsX1269) HEK293T cells. Total protein expression in the pCMV-EYFP-3710NaV1.1 (F1237fsX1269) HEK293T cells was obviously deceased as compared with that in the pCMV-EYFP-SCN1A-WT HEK293T cells (P<0.05), and the level of surface protein expression in the pCMV-EYFP-3710NaV1.1 (F1237fsX1269) HEK293T cells only enjoyed 43 % of that in the pCMV-EYFP-SCN1A-WT HEK293T cells (P<0.05). Normal sodium current created by pCMV-EYFP-SCN1A-WT protein (electric current density: [-149.0±7.7] pA/pF) was noted, while no in the pCMV-EYFP-3710 proteins Conclusion The reduction of surface expression of F1237fsX1269 truncated protein indicates impaired trafficking, but the residual surface expression may have potential pathogenicity by dominant negative effect. Key words: Dravet syndrome; SCN1A; NaV1.1; Truncated mutation; Surface protein