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.

Na _v 1.5, the cardiac isoform of the voltage-gated Na ⁺ channel, is critical to heart excitability and conduction. However, the mechanisms regulating its expression at the cell membrane are poorly understood. The Na _v 1.5 C-terminus contains a PY-motif (xPPxY) that is known to act as binding site for Nedd4/Nedd4-like ubiquitin-protein ligases. Because Nedd4-2 is well expressed in the heart, we investigated its role in the ubiquitination and regulation of Na _v 1.5. Yeast two-hybrid and GST-pulldown experiments revealed an interaction between Na _v 1.5 C-terminus and Nedd4-2, which was abrogated by mutating the essential tyrosine of the PY-motif. Ubiquitination of Na _v 1.5 was detected in both transfected HEK cells and heart extracts. Furthermore, Nedd4-2–dependent ubiquitination of Na _v 1.5 was observed. To test for a functional role of Nedd4-2, patch-clamp experiments were performed on HEK cells expressing wild-type and mutant forms of both Na _v 1.5 and Nedd4-2. Na _v 1.5 current density was decreased by 65% upon Nedd4-2 cotransfection, whereas the PY-motif mutant channels were not affected. In contrast, a catalytically inactive Nedd4-2 had no effect, indicating that ubiquitination mediates this downregulation. However, Nedd4-2 did not alter the whole-cell or the single channel biophysical properties of Na _v 1.5. Consistent with the functional findings, localization at the cell periphery of Na _v 1.5-YFP fusion proteins was reduced upon Nedd4-2 coexpression. The Nedd4-1 isoform did not regulate Na _v 1.5, suggesting that Nedd4-2 is a specific regulator of Na _v 1.5. These results demonstrate that Na _v 1.5 can be ubiquitinated in heart tissues and that the ubiquitin-protein ligase Nedd4-2 acts on Na _v 1.5 by decreasing the channel density at the cell surface.

Short QT syndrome (SQTS) is a genetically determined ion-channel disorder, which may cause malignant tachyarrhythmias and sudden cardiac death. Thus far, mutations in five different genes encoding potassium and calcium channel subunits have been reported. We present, for the first time, a novel loss-of-function mutation coding for an L-type calcium channel subunit. The electrocardiogram of the affected member of a single family revealed a QT interval of 317 ms (QTc 329 ms) with tall, narrow, and symmetrical T-waves. Invasive electrophysiological testing showed short ventricular refractory periods and increased vulnerability to induce ventricular fibrillation. DNA screening of the patient identified no mutation in previously known SQTS genes; however, a new variant at a heterozygous state was identified in the CACNA2D1 gene (nucleotide c.2264G > C; amino acid p.Ser755Thr), coding for the Ca_vα₂δ-1 subunit of the L-type calcium channel. The pathogenic role of the p.Ser755Thr variant of the CACNA2D1 gene was analysed by using co-expression of the two other L-type calcium channel subunits, Ca_v1.2α1 and Ca_vβ_2b, in HEK-293 cells. Barium currents (I_Ba) were recorded in these cells under voltage-clamp conditions using the whole-cell configuration. Co-expression of the p.Ser755Thr Ca_vα₂δ-1 subunit strongly reduced the I_Ba by more than 70% when compared with the co-expression of the wild-type (WT) variant. Protein expression of the three subunits was verified by performing western blots of total lysates and cell membrane fractions of HEK-293 cells. The p.Ser755Thr variant of the Ca_vα₂δ-1 subunit was expressed at a similar level compared with the WT subunit in both fractions. Since the mutant Ca_vα₂δ-1 subunit did not modify the expression of the pore-forming subunit of the L-type calcium channel, Ca_v1.2α1, it suggests that single channel biophysical properties of the L-type channel are altered by this variant. 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.

The movement of ions across specific channels embedded on the membrane of individual cardiomyocytes is crucial for the generation and propagation of the cardiac electric impulse. Emerging evidence over the past 20 years strongly suggests that the normal electric function of the heart is the result of dynamic interactions of membrane ion channels working in an orchestrated fashion as part of complex molecular networks. Such networks work together with exquisite temporal precision to generate each action potential and contraction. Macromolecular complexes play crucial roles in transcription, translation, oligomerization, trafficking, membrane retention, glycosylation, post-translational modification, turnover, function, and degradation of all cardiac ion channels known to date. In addition, the accurate timing of each cardiac beat and contraction demands, a comparable precision on the assembly and organizations of sodium, calcium, and potassium channel complexes within specific subcellular microdomains, where physical proximity allows for prompt and efficient interaction. This review article, part of the Compendium on Sudden Cardiac Death, discusses the major issues related to the role of ion channel macromolecular assemblies in normal cardiac electric function and the mechanisms of arrhythmias leading to sudden cardiac death. It provides an idea of how these issues are being addressed in the laboratory and in the clinic, which important questions remain unanswered, and what future research will be needed to improve knowledge and advance therapy.

The voltage-gated Na⁺ channels (Na_v) form a family composed of 10 genes. The COOH termini of Na_v contain a cluster of amino acids that are nearly identical among 7 of the 10 members. This COOH-terminal sequence, PPSYDSV, is a PY motif known to bind to WW domains of E3 protein-ubiquitin ligases of the Nedd4 family. We recently reported that cardiac Na_v1.5 is regulated by Nedd4-2. In this study, we further investigated the molecular determinants of regulation of Na_v proteins. When expressed in HEK-293 cells and studied using whole cell voltage clamping, the neuronal Na_v1.2 and Na_v1.3 were also downregulated by Nedd4-2. Pull-down experiments using fusion proteins bearing the PY motif of Na_v1.2, Na_v1.3, and Na_v1.5 indicated that mouse brain Nedd4-2 binds to the Na_v PY motif. Using intrinsic tryptophan fluorescence imaging of WW domains, we found that Na_v1.5 PY motif binds preferentially to the fourth WW domain of Nedd4-2 with a K_d of ∼55 μM. We tested the binding properties and the ability to ubiquitinate and downregulate Na_v1.5 of three Nedd4-like E3s: Nedd4-1, Nedd4-2, and WWP2. Despite the fact that along with Nedd4-2, Nedd4-1 and WWP2 bind to Na_v1.5 PY motif, only Nedd4-2 robustly ubiquitinated and downregulated Na_v1.5. Interestingly, coexpression of WWP2 competed with the effect of Nedd4-2. Finally, using brefeldin A, we found that Nedd4-2 accelerated internalization of Na_v1.5 stably expressed in HEK-293 cells. This study shows that Nedd4-dependent ubiquitination of Na_v channels may represent a general mechanism regulating the excitability of neurons and myocytes via modulation of channel density at the plasma membrane.