Voltage-gated Na v channels are required for normal electrical activity in neurons, skeletal muscle, and cardiomyocytes. In the heart, Na v 1.5 is the predominant Na v channel, and Na v 1.5-dependent activity regulates rapid upstroke of the cardiac action potential. Na v 1.5 activity requires precise localization at specialized cardiomyocyte membrane domains. However, the molecular mechanisms underlying Na v channel trafficking in the heart are unknown. In this paper, we demonstrate that ankyrin-G is required for Na v 1.5 targeting in the heart. Cardiomyocytes with reduced ankyrin-G display reduced Na v 1.5 expression, abnormal Na v 1.5 membrane targeting, and reduced Na + channel current density. We define the structural requirements on ankyrin-G for Na v 1.5 interactions and demonstrate that loss of Na v 1.5 targeting is caused by the loss of direct Na v 1.5–ankyrin-G interaction. These data are the first report of a cellular pathway required for Na v channel trafficking in the heart and suggest that ankyrin-G is critical for cardiac depolarization and Na v channel organization in multiple excitable tissues.

We recently showed that the COOH terminus of the cystic fibrosis transmembrane conductance regulator associates with the submembranous scaffolding protein EBP50 (ERM-binding phosphoprotein 50 kD; also called Na + /H + exchanger regulatory factor). Since EBP50 associates with ezrin, this interaction links the cystic fibrosis transmembrane conductance regulator (CFTR) to the cortical actin cytoskeleton. EBP50 has two PDZ domains, and CFTR binds with high affinity to the first PDZ domain. Here, we report that Yes-associated protein 65 (YAP65) binds with high affinity to the second EBP50 PDZ domain. YAP65 is concentrated at the apical membrane in airway epithelia and interacts with EBP50 in cells. The COOH terminus of YAP65 is necessary and sufficient to mediate association with EBP50. The EBP50–YAP65 interaction is involved in the compartmentalization of YAP65 at the apical membrane since mutant YAP65 proteins lacking the EBP50 interaction motif are mislocalized when expressed in airway epithelial cells. In addition, we show that the nonreceptor tyrosine kinase c-Yes is contained within EBP50 protein complexes by association with YAP65. Subapical EBP50 protein complexes, containing the nonreceptor tyrosine kinase c-Yes, may regulate apical signal transduction pathways leading to changes in ion transport, cytoskeletal organization, or gene expression in epithelial cells.