title: Dynamitin affects cell-surface expression of voltage-gated sodium channel Nav1.5.
abstract: The major cardiac voltage-gated sodium channel Nav1.5 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 Nav1.5-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 Nav1.5. Co-immunoprecipitation assays confirmed the association between Nav1.5 and dynamitin in mouse heart extracts. Immunostaining experiments showed that dynamitin and Nav1.5 co-localize at intercalated discs of mouse cardiomyocytes. The whole-cell patch-clamp technique was applied to test the functional link between Nav1.5 and dynamitin. Dynamitin overexpression in HEK-293 (human embryonic kidney 293) cells expressing Nav1.5 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 Nav1.5 at the cell surface when dynactin-dependent transport was inhibited. The present study strongly suggests that dynamitin is involved in the regulation of Nav1.5 cell-surface density.
authors.forename: Benoît
authors.initials: B
authors.lastname: Chatin
authors.forename: Pauline
authors.initials: P
authors.lastname: Colombier
authors.forename: Anne Laure
authors.initials: AL
authors.lastname: Gamblin
authors.forename: Marie
authors.initials: M
authors.lastname: Allouis
authors.forename: Françoise
authors.initials: F
authors.lastname: Le Bouffant
mesh_terms: D000818:Animals; D001665:Binding Sites; D000072159:Dynactin Complex; D057809:HEK293 Cells; D006801:Humans; D008807:Mice, Inbred BALB C; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000378:metabolism; D062554:NAV1.5 Voltage-Gated Sodium Channel / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D020798:Two-Hybrid System Techniques
chemical_list: C000606361:DCTN2 protein, human; C000606365:Dctn2 protein, mouse; D000072159:Dynactin Complex; D008869:Microtubule-Associated Proteins; D062554:NAV1.5 Voltage-Gated Sodium Channel; C568320:SCN5A protein, human