Abstract 14602: Physical and Biophysical Coupling of the Cardiac Sodium Channel Involves 14-3-3

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Jerome C Clatot ◽  
Malcolm Hoshi ◽  
Haiyan Liu ◽  
Xiaoping Wan ◽  
Krekwit Shinlapawittayatorn ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Sodium Channels ◽  
Dominant Negative ◽  
Hek293 Cells ◽  
Alpha Subunit ◽  
Gene Encoding ◽  
Cardiac Sodium Channel ◽  
Channel Assembly ◽  
Cardiac Sodium Channels ◽  
Biophysical Interactions

Introduction: Mutations in SCN5A, the gene encoding for the cardiac sodium channel, produce alterations of the cardiac action potential that lead to life-threatening arrhythmias such as Long QT Syndrome (LQT3) and Brugada Syndrome (BrS). The conventional wisdom that sodium channels exist in complexes containing a single alpha-subunit has been challenged by the existence of dominant-negative (DN) mutations in BrS and the presence of polymorphisms that can restore trafficking and gating deficiencies of mutant channels in LQT and BrS. In fact, we have previously demonstrated that SCN5A subunits can interact with each other. Here we hypothesized that the physical and biophysical interactions between SCN5A alpha-subunits involve the partner protein 14-3-3, known to form dimers. Methods: SCN5A DN-BrS mutants and LQT3 gating deficient mutants were expressed in HEK293 cells and in commercially available iPS-derived cardiomyocytes, iCells©, in presence or absence of 14-3-3 inhibition. Resulting currents were measured using patch-clamp. Results: In order to investigate if the DN-effect seen by some BrS mutants is due to interaction of the sodium channel with the protein 14-3-3 which in turn would be involved in the alpha-alpha interaction, we expressed two different BrS DN-mutants in HEK293 cells with and without difopein, a specific 14-3-3 inhibitor. The presence of difopein abolished the DN-effect of both mutants. The DN-effect was also abolished when we mutated the putative 14-3-3 binding site on SCN5A and expressed the DN-mutants either in HEK293 cells or in iCells©. Inhibition of 14-3-3 also impaired the biophysical coupling observed in presence of SCN5A gating deficient mutants that affect either activation or inactivation of not only the mutants but also of the wild-type channel. Conclusions: Our results suggest that binding of 14-3-3 to the cardiac sodium channel alpha-subunit is involved in the alpha-alpha interaction and biophysical coupling of the channel. This study not only shifts paradigms in regards to sodium channel assembly and structure, but also puts forward the idea that physical and biophysical uncoupling of cardiac sodium channels could be a new therapy target for cardiac arrhythmias caused by SCN5A mutations.

scholarly journals NMR solution structure and analysis of isolated S3b-S4a motif of repeat IV of the human cardiac sodium channel

2021 ◽  
Author(s):  
Adel K Hussein ◽  
Mohammed H Bhuiyan ◽  
Jianqin Zhuang ◽  
Sébastien F Poget
Keyword(s):  
Sodium Channel ◽  
Sodium Channels ◽  
Solution Structure ◽  
Structural Changes ◽  
Nmr Relaxation ◽  
Nmr Studies ◽  
Loop Region ◽  
C Terminus ◽  
Cardiac Sodium Channel ◽  
Cardiac Sodium Channels

Voltage-gated sodium channels are membrane proteins that play an important role in the propagation of electrical signals by mediating the rising phase of an action potential. Numerous diseases, including epilepsy, extreme pain, and certain cardiac arrhythmias have been linked to defects in these channels. The S3b-S4a helix-turn-helix motif (paddle motif) is a region of the channel that is involved in voltage sensing and undergoes significant structural changes during gating. It is also the binding site for many gating-modifier toxins. We determined the solution structure of the paddle motif from the fourth repeat of NaV1.5 in dodecylphosphocholine micelles by NMR spectroscopy and investigated its dynamics and micelle interactions. The structure displays a helix hairpin with a short connecting loop, and likely represents the activated conformation with three of the first four gating charges facing away from S3. Furthermore, paramagnetic relaxation measurements showed that the paddle motif is mainly interacting with the interface region of the micelle. NMR relaxation studies revealed that the paddle motif is mostly rigid, with some residues around the loop region and the last 4 residues on the C-terminus displaying heightened mobility. The structural findings reported here allowed the interpretation of three disease-causing mutations in this region of the human cardiac sodium channel, S1609W, F1617del and T1620M. The establishment of this model system for NMR studies of the paddle region offers a promising platform for future toxin interaction studies in the cardiac sodium channels, and similar approaches may be applied to other sodium channel isoforms.

Abstract 1503: Knockdown of Mog1 Expression Reduces Sodium Currents by Reducing the Trafficking of Cardiac Sodium Channel Na V 1.5 to the Cell Membrane

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Susmita Chakrabarti ◽  
Sandro Yong ◽  
Shin Yoo ◽  
Ling Wu ◽  
Qing Kenneth Wang
Keyword(s):  
Sodium Channel ◽  
Ventricular Myocytes ◽  
Sodium Current ◽  
Expression System ◽  
Heart Association ◽  
Hek293 Cells ◽  
Similar Reduction ◽  
Mammalian Expression ◽  
Ventricular Cells ◽  
Cardiac Sodium Channel

The cardiac sodium channel (Na v 1.5) plays a significant role in cardiac physiology and leads to cardiac arrhythmias and sudden death when mutated. Modulation of Na v 1.5 activity can also arise from changes to accessory subunits or proteins. Our laboratory has recently reported that MOG1, a small protein that is highly conserved from yeast to humans, is a co-factor of Na v 1.5. Increased MOG1 expression has been shown to increase Na v 1.5 current density. In adult mouse ventricular myocytes, these two proteins were found to be co-localized at the intercalated discs. Here, we further characterize the regulatory role of MOG1 using the RNA interference technique. Sodium current was recorded in voltage-clamp mode from a holding potential of −100 mV and activated to −20 mV. In 3-day old mouse neonatal ventricular cells transfected with siRNA against mouse MOG1 decreased sodium current densities (pA/pF) compared to control or scramble siRNA treated cells (−10.2±3.3, n=11 vs. −165±16, n=20 or −117.9±11.7, n=11). A similar reduction in sodium current was observed in mammalian expression system consisting of HEK293 cells stably expressing human Na v 1.5, by transfecting siRNAs against either human or mouse MOG1 (−41.7±8.3, n=7 or, −82.6±9.6, n=7 vs. −130.6±11.5, n=7; −111.5±8.5, n=7, respectively). Immunocytochemistry revealed that the expression of MOG1 and Na v 1.5 were decreased in both HEK and neonatal cells when compared to scramble siRNAs or control groups. These results show that MOG1 is an essential co-factor for Na v 1.5 by way of a channel trafficking. Such interactions between MOG1 and Na v 1.5 suggest that early localization of MOG1 on the membrane of neonatal cardiomyocytes may be necessary for proper localization and the distribution of Na v 1.5 during cardiac development. This research has received full or partial funding support from the American Heart Association, AHA National Center.

Abstract 15420: Resolving a Controversy: Inhibition of Cardiac Ryanodine Receptors is the Principal Mechanism of Antiarrhythmic Action of Flecainide in Catecholaminergic Polymorphic Ventricular Tachycardia

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Dmytro O Kryshtal ◽  
Daniel J Blackwell ◽  
Christian L Egly ◽  
Abigail N Smith ◽  
Suzanne M Batiste ◽  
...  
Keyword(s):  
Ventricular Tachycardia ◽  
Sodium Channel ◽  
Sodium Channels ◽  
Catecholaminergic Polymorphic Ventricular Tachycardia ◽  
Polymorphic Ventricular Tachycardia ◽  
Antiarrhythmic Action ◽  
Channel Block ◽  
Principal Mechanism ◽  
Cardiac Sodium Channels

Rationale: The class Ic antiarrhythmic drug flecainide prevents ventricular tachyarrhythmia in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), a disease caused by hyperactive cardiac ryanodine receptor (RyR2) calcium (Ca) release. Although flecainide inhibits single RyR2 channels in vitro , reports have claimed that RyR2 inhibition by flecainide is not relevant for its mechanism of antiarrhythmic action and concluded that sodium channel block alone is responsible for flecainide’s efficacy in CPVT. Objective: To determine whether RyR2 block independently contributes to flecainide’s efficacy for suppressing spontaneous sarcoplasmic reticulum (SR) Ca release and for preventing ventricular tachycardia in vivo . Methods and Results: We synthesized N -methyl flecainide analogues (QX-FL and NM-FL) and showed that N -methylation reduces flecainide’s inhibitory potency on RyR2 channels but not on cardiac sodium channels. Antiarrhythmic efficacy was tested utilizing a calsequestrin knockout (Casq2-/-) CPVT mouse model. In membrane-permeabilized Casq2-/- cardiomyocytes — lacking intact sarcolemma and devoid of sodium channel contribution — flecainide, but not its analogues, suppressed RyR2-mediated Ca release at clinically relevant concentrations. In voltage-clamped, intact Casq2-/- cardiomyocytes pretreated with tetrodotoxin (TTX) to inhibit sodium channels and isolate the effect of flecainide on RyR2, flecainide significantly reduced the frequency of spontaneous SR Ca release, while QX-FL and NM-FL did not. In vivo , flecainide effectively suppressed catecholamine-induced ventricular tachyarrhythmias in Casq2-/- mice, whereas NM-FL did not, despite comparable sodium channel block. Conclusions: Flecainide remains an effective inhibitor of RyR2-mediated arrhythmogenic Ca release even when cardiac sodium channels are blocked. In mice with CPVT, sodium channel block alone was not enough to prevent arrhythmias. Hence, RyR2 inhibition by flecainide is critical for its mechanism of antiarrhythmic action.

scholarly journals Characterization of an N-terminal Nav1.5 channel variant – a potential risk factor for arrhythmias and sudden death?

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Stefanie Scheiper-Welling ◽  
Paolo Zuccolini ◽  
Oliver Rauh ◽  
Britt-Maria Beckmann ◽  
Christof Geisen ◽  
...  
Keyword(s):  
Sudden Death ◽  
Functional Characterization ◽  
Missense Variant ◽  
Hek293 Cells ◽  
Patch Clamp Technique ◽  
Gene Encoding ◽  
Heterozygous Variant ◽  
Cardiac Sodium Channel ◽  
Electrophysiological Measurements

Abstract Background Alterations in the SCN5A gene encoding the cardiac sodium channel Nav1.5 have been linked to a number of arrhythmia syndromes and diseases including long-QT syndrome (LQTS), Brugada syndrome (BrS) and dilative cardiomyopathy (DCM), which may predispose to fatal arrhythmias and sudden death. We identified the heterozygous variant c.316A > G, p.(Ser106Gly) in a 35-year-old patient with survived cardiac arrest. In the present study, we aimed to investigate the functional impact of the variant to clarify the medical relevance. Methods Mutant as well as wild type GFP tagged Nav1.5 channels were expressed in HEK293 cells. We performed functional characterization experiments using patch-clamp technique. Results Electrophysiological measurements indicated, that the detected missense variant alters Nav1.5 channel functionality leading to a gain-of-function effect. Cells expressing S106G channels show an increase in Nav1.5 current over the entire voltage window. Conclusion The results support the assumption that the detected sequence aberration alters Nav1.5 channel function and may predispose to cardiac arrhythmias and sudden cardiac death.

scholarly journals RYR2 Channel Inhibition Is the Principal Mechanism 0f Flecainide Action in CPVT

2020 ◽  
Author(s):  
Dmytro O Kryshtal ◽  
Daniel Blackwell ◽  
Christian Egly ◽  
Abigail N Smith ◽  
Suzanne M Batiste ◽  
...  
Keyword(s):  
Ventricular Tachycardia ◽  
Sodium Channel ◽  
Sodium Channels ◽  
Antiarrhythmic Action ◽  
Channel Block ◽  
Principal Mechanism ◽  
Channel Inhibition ◽  
Ryr2 Channel ◽  
Cardiac Sodium Channels

Rationale: The class Ic antiarrhythmic drug flecainide prevents ventricular tachyarrhythmia in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), a disease caused by hyperactive cardiac ryanodine receptor (RyR2) calcium (Ca) release. Although flecainide inhibits single RyR2 channels in vitro, reports have claimed that RyR2 inhibition by flecainide is not relevant for its mechanism of antiarrhythmic action and concluded that sodium channel block alone is responsible for flecainide's efficacy in CPVT. Objective: To determine whether RyR2 block independently contributes to flecainide's efficacy for suppressing spontaneous sarcoplasmic reticulum (SR) Ca release and for preventing ventricular tachycardia in vivo. Methods and Results: We synthesized N-methylated flecainide analogues (QX-FL and NM-FL) and showed that N-methylation reduces flecainide's inhibitory potency on RyR2 channels incorporated into artificial lipid bilayers. N-Methylation did not alter flecainide's inhibitory activity on human cardiac sodium channels expressed in HEK293T cells. Antiarrhythmic efficacy was tested utilizing a calsequestrin knockout (Casq2-/-) CPVT mouse model. In membrane-permeabilized Casq2-/- cardiomyocytes — lacking intact sarcolemma and devoid of sodium channel contribution — flecainide, but not its analogues, suppressed RyR2-mediated Ca release at clinically relevant concentrations. In voltage-clamped, intact Casq2-/- cardiomyocytes pretreated with tetrodotoxin (TTX) to inhibit sodium channels and isolate the effect of flecainide on RyR2, flecainide significantly reduced the frequency of spontaneous SR Ca release, while QX-FL and NM-FL did not. In vivo, flecainide effectively suppressed catecholamine-induced ventricular tachyarrhythmias in Casq2-/- mice, whereas NM-FL had no significant effect on arrhythmia burden, despite comparable sodium channel block. Conclusions: Flecainide remains an effective inhibitor of RyR2-mediated arrhythmogenic Ca release even when cardiac sodium channels are blocked. In mice with CPVT, sodium channel block alone did not prevent ventricular tachycardia. Hence, RyR2 channel inhibition likely constitutes the principal mechanism of antiarrhythmic action of flecainide in CPVT.

scholarly journals Activity-induced internalization and rapid degradation of sodium channels in cultured fetal neurons.

1996 ◽  
Vol 134 (2) ◽  
pp. 499-509 ◽  
Author(s):  
C Paillart ◽  
J L Boudier ◽  
J A Boudier ◽  
H Rochat ◽  
F Couraud ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Amphotericin B ◽  
Sodium Channels ◽  
Time Dependent ◽  
Alpha Subunit ◽  
Dependent Manner ◽  
Down Regulation ◽  
Proteolytic Fragment ◽  
Channel Density ◽  
Specific Distribution

A regulatory mechanism for neuronal excitability consists in controlling sodium channel density at the plasma membrane. In cultured fetal neurons, activation of sodium channels by neurotoxins, e.g., veratridine and alpha-scorpion toxin (alpha-ScTx) that enhance the channel open state probability induced a rapid down-regulation of surface channels. Evidence that the initial step of activity-induced sodium channel down-regulation is mediated by internalization was provided by using 125I-alpha-ScTx as both a channel probe and activator. After its binding to surface channels, the distribution of 125I-alpha-ScTx into five subcellular compartments was quantitatively analyzed by EM autoradiography. 125I-alpha-ScTx was found to accumulate in tubulovesicular endosomes and disappear from the cell surface in a time-dependent manner. This specific distribution was prevented by addition of tetrodotoxin (TTX), a channel blocker. By using a photoreactive derivative to covalently label sodium channels at the surface of cultured neurons, we further demonstrated that they are degraded after veratridine-induced internalization. A time-dependent decrease in the amount of labeled sodium channel alpha subunit was observed after veratridine treatment. After 120 min of incubation, half of the alpha subunits were cleaved. This degradation was prevented totally by TTX addition and was accompanied by the appearance of an increasing amount of a 90-kD major proteolytic fragment that was already detected after 45-60 min of veratridine treatment. Exposure of the photoaffinity-labeled cells to amphotericin B, a sodium ionophore, gave similar results. In this case, degradation was prevented when Na+ ions were substituted by choline ions and not blocked by TTX. After veratridine- or amphotericin B-induced internalization of sodium channels, breakdown of the labeled alpha subunit was inhibited by leupeptin, while internalization was almost unaffected. Thus, cultured fetal neurons are capable of adjusting sodium channel density by an activity-dependent endocytotic process that is triggered by Na+ influx.

scholarly journals Modulation of the Cardiac Sodium Channel NaV1.5 Peak and Late Currents by NAD+ Precursors

2020 ◽  
Author(s):  
Daniel S. Matasic ◽  
Jin-Young Yoon ◽  
Jared M. McLendon ◽  
Haider Mehdi ◽  
Mark S. Schmidt ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Membrane Trafficking ◽  
Cardiac Electrophysiology ◽  
Phosphorylation Site ◽  
Sirtuin 1 ◽  
Hek293 Cells ◽  
Mutant Form ◽  
Wild Type ◽  
Cardiac Sodium Channel

ABSTRACTRationaleThe cardiac sodium channel NaV1.5, encoded by SCN5A, produces the rapidly inactivating depolarizing current INa that is responsible for the initiation and propagation of the cardiac action potential. Acquired and inherited dysfunction of NaV1.5 results in either decreased peak INa or increased residual late INa (INa,L), leading to tachy/bradyarrhythmias and sudden cardiac death. Previous studies have shown that increased cellular NAD+ and NAD+/NADH ratio increase INa through suppression of mitochondrial reactive oxygen species and PKC-mediated NaV1.5 phosphorylation. In addition, NAD+-dependent deacetylation of NaV1.5 at K1479 by Sirtuin 1 increases NaV1.5 membrane trafficking and INa. The role of NAD+ precursors in modulating INa remains unknown.ObjectiveTo determine whether and by which mechanisms the NAD+ precursors nicotinamide riboside (NR) and nicotinamide (NAM) affect peak INa and INa,Lin vitro and cardiac electrophysiology in vivo.Methods and ResultsThe effects of NAD+ precursors on the NAD+ metabolome and electrophysiology were studied using HEK293 cells expressing wild-type and mutant NaV1.5, rat neonatal cardiomyocytes (RNCMs), and mice. NR increased INa in HEK293 cells expressing NaV1.5 (500 μM: 51 ± 18%, p=0.02, 5 mM: 59 ± 22%, p=0.03) and RNCMs (500 µM: 60 ± 26%, p=0.02, 5 mM: 75 ± 39%, p=0.03) while reducing INa,L at the higher concentration (RNCMs, 5 mM: −45 ± 11%, p=0.04). NR (5 mM) decreased NaV1.5 K1479 acetylation but increased INa in HEK293 cells expressing a mutant form of NaV1.5 with disruption of the acetylation site (NaV1.5-K1479A). Disruption of the PKC phosphorylation site abolished the effect of NR on INa. Furthermore, NAM (5 mM) had no effect on INa in RNCMs or in HEK293 cells expressing wild-type NaV1.5, but increased INa in HEK293 cells expressing NaV1.5-K1479A. Dietary supplementation with NR for 10-12 weeks decreased QTc in C57BL/6J mice (0.35% NR: −4.9 ± 2.0%, p=0.26; 1.0% NR: −9.5 ± 2.8%, p=0.01).ConclusionsNAD+ precursors differentially regulate NaV1.5 via multiple mechanisms. NR increases INa, decreases INa,L, and warrants further investigation as a potential therapy for arrhythmic disorders caused by NaV1.5 deficiency and/or dysfunction.

scholarly journals The β1-Subunit of NaV1.5 Cardiac Sodium Channel is required for a Dominant Negative Effect through α-α Interaction

2013 ◽  
Vol 104 (2) ◽  
pp. 133a
Author(s):  
Aurelie Mercier ◽  
Romain Clément ◽  
Thomas Harnois ◽  
Nicolas Bourmeyster ◽  
Jean-François Faivres ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Cardiac Sodium Channel ◽  
Negative Effect

Abstract 16722: Perinexal Width Modulates Sodium Channel Recruitment During Extracellular Stimulation

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Katrina Colucci Chang ◽  
Xiaobo Wu ◽  
Grace Blair ◽  
Alicia Lozano ◽  
Alexandra Hanlon ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Sodium Channels ◽  
Voltage Divider ◽  
Extracellular Calcium ◽  
Extracellular Potassium ◽  
Intercalated Disc ◽  
Activation Threshold ◽  
Voltage Gated Sodium Channels ◽  
Cardiac Sodium Channels ◽  
Ephaptic Coupling

Excitability in cardiomyocytes is dependent on the subthreshold current required to raise transmembrane potential to the activation threshold of voltage gated sodium channels and sodium channel recruitment to trigger an action potential. Cardiac sodium channels are densely expressed in the intercalated disc within the perinexal nanodomain, which is 2 orders of magnitude narrower than bulk extracellular interstitium. We hypothesized that perinexal narrowing reduces extracellular induced excitability because the perinexus functions as a voltage divider. Methods: Excitability with an extracellular stimulus was quantified in isolated Langendorff perfused male retired breeder guinea pig hearts by strength duration curves using the Lapicque method. Interventions included changing extracellular potassium (K+: 3, 4.5, and 10 mM), inhibiting sodium channels (90-uM Flecainide), and narrowing the perinexus by increasing extracellular calcium (Ca2+: 1.25 to 2.5 mM). Results: Consistent with previous studies, decreasing K+ from 4.56 to 3 mM depressed excitability with 2.5 mM Ca2+ but not 1.25 mM Ca2+, and conduction velocity (CV) decreased by 10.5 % with both 1.25 and 2.5 mM Ca2+. When K+ was raised from 4.56 to 10 mM, no change was seen in excitability with both Ca2+ concentrations. However, CV decreased by 16% with both Ca2+ concentrations. Flecainide depressed excitability only with 2.5 but not 1.25 mM Ca2+. Meanwhile CV decreased by 13% with 1.25 but CV did not change with 2.5 mM Ca2+. Finally, raising Ca2+ alone at baseline decreased excitability, without substantially changing conduction. Conclusions: Elevating extracellular calcium to narrow perinexi reduces excitability measured by extracellular stimulation consistent with a hypothesis that sodium channels in the intercalated disc are electrically isolated from the bulk interstitium. Furthermore, excitability and conduction do not correlate in response to similar K+ changes when Ca2+ also varies, suggesting cardiac excitability and propagation are independent mechanisms when the excitatory current occurs through regenerative propagation as occurs through gap junctions or arrives via an extracellular field as occurs with pacing and ephaptic coupling.

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