SAP97 and dystrophin macromolecular complexes determine two pools of cardiac sodium channels Nav1.5 in cardiomyocytes

Rationale: The cardiac sodium channel Nav1.5 plays a key role in excitability and conduction. The 3 last residues of Nav1.5 (Ser-Ile-Val) constitute a PDZ-domain binding motif that interacts with the syntrophin-dystrophin complex. As dystrophin is absent at the intercalated discs, Nav1.5 could potentially interact with other, yet unknown, proteins at this site. Objective: The aim of this study was to determine whether Nav1.5 is part of distinct regulatory complexes at lateral membranes and intercalated discs. Methods and Results: Immunostaining experiments demonstrated that Nav1.5 localizes at lateral membranes of cardiomyocytes with dystrophin and syntrophin. Optical measurements on isolated dystrophin-deficient mdx hearts revealed significantly reduced conduction velocity, accompanied by strong reduction of Nav1.5 at lateral membranes of mdx cardiomyocytes. Pull-down experiments revealed that the MAGUK protein SAP97 also interacts with the SIV motif of Nav1.5, an interaction specific for SAP97 as no pull-down could be detected with other cardiac MAGUK proteins (PSD95 or ZO-1). Furthermore, immunostainings showed that Nav1.5 and SAP97 are both localized at intercalated discs. Silencing of SAP97 expression in HEK293 and rat cardiomyocytes resulted in reduced sodium current (I_Na) measured by patch-clamp. The INa generated by Nav1.5 channels lacking the SIV motif was also reduced. Finally, surface expression of Nav1.5 was decreased in silenced cells, as well as in cells transfected with SIV-truncated channels. Conclusions: These data support a model with at least 2 coexisting pools of Nav1.5 channels in cardiomyocytes: one targeted at lateral membranes by the syntrophin-dystrophin complex, and one at intercalated discs by SAP97.