Nav1.5 targeting requires direct interaction with 190-kD ankyrin-G. (A and B) Immunolocalization of ankyrin-G and Nav1.5 in control (nontransduced) and rat ankyrin-G shRNA virally transduced neonatal cardiomyocytes. Note the localization of Nav1.5 in the perinuclear region of shRNA-transduced myocytes (white arrows). Yellow arrows denote remaining ankyrin-G staining in transduced myocytes, and asterisks mark sites of complete knockdown. Virally transduced myocytes in the figure are denoted by positive YFP fluorescence (pseudocolored in blue). (C) Cardiomyocytes expressing rat-specific ankyrin-G shRNA (note the blue color denoting YFP expression) were transfected with GFP-labeled human ankyrin-G cDNA and immunolabeled with Nav1.5 and ankyrin-G antibodies. Note that human GFP–ankyrin-G restores the localization of Nav1.5 to normal (compare Nav1.5 in B and C). (D and E) GFP-human ankyrin-G mutants (R14 and R15) that lack binding activity for Nav1.5 (see Fig. 6) are unable to rescue to normal the aberrant localization of Nav1.5 (note perinuclear distribution; arrows) in myocytes stably transduced with rat ankyrin-G shRNA (note positive YFP fluorescence). Bars, 10 μm.
