SAP97 and Dystrophin Macromolecular Complexes Determine Two Pools of Cardiac Sodium Channels Na _v 1.5 in Cardiomyocytes

Abstract

Rationale:The cardiac sodium channel Na v 1.5 plays a key role in excitability and conduction. The 3 last residues of Na v 1.5 (Ser-Ile-Val) constitute a PDZ-domain binding motif that interacts with the syntrophin-dystrophin complex. As dystrophin is absent at the intercalated discs, Na v 1.5 could potentially interact with other, yet unknown, proteins at this site.Objective: The aim of this study was to determine whether Na v 1.5 is part of distinct regulatory complexes at lateral membranes and intercalated discs. Methods and Results:Immunostaining experiments demonstrated that Na v 1.5 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 Na v 1.5 at lateral membranes of mdx cardiomyocytes. Pull-down experiments revealed that the MAGUK protein SAP97 also interacts with the SIV motif of Na v 1.5, 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 Na v 1.5 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 I Na generated by Na v 1.5 channels lacking the SIV motif was also reduced. Finally, surface expression of Na v 1.5 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 Na v 1.5 channels in cardiomyocytes: one targeted at lateral membranes by the syntrophin-dystrophin complex, and one at intercalated discs by SAP97. (Circ Res. 2011;108:294-304.) Key Words: sodium channel Ⅲ Na v 1.5 Ⅲ MAGUK proteins Ⅲ SAP97 Ⅲ dystrophin T he cardiac sodium channel Na v 1.5 initiates the cardiac action potential, thus playing a key role in cardiac excitability and impulse propagation. The physiological importance of this channel is illustrated by numerous cardiac pathologies caused by hundreds of mutations identified in SCN5A, the gene encoding Na v 1.5. 1 The Na v 1.5 channel is composed of one 220-kDa ␣-subunit that constitutes a functional channel, and 30-kDa ␤-subunits. In addition to these accessory ␤-subunits, several proteins have been shown to regulate and interact with Na v 1.5. 1,2 In most cases, the physiological relevance of these interactions is poorly understood, mainly because of a lack of appropriate animal models. Many of the interacting proteins bind to the C terminus of Na v 1.5, where several protein-protein interaction motifs are located. 1,2 We have shown that the ubiquitin-protein ligase Nedd4-2 binds the PY motif of Na v 1.5 and reduces the sodium current (I Na ) in HEK293 cells by promoting its internalization. 3 We have also demonstrated that Na v 1.5 associates with the dystrophin-syntrophin multiprotein complex (DMC) in cardiac cells. 4 In dystrophin-deficient mice (m...

The Interaction of Caveolin 3 Protein with the Potassium Inward Rectifier Channel Kir2.1

“…This is derived from this work and recent work by Abriel and colleagues (27), who demonstrated that Na V 1.5 associates with syntrophin and SAP97, Makielski and colleagues (5,23), who established that Na V 1.5 coimmunoprecipitates with Cav3 and syntrophin, Vandenberg and colleagues, who showed that Kir2.1 coimmunoprecipitates with syntrophin (11), and Anumonwo and colleagues (28), who assert that Kir2.1 associates with SAP97. This is also in agreement with a recent study by Jalife and colleagues (29), who propose reciprocal modulation of Kir2.1 and Na V 1.5 and suggest that these ion channels are in the same macromolecular complex.…”

SAP97 promotes the stability of Nax channels at the plasma membrane

“…Among voltage-gated sodium channels, Na v 1.5 has also been reported to interact with SAP97 via its C-terminal PDZ-binding motif, and the amount of Na v 1.5 on the cell surface is decreased in SAP97-silenced myocytes, although the mechanism remains unclear [17].…”

Cardiomyocyte ATP release through pannexin 1 aids in early fibroblast activation

“…Originally described in the brain at neuronal synapses (75), there is increasing evidence that these scaffolds play a similar role in the heart (1,7,27,39,45,46,53,61,63,81). It has been shown that interaction of cardiac ion channels with SAP97 occurs with a number of potassium channels including Kv1.5, Kv4, Kir2.2 and 2.3, and the hyperpolarization-activated, cyclic nucleotidegated channels (1,7,27,45,46,61,81).…”