Cardiac Sodium Channel Na _v 1.5 Is Regulated by a Multiprotein Complex Composed of Syntrophins and Dystrophin

Abstract

Abstract-The cardiac sodium channel Na v 1.5 plays a key role in cardiac excitability and conduction. The purpose of this study was to elucidate the role of the PDZ domain-binding motif formed by the last three residues (Ser-Ile-Val) of the Na v 1.5 C-terminus. Pull-down experiments were performed using Na v 1.5 C-terminus fusion proteins and human or mouse heart protein extracts, combined with mass spectrometry analysis. These experiments revealed that the C-terminus associates with dystrophin, and that this interaction was mediated by alpha-and beta-syntrophin proteins.Truncation of the PDZ domain-binding motif abolished the interaction. We used dystrophin-deficient mdx 5cv mice to study the role of this protein complex in Na v 1.5 function. Western blot experiments revealed a 50% decrease in the Na v 1.5 protein levels in mdx 5cv hearts, whereas Na v 1.5 mRNA levels were unchanged. Patch-clamp experiments showed a 29% decrease of sodium current in isolated mdx 5cv cardiomyocytes. Finally, ECG measurements of the mdx 5cv mice exhibited a 19% reduction in the P wave amplitude, and an 18% increase of the QRS complex duration, compared with controls. These results indicate that the dystrophin protein complex is required for the proper expression and function of Na v 1.5. In the absence of dystrophin, decreased sodium current may explain the alterations in cardiac conduction observed in patients with dystrophinopathies. Key Words: Duchenne dystrophy Ⅲ dystrophin Ⅲ ECG Ⅲ mouse Ⅲ sodium channels Ⅲ syntrophin T he main cardiac voltage-gated sodium channel, Na v 1.5, generates the fast depolarization of the cardiac action potential, and plays a key role in cardiac conduction. Its importance for normal cardiac function has been exemplified by the description of numerous naturally occurring genetic variants of the gene SCN5A, which encodes Na v 1.5, that are linked to various cardiac diseases. 1 Among them, the congenital long QT syndrome type-3 and the Brugada syndrome are caused by gain or loss-of-function of Na v 1.5, respectively. 1 Na v 1.5 is the pore-forming ␣-subunit protein of the cardiac sodium channel. It has a molecular weight of Ϸ220 kDa, and may be associated with at least 4 types of auxiliary small (30 to 35 kDa) ␤-subunits. Recently, several proteins that bind directly to Na v 1.5 have been described. 2 However, in most cases the physiological relevance of these interactions remains poorly understood, mainly because of a lack of appropriate animal models. With the exception of ankyrin-G, 3 all these partner proteins interact with the 243-residues-long intracellular C-terminal domain of the channel which contains several protein-protein interaction motifs. 2 Among them, the last three residues of Na v 1.5 (2014-Ser-Ile-Val-2016) constitute a PDZ domain-binding motif to which syntrophins and dystrophin have been shown to interact directly or indirectly, respectively. 4 -6 However, thus far, the role of these interacting proteins in the heart has never been investigated.In this study, by performing mass spe...

Regulation of the Cardiac Sodium Channel Nav1.5 by Utrophin in Dystrophin Deficient Mice

“…In order to verify that the decrease in functional Na v 1.5 found in mdx5cv was not strain-specific, we assessed the sodium peak current in mdx mice. We found a decrease similar to that previously observed in mdx5cv, 11 suggesting that down-regulation of the channel in the absence of dystrophin is a general phenomenon in mice and occurs despite utrophin over-expression. 4.3 Consequences of decreased Na v 1.5 protein and sodium current in DKO mice…”

“…11 The direct interaction between Na v 1.5 and syntrophins takes place at the last three Cterminal amino acids (SIV) of Na v 1.5, a PDZ domain-binding motif. 12 Utrophin, a homologue protein of dystrophin, is also present in the DMC 16 and was similarly shown to interact with syntrophins.…”

“…11,12 We demonstrated that this interaction occurs via the PDZ domain-binding motif (SIV) of Na v 1.5 and syntrophins. 11 There are five members (a 1 , b 1 , b 2 , g 1 , and g 2 ) of the syntrophin family that are differentially distributed in skeletal muscle and other tissues.…”

Sodium Channel Remodeling in Subcellular Microdomains of Murine Failing Cardiomyocytes

“…In the intercalated disc (ID) region, Na V 1.5 colocalizes with N‐cadherin, ankyrin G, plakophilin‐2, and synapse‐associated protein 97, whereas Na V 1.5‐based channels located at the lateral membrane (LM) of the cardiomyocyte associate primarily with the dystrophin‐syntrophin complex 8, 9. Remodeling of region‐specific proteins may affect Na V 1.5 locally and lead to disturbances in I Na , conduction abnormalities, and arrhythmias 10, 11, 12, 13…”

Development of a Patient-Derived Induced Pluripotent Stem Cell Model for the Investigation of <i>SCN5A</i>-D1275N-Related Cardiac Sodium Channelopathy

“…Furthermore, several proteins reportedly interact with and regulate the expression of NaV1.5 protein in CMs; MOG1 and ankyrin-G are associated with NaV1.5 cell surface expression, 28,29 Nedd4-2 catalyzes NaV1.5 ubiquitination 30 and degradation, 27 and dystrophin mediates NaV1.5 stability via syntrophin adaptor proteins. 31 Moreover, a study in a HEK 293 system showed that the neuronal sodium channel protein, NaV1.8, encoded by SCN10A, participates in NaV1.5 regulation via direct protein-protein interactions. 32 In this study, the reduced expression of NaV1.5 in D1275N-hiPSC-CMs was restored by the proteasome inhibitor, MG132, treatment suggesting that ubiquitin-dependent proteolysis might be the major underlying mechanism resulting in NaV1.5 loss-of-function in D1275N channels.…”