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...

Peripheral neuropathic pain is a disabling condition resulting from nerve injury. It is characterized by the dysregulation of voltage-gated sodium channels (Na v s) expressed in dorsal root ganglion (DRG) sensory neurons. The mechanisms underlying the altered expression of Na v s remain unknown. This study investigated the role of the E3 ubiquitin ligase NEDD4-2, which is known to ubiquitylate Na v s, in the pathogenesis of neuropathic pain in mice. The spared nerve injury (SNI) model of traumatic nerve injury-induced neuropathic pain was used, and an Na v 1.7-specific inhibitor, ProTxII, allowed the isolation of Na v 1.7-mediated currents. SNI decreased NEDD4-2 expression in DRG cells and increased the amplitude of Na v 1.7 and Na v 1.8 currents. The redistribution of Na v 1.7 channels toward peripheral axons was also observed. Similar changes were observed in the nociceptive DRG neurons of Nedd4L knockout mice (SNS-Nedd4L -/-). SNS-Nedd4L -/-mice exhibited thermal hypersensitivity and an enhanced second pain phase after formalin injection. Restoration of NEDD4-2 expression in DRG neurons using recombinant adenoassociated virus (rAAV2/6) not only reduced Na v 1.7 and Na v 1.8 current amplitudes, but also alleviated SNI-induced mechanical allodynia. These findings demonstrate that NEDD4-2 is a potent posttranslational regulator of Na v s and that downregulation of NEDD4-2 leads to the hyperexcitability of DRG neurons and contributes to the genesis of pathological pain.

T he cardiac action potential (AP) is initiated by the Na + channel Na V 1.5, an established key element for cardiac excitability and impulse propagation. The importance of Na V 1.5 is exemplified by the myriad of cardiac disorders caused by hundreds of mutations identified in SCN5A, the gene coding for Na V 1.5.1 For some SCN5A mutation carriers, cardiac conduction slowing or block, secondary to reduced Na + channel function, predisposes them to ventricular arrhythmias and sudden cardiac death. Editorial see p 132 Clinical Perspective on p 160The cardiac Na + channel is composed of a 220-kDa α-subunit, Na V 1.5, constituting the pore of the channel, which is known to associate with four ≈30-kDa β-subunits. Recent studies have demonstrated that many proteins interact with and regulate Na V 1.5. 2 The physiological relevance of these interactions, however, is poorly understood, mainly due to a lack of in vivo studies. Many protein-protein interaction motifs for these regulatory proteins are located at the C-terminus of Na V 1.5. 2 In particular, we have previously demonstrated that Na V 1.5 associates with the dystrophinsyntrophin multiprotein complex (DMC) in cardiac cells.3 InBackground-Sodium channel Na V 1.5 underlies cardiac excitability and conduction. The last 3 residues of Na V 1.5 (Ser-IleVal) constitute a PDZ domain-binding motif that interacts with PDZ proteins such as syntrophins and SAP97 at different locations within the cardiomyocyte, thus defining distinct pools of Na V 1.5 multiprotein complexes. Here, we explored the in vivo and clinical impact of this motif through characterization of mutant mice and genetic screening of patients. Methods and Results-To investigate in vivo the regulatory role of this motif, we generated knock-in mice lacking the SIV domain (∆SIV). ∆SIV mice displayed reduced Na V 1.5 expression and sodium current (I Na ), specifically at the lateral myocyte membrane, whereas Na V 1.5 expression and I Na at the intercalated disks were unaffected. Optical mapping of ∆SIV hearts revealed that ventricular conduction velocity was preferentially decreased in the transversal direction to myocardial fiber orientation, leading to increased anisotropy of ventricular conduction. Internalization of wild-type and ΔSIV channels was unchanged in HEK293 cells. However, the proteasome inhibitor MG132 rescued ΔSIV I Na , suggesting that the SIV motif is important for regulation of Na V 1.5 degradation. A missense mutation within the SIV motif (p.V2016M) was identified in a patient with Brugada syndrome. The mutation decreased Na V 1.5 cell surface expression and I Na when expressed in HEK293 cells. Conclusions-Our results demonstrate the in vivo significance of the PDZ domain-binding motif in the correct expression of Na V 1.5 at the lateral cardiomyocyte membrane and underline the functional role of lateral Na V 1.5 in ventricular conduction. Recherche 1087, L'Institut du Thorax, Nantes, France (R.R.); Centre National de la Recherche Scientifique Unité Mixte de Recherche 6291, Nantes, France...

In the present study, we report the first pathogenic mutation in the CACNA2D1 gene in humans, which causes a new variant of SQTS. It remains to be determined whether mutations in this gene lead to other manifestations of the J-wave syndrome.

Utrophin plays a central role in the regulation of Na(v)1.5 in mdx mice. These findings provide support for therapeutic strategies aimed at overexpressing utrophin in the hopes of reducing cardiac pathology in DMD patients.

Neuronal precursor cell-expressed developmentally down-regulated 4 (Nedd4) proteins are ubiquitin ligases, which attach ubiquitin moieties to their target proteins, a post-translational modification that is most commonly associated with protein degradation. Nedd4 ubiquitin ligases have been shown to down-regulate both potassium and sodium channels. In this study, we investigated whether Nedd4 ubiquitin ligases also regulate Cav calcium channels.We expressed three Nedd4 family members, Nedd4-1, Nedd4-2, and WWP2, together with Cav1.2 channels in tsA-201 cells. We found that Nedd4-1 dramatically decreased Cav whole-cell currents, whereas Nedd4-2 and WWP2 failed to regulate the current. Surface biotinylation assays revealed that Nedd4-1 decreased the number of channels inserted at the plasma membrane. Western blots also showed a concomitant decrease in the total expression of the channels. Surprisingly, however, neither the Cav pore-forming α1 subunit nor the associated Cavβ and Cavα2δ subunits were ubiquitylated by Nedd4-1. The proteasome inhibitor MG132 prevented the degradation of Cav channels, whereas monodansylcadaverine and chloroquine partially antagonized the Nedd4-1-induced regulation of Cav currents. Remarkably, the effect of Nedd4-1 was fully prevented by brefeldin A. These data suggest that Nedd4-1 promotes the sorting of newly synthesized Cav channels for degradation by both the proteasome and the lysosome. Most importantly, Nedd4-1-induced regulation required the co-expression of Cavβ subunits, known to antagonize the retention of the channels in the endoplasmic reticulum. Altogether, our results suggest that Nedd4-1 interferes with the chaperon role of Cavβ at the endoplasmic reticulum/Golgi level to prevent the delivery of Cav channels at the plasma membrane.

No abstract

Cardiac ion channels play an essential role in the generation of the action potential of cardiomyocytes. Over the past 15 years, a new field of research called channelopathies has emerged; it regroups all diseases caused by ion channel dysfunction. Investigators have largely determined the physiological roles of cardiac ion channels, but little is known about the molecular determinants of their regulation. Two posttranslational mechanisms that are crucial in determining the fate of proteins are the ubiquitylation and the SUMOylation pathways, which lead to the degradation and/or regulation of modified proteins. Recently, several groups have investigated the physiological impacts of these mechanisms on the regulation of different classes of cardiac ion channels. The objective of this review was to summarize and briefly discuss these results.