Dynamitin affects cell-surface expression of voltage-gated sodium channel Na_v1.5

The major cardiac voltage-gated sodium channel Na_v1.5 associates with proteins that regulate its biosynthesis, localization, activity and degradation. Identification of partner proteins is crucial for a better understanding of the channel regulation. Using a yeast two-hybrid screen, we identified dynamitin as a Na_v1.5-interacting protein. Dynamitin is part of the microtubule-binding multiprotein complex dynactin. When overexpressed it is a potent inhibitor of dynein/kinesin-mediated transport along the microtubules by disrupting the dynactin complex and dissociating cargoes from microtubules. The use of deletion constructs showed that the C-terminal domain of dynamitin is essential for binding to the first intracellular interdomain of Na_v1.5. Co-immunoprecipitation assays confirmed the association between Na_v1.5 and dynamitin in mouse heart extracts. Immunostaining experiments showed that dynamitin and Na_v1.5 co-localize at intercalated discs of mouse cardiomyocytes. The whole-cell patch-clamp technique was applied to test the functional link between Na_v1.5 and dynamitin. Dynamitin overexpression in HEK-293 (human embryonic kidney 293) cells expressing Na_v1.5 resulted in a decrease in sodium current density in the membrane with no modification of the channel-gating properties. Biotinylation experiments produced similar information with a reduction in Na_v1.5 at the cell surface when dynactin-dependent transport was inhibited. The present study strongly suggests that dynamitin is involved in the regulation of Na_v1.5 cell-surface density.