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 15925: Localization of Nav1.5 at the Intercellular Junctions Resolved by Diffraction-Unlimited Microscopy in Three Dimensions and by Correlative Light-Electron Microscopy

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Alejandra Leo-Macias ◽  
Esperanza Agullo-Pascual ◽  
Eli Rothenberg ◽  
Mario Delmar
Keyword(s):  
Electron Microscopy ◽  
Sodium Channel ◽  
Ventricular Myocytes ◽  
Sodium Current ◽  
Intercellular Junctions ◽  
Three Dimensions ◽  
Macromolecular Complex ◽  
Adult Heart ◽  
Mechanical Coupling ◽  
Cardiac Sodium Channel

Sodium current amplitude, kinetics and regulation depend on the properties of the pore-forming protein (mostly NaV1.5 in adult heart) and on the specific molecular partners with which the channel protein associates. The composition of the voltage-gated sodium channel macromolecular complex is location-specific; yet, the exact position of NaV1.5 in the subcellular landscape of the intercalated disc (ID), remains unclear. We implemented diffraction unlimited microscopy (direct stochastic optical reconstruction microscopy, or “dSTORM”) to localize the pore-forming subunit of the cardiac sodium channel NaV1.5 with a resolution of 20nm on the XY plane. In isolated adult ventricular myocytes, NaV1.5 was found in distinct semi-circular clusters. When the entire population of clusters within a 500 nm window from the ID was considered (more than 350 individual clusters analyzed), 75% of them localized to N-cadherin rich sites. NaV1.5-distal clusters were found at an average 313±15 nm from the cell end. Introducing an astigmatic lens in the light path allowed us to solve cluster location in three dimensions, at resolutions of 20 nm in XY and 40 nm in the z plane. Three-dimensional images confirmed the preferential localization at or near N-cadherin plaques, and further suggested that NaV1.5 arrives to the membrane via N-cadherin-anchored paths, most likely microtubules. In additional experiments, we developed a novel approach to correlate the image of NaV1.5 clusters by dSTORM with the cellular ultrastructure as resolved by electron microscopy on the same sample. This “correlative light-electron microscopy” method confirmed the preference of NaV1.5 clusters at sites of mechanical coupling. Overall, we provide the first ultrastructural description of NaV1.5 at the cardiac ID and its relation with the major electron-dense domains of the adult heart. Our data support a model by which microtubule-mediated delivery of NaV1.5 anchors at N-cadherin-rich sites, likely “mixed junctions” also containing desmosomal molecules (such as plakophilin-2; see Cerrone et al; Circulation 129:1092-1103, 2014) and connexin43. These findings have major implications to the understanding of sodium current disruption in diseases affecting the integrity of the ID.

scholarly journals GPD1L links redox state to cardiac excitability by PKC-dependent phosphorylation of the sodium channel SCN5A

2009 ◽  
Vol 297 (4) ◽  
pp. H1446-H1452 ◽  
Author(s):  
Carmen R. Valdivia ◽  
Kazuo Ueda ◽  
Michael J. Ackerman ◽  
Jonathan C. Makielski
Keyword(s):  
Sodium Channel ◽  
Sodium Current ◽  
Expression System ◽  
Sudden Cardiac Arrest ◽  
Sudden Infant ◽  
Cell Physiology ◽  
Glycerol Phosphate ◽  
Enzymatic Function ◽  
Cardiac Sodium Channel ◽  
Glycerol Phosphate Dehydrogenase

The SCN5A-encoded cardiac sodium channel underlies excitability in the heart, and dysfunction of sodium current ( INa) can cause fatal ventricular arrhythmia in maladies such as long QT syndrome, Brugada syndrome (BrS), and sudden infant death syndrome (SIDS). The gene GPD1L encodes the glycerol phosphate dehydrogenase 1-like protein with homology to glycerol phosphate dehydrogenase (GPD1), but the function for this enzyme is unknown. Mutations in GPD1L have been associated with BrS and SIDS and decrease INa through an unknown mechanism. Using a heterologous expression system, we show that GPD1L associated with SCN5A and that the BrS- and SIDS-related mutations in GPD1L caused a loss of enzymatic function resulting in glycerol-3-phosphate PKC-dependent phosphorylation of SCN5A at serine 1503 (S1503) through a GPD1L-dependent pathway. The direct phosphorylation of S1503 markedly decreased INa. These results show a function for GPD1L in cell physiology and a mechanism linking mutations in GPD1L to sudden cardiac arrest. Because the enzymatic step catalyzed by GPD1L depends upon nicotinamide adenine dinucleotide, this GPD1L pathway links the metabolic state of the cell to INa and excitability and may be important more generally in cardiac ischemia and heart failure.

scholarly journals Electrophysiological properties of mouse and epitope-tagged human cardiac sodium channel Nav1.5 expressed in HEK293 cells

F1000Research ◽  
2013 ◽  
Vol 2 ◽  
pp. 48 ◽  
Author(s):  
Katja Reinhard ◽  
Jean-Sébastien Rougier ◽  
Jakob Ogrodnik ◽  
Hugues Abriel
Keyword(s):  
Sodium Channel ◽  
Fluorescent Protein ◽  
Sodium Current ◽  
Genetically Modified ◽  
Hek293 Cells ◽  
Biophysical Properties ◽  
Electrophysiological Properties ◽  
Cardiac Sodium Channel ◽  
N Terminus ◽  
Green Fluorescent

Background: The pore-forming subunit of the cardiac sodium channel, Nav1.5, has been previously found to be mutated in genetically determined arrhythmias. Nav1.5 associates with many proteins that regulate its function and cellular localisation. In order to identify more in situ Nav1.5 interacting proteins, genetically-modified mice with a high-affinity epitope in the sequence of Nav1.5 can be generated.Methods: In this short study, we (1) compared the biophysical properties of the sodium current (INa) generated by the mouse Nav1.5 (mNav1.5) and human Nav1.5 (hNav1.5) constructs that were expressed in HEK293 cells, and (2) investigated the possible alterations of the biophysical properties of the human Nav1.5 construct that was modified with specific epitopes.Results: The biophysical properties of mNav1.5 were similar to the human homolog. Addition of epitopes either up-stream of the N-terminus of hNav1.5 or in the extracellular loop between the S5 and S6 transmembrane segments of domain 1, significantly decreased the amount of INa and slightly altered its biophysical properties. Adding green fluorescent protein (GFP) to the N-terminus did not modify any of the measured biophysical properties of hNav1.5.Conclusions: These findings have to be taken into account when planning to generate genetically-modified mouse models that harbour specific epitopes in the gene encoding mNav1.5.

Larger late sodium current density as well as greater sensitivities to ATX II and ranolazine in rabbit left atrial than left ventricular myocytes

2014 ◽  
Vol 306 (3) ◽  
pp. H455-H461 ◽  
Author(s):  
Antao Luo ◽  
Jihua Ma ◽  
Yejia Song ◽  
Chunping Qian ◽  
Ying Wu ◽  
...  
Keyword(s):  
Left Atrial ◽  
Ventricular Myocytes ◽  
Sodium Current ◽  
Left Ventricular ◽  
Atrial Myocytes ◽  
Open Probability ◽  
Late Sodium Current ◽  
Ventricular Cells ◽  
Open Time ◽  
Hill Slope

An increase of cardiac late sodium current ( INa.L) is arrhythmogenic in atrial and ventricular tissues, but the densities of INa.L and thus the potential relative contributions of this current to sodium ion (Na+) influx and arrhythmogenesis in atria and ventricles are unclear. In this study, whole-cell and cell-attached patch-clamp techniques were used to measure INa.L in rabbit left atrial and ventricular myocytes under identical conditions. The density of INa.L was 67% greater in left atrial (0.50 ± 0.09 pA/pF, n = 20) than in left ventricular cells (0.30 ± 0.07 pA/pF, n = 27, P < 0.01) when elicited by step pulses from −120 to −20 mV at a rate of 0.2 Hz. Similar results were obtained using step pulses from −90 to −20 mV. Anemone toxin II (ATX II) increased INa.L with an EC50 value of 14 ± 2 nM and a Hill slope of 1.4 ± 0.1 ( n = 9) in atrial myocytes and with an EC50 of 21 ± 5 nM and a Hill slope of 1.2 ± 0.1 ( n = 12) in ventricular myocytes. Na+ channel open probability (but not mean open time) was greater in atrial than in ventricular cells in the absence and presence of ATX II. The INa.L inhibitor ranolazine (3, 6, and 9 μM) reduced INa.L more in atrial than ventricular myocytes in the presence of 40 nM ATX II. In summary, rabbit left atrial myocytes have a greater density of INa.L and higher sensitivities to ATX II and ranolazine than rabbit left ventricular myocytes.

Alternatively spliced human tissue factor (asHTF) is not pro-coagulant

2007 ◽  
Vol 97 (01) ◽  
pp. 11-14 ◽  
Author(s):  
Petra Censarek ◽  
Anett Bobbe ◽  
Maria Grandoch ◽  
Karsten Schrör ◽  
Artur-Aron Weber
Keyword(s):  
Smooth Muscle ◽  
Tissue Factor ◽  
Human Tissue ◽  
Thrombin Generation ◽  
Expression System ◽  
Hek293 Cells ◽  
Transfected Cells ◽  
Mammalian Expression ◽  
Coagulant Activity ◽  
Alternatively Spliced

SummaryIt has been proposed that alternatively-spliced human tissue factor (asHTF) is pro-coagulant. We have evaluated the function of asHTF in a mammalian expression system. Full-length human tissue factor (HTF) and asHTF were cloned from smooth muscle cells and over-expressed in HEK293 cells. As expected, a marked pro-coagulant activity (FX activation, thrombin generation) was observed on the surface, in lysates, and on microparticles from HTF transfected cells. In contrast, no pro-coagulant activity of as HTF was observed.

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.

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 Contribution of sodium channel neuronal isoform Nav1.1 to late sodium current in ventricular myocytes from failing hearts

2014 ◽  
Vol 593 (6) ◽  
pp. 1409-1427 ◽  
Author(s):  
Sudhish Mishra ◽  
Vitaliy Reznikov ◽  
Victor A. Maltsev ◽  
Nidas A. Undrovinas ◽  
Hani N. Sabbah ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Ventricular Myocytes ◽  
Sodium Current ◽  
Late Sodium Current

scholarly journals Characterization of the cardiac sodium channel SCN5A mutation, N1325S, in single murine ventricular myocytes

2007 ◽  
Vol 352 (2) ◽  
pp. 378-383 ◽  
Author(s):  
Sandro L. Yong ◽  
Ying Ni ◽  
Teng Zhang ◽  
David J. Tester ◽  
Michael J. Ackerman ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Ventricular Myocytes ◽  
Cardiac Sodium Channel ◽  
Scn5a Mutation

scholarly journals MOG1 restores the expression and function of SCN5A-p.R104W through sec23a-mediated forward trafficking

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
L Luo ◽  
Y Wang ◽  
Y Du ◽  
C Dong ◽  
A Ma ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Sodium Current ◽  
Site Directed Mutagenesis ◽  
Hek293 Cells ◽  
Funding Source ◽  
Inherited Disease ◽  
Patch Clamp Recording ◽  
Knock Out ◽  
Cardiac Sodium Channel ◽  
And Function

Abstract Background Brugada syndrome (BrS) is an inherited disease which causes fatal arrhythmias and sudden cardiac death. Mutations in SCN5A gene, which encoding cardiac sodium channel (NaV1.5), are the most common genotype of BrS patients. Some SCN5A-related variants were reported to retain NaV1.5 in endoplasmic reticulum (ER) due to trafficking deficiency. MOG1 was previously reported to interact with NaV1.5 and increased sodium current (INa) through enhancing the trafficking. However, its molecular mechanisms are still unclear. Coat protein complex II (COPII) is responsible for the ER to Golgi transport. Sec23 forms the inner coat of COPII and participates in cargo proteins selection. Purpose To demonstrate that MOG1 rescues SCN5A-related variants by enhancing the forward trafficking through Sec23a-NaV1.5 interaction. Methods Site directed mutagenesis, immunofluorescence staining, biotinylation assay, Western blot analysis and whole-cell patch clamp recording were used. CRISPR/Cas9 was used to knock out Sec23a expression in HEK293 cells. Results We found that SCN5A-p.R104W was characterized as reduced NaV1.5 level and lack of INa. The variant SCN5A-p.R104W was mainly distributed in ER. MOG1 could rescue the total and surface expression of SCN5A-p.R104W but could not restore INa (Figure 1a). Considering that most patients are heterozygous, co-transfection of SCN5A-WT and SCN5A-p.R104W were obtained. We found MOG1 could increase both NaV1.5 level and INa of heterozygous expressed SCN5A-p.R104W. We further revealed an interaction between NaV1.5 and Sec23a by co-immunoprecipitation (Co-IP) assay. The interaction between NaV1.5 and Sec23a was increased by MOG1, which indicates that Sec23a participates in MOG1-mediated increase in NaV1.5 level (Figure 1b). Knockout of Sec23a reduced cell surface, but not total, NaV1.5 level (Figure 1c and 1d). Next, the Sec23a knockout HEK293 cells were co-transfected with SCN5A-p.R104W and pcDNA3 or MOG1. MOG1 could not increase SCN5A-p.R104W protein level in Sec23a knockout cells. Conclusion Our data demonstrated a novel mechanism that MOG1 restores the expression and function of SCN5A-p.R104W by enhancing its forward trafficking through Sec23a-NaV1.5 interaction. Figure 1 Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Natural Science Foundation of China

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