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Hugues Abriel - One of the best experts on this subject based on the ideXlab platform.
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Single-Molecule Localization of the Cardiac Voltage-Gated Sodium Channel Reveals Different Modes of Reorganization at Cardiomyocyte Membrane Domains.
Circulation. Arrhythmia and electrophysiology, 2020Co-Authors: Sarah H. Vermij, Mario Delmar, Jean-sébastien Rougier, Esperanza Agullo-pascual, Eli Rothenberg, Hugues AbrielAbstract:Background: Mutations in the gene encoding the cardiac voltage-gated Sodium Channel Nav1.5 cause various cardiac arrhythmias. This variety may arise from different determinants of Nav1.5 expression...
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Calmodulin binds to the N-terminal domain of the cardiac Sodium Channel Nav1.5
bioRxiv, 2020Co-Authors: Zizun Wang, Jean-sébastien Rougier, Sarah H. Vermij, Valentin Sottas, Anna Shestak, Daniela Ross-kaschitza, E. V. Zaklyazminskaya, Andy Hudmon, Hugues AbrielAbstract:ABSTRACT The cardiac voltage-gated Sodium Channel Nav1.5 conducts the rapid inward Sodium current crucial for cardiomyocyte excitability. Loss-of-function mutations in its gene SCN5A are linked to cardiac arrhythmias such as Brugada Syndrome (BrS). Several BrS-associated mutations in the Nav1.5 N-terminal domain exert a dominant-negative effect (DNE) on wild-type Channel function, for which mechanisms remain poorly understood. We aim to contribute to the understanding of BrS pathophysiology by characterizing three mutations in the Nav1.5 N-terminal domain (NTD): Y87C–here newly identified–, R104W and R121W. In addition, we hypothesize that the calcium sensor protein calmodulin is a new NTD binding partner. Recordings of whole-cell Sodium currents in TsA-201 cells expressing WT and variant Nav1.5 showed that Y87C and R104W but not R121W exert a DNE on WT Channels. Biotinylation assays revealed reduction in fully glycosylated Nav1.5 at the cell surface and in whole-cell lysates. Localization of Nav1.5 WT Channel with the ER however did not change in the presence of variants, shown by transfected and stained rat neonatal cardiomyocytes. We next demonstrated that calmodulin binds Nav1.5 N-terminus using in silico modeling, SPOTS, pull-down and proximity ligation assays. This binding is impaired in the R121W variant and in a Nav1.5 construct missing residues 80-105, a predicted calmodulin binding site. In conclusion, we present the first evidence that calmodulin binds to the Nav1.5 NTD, which seems to be a determinant for the DNE.
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Biophysical Characterization of Epigallocatechin-3-Gallate Effect on the Cardiac Sodium Channel Nav1.5.
Molecules, 2020Co-Authors: Mohamed Yassine Amarouch, Han Kurt, Lucie Delemotte, Hugues AbrielAbstract:Epigallocatechin-3-Gallate (EGCG) has been extensively studied for its protective effect against cardiovascular disorders. This effect has been attributed to its action on multiple molecular pathways and transmembrane proteins, including the cardiac Nav1.5 Channels, which are inhibited in a dose-dependent manner. However, the molecular mechanism underlying this effect remains to be unveiled. To this aim, we have characterized the EGCG effect on Nav1.5 using electrophysiology and molecular dynamics (MD) simulations. EGCG superfusion induced a dose-dependent inhibition of Nav1.5 expressed in tsA201 cells, negatively shifted the steady-state inactivation curve, slowed the inactivation kinetics, and delayed the recovery from fast inactivation. However, EGCG had no effect on the voltage-dependence of activation and showed little use-dependent block on Nav1.5. Finally, MD simulations suggested that EGCG does not preferentially stay in the center of the bilayer, but that it spontaneously relocates to the membrane headgroup region. Moreover, no sign of spontaneous crossing from one leaflet to the other was observed, indicating a relatively large free energy barrier associated with EGCG transport across the membrane. These results indicate that EGCG may exert its biophysical effect via access to its binding site through the cell membrane or via a bilayer-mediated mechanism.
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Calmodulin binds to the N-terminal domain of the cardiac Sodium Channel Nav1.5.
Channels, 2020Co-Authors: Zizun Wang, Jean-sébastien Rougier, Sarah H. Vermij, Valentin Sottas, Anna Shestak, Daniela Ross-kaschitza, E. V. Zaklyazminskaya, Andy Hudmon, Hugues AbrielAbstract:The cardiac voltage-gated Sodium Channel Nav1.5 conducts the rapid inward Sodium current crucial for cardiomyocyte excitability. Loss-of-function mutations in its gene SCN5A are linked to cardiac arrhythmias such as Brugada Syndrome (BrS). Several BrS-associated mutations in the Nav1.5 N-terminal domain (NTD) exert a dominant-negative effect (DNE) on wild-type Channel function, for which mechanisms remain poorly understood. We aim to contribute to the understanding of BrS pathophysiology by characterizing three mutations in the Nav1.5 NTD: Y87C-here newly identified-, R104W, and R121W. In addition, we hypothesize that the calcium sensor protein calmodulin is a new NTD binding partner. Recordings of whole-cell Sodium currents in TsA-201 cells expressing WT and variant Nav1.5 showed that Y87C and R104W but not R121W exert a DNE on WT Channels. Biotinylation assays revealed reduction in fully glycosylated Nav1.5 at the cell surface and in whole-cell lysates. Localization of Nav1.5 WT Channel with the ER did not change in the presence of variants, as shown by transfected and stained rat neonatal cardiomyocytes. We demonstrated that calmodulin binds the Nav1.5 NTD using in silico modeling, SPOTS, pull-down, and proximity ligation assays. Calmodulin binding to the R121W variant and to a Nav1.5 construct missing residues 80-105, a predicted calmodulin-binding site, is impaired. In conclusion, we describe the new natural BrS Nav1.5 variant Y87C and present first evidence that calmodulin binds to the Nav1.5 NTD, which seems to be a determinant for the DNE.
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Calmodulin binds to the N-terminal domain of the cardiac Sodium Channel Nav1.5
Channels (Austin Tex.), 2020Co-Authors: Zizun Wang, Geoffrey S. Pitt, Jean-sébastien Rougier, Sarah H. Vermij, Valentin Sottas, Anna Shestak, Daniela Ross-kaschitza, E. V. Zaklyazminskaya, Andy Hudmon, Hugues AbrielAbstract:The cardiac voltage-gated Sodium Channel Nav1.5 conducts the rapid inward Sodium current crucial for cardiomyocyte excitability. Loss-of-function mutations in its gene SCN5A are linked to cardiac a...
Peter C. Ruben - One of the best experts on this subject based on the ideXlab platform.
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Proton modulation of cardiac I Na: a potential arrhythmogenic trigger.
Handbook of experimental pharmacology, 2014Co-Authors: David K. Jones, Peter C. RubenAbstract:Voltage-gated Sodium (NaV) Channels generate the upstroke and mediate duration of the ventricular action potential, thus they play a critical role in mediating cardiac excitability. Cardiac ischemia triggers extracellular pH to drop as low as pH 6.0, within just 10 min of its onset. Heightened proton concentrations reduce Sodium conductance and alter the gating parameters of the cardiac-specific voltage-gated Sodium Channel, Nav1.5. Most notably, acidosis destabilizes fast inactivation, which plays a critical role in regulating action potential duration. The changes in Nav1.5 Channel gating contribute to cardiac dysfunction during ischemia that can cause syncope, cardiac arrhythmia, and even sudden cardiac death. Understanding NaV Channel modulation by protons is paramount to treatment and prevention of the deleterious effects of cardiac ischemia and other triggers of cardiac acidosis.
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Proton-dependent inhibition of the cardiac Sodium Channel Nav1.5 by ranolazine
Frontiers in Pharmacology, 2013Co-Authors: Stanislav Sokolov, Colin H. Peters, Sridharan Rajamani, Peter C. RubenAbstract:Ranolazine is clinically approved for treatment of angina pectoris and is a potential candidate for antiarrhythmic, antiepileptic and analgesic applications. These therapeutic effects of ranolazine hinge on its ability to inhibit persistent or late Na+ currents in a variety of voltage-gated Sodium Channels. Extracellular acidosis, typical of ischemic events, may alter the efficiency of drug/Channel interactions. In this study, we examined pH modulation of ranolazine’s interaction with the cardiac Sodium Channel, Nav1.5. We performed whole-cell path clamp experiments at extracellular pH 7.4 and 6.0 on Nav1.5 transiently expressed in HEK293 cell line. Consistent with previous studies, we found that ranolazine induced a stable conformational state in the cardiac Sodium Channel with onset/recovery kinetics and voltage-dependence resembling intrinsic slow inactivation. This interaction diminished the availability of the Channels in a voltage- and use-dependent manner. Low extracellular pH impaired inactivation states leading to an increase in late Na+ currents. Ranolazine interaction with the Channel was also slowed 4-5 fold. However, ranolazine restored the voltage-dependent steady-state availability profile, thereby reducing window/persistent currents at pH 6.0 in a manner comparable to pH 7.4. These results suggest that ranolazine is effective at therapeutically relevant concentrations (10µM), in acidic extracellular pH, where it compensates for impaired native slow inactivation.
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Proton Modulation of Gating Currents in the Cardiac Voltage-Gated Sodium Channel, Nav1.5
Biophysical Journal, 2012Co-Authors: David K. Jones, Thomas W. Claydon, Peter C. RubenAbstract:Low pH reduces single Channel conductance and destabilizes the fast-inactivated state of the cardiac voltage-gated Sodium Channel (Nav1.5) by increasing both window and persistent currents (Jones et al., 2011, Biophys. J. 101(7)). Since fast inactivation is tightly coupled to NaV Channel voltage sensor activation, we hypothesized that alterations in the kinetics of voltage sensor movement may underlie the destabilization of the fast-inactivated state in Nav1.5. To test this, we expressed Nav1.5 Channels in Xenopus oocytes and recorded gating currents using a cut-open voltage clamp with extracellular solution titrated to either pH 7.4 (control) or pH 6.0. At pH 6.0, compared to pH 7.4, the V1/2 of the Q(V) curve was significantly depolarized (from −57.8±4.3 mV to −40.8±5.1 mV). Additionally, the slow time constant of charge recovery was significantly reduced from 16.1±5.0 ms at pH 7.4 to 9.7±4.2 ms at pH 6.0. These data suggest a molecular basis for the increased persistent and window currents previously shown in Nav1.5 Channels at reduced extracellular pH. Specifically, protons may electrostatically affect the rate of voltage sensor movement, either by directly binding to extracellular residues (e.g. H880) or indirectly by binding to carboxylates in the pore domain (Kahn et al., J Physiol. 2002, 543). (Supported by an NSERC Discovery Grant to PCR, a CFI Infrastructure grant to PCR and TC, and a CIHR Vanier Scholarship to DKJ.)
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Extracellular proton modulation of the cardiac voltage-gated Sodium Channel, Nav1.5.
Biophysical Journal, 2011Co-Authors: David K. Jones, Colin H. Peters, Thomas W. Claydon, S.a. Tolhurst, Peter C. RubenAbstract:Low pH depolarizes the voltage dependence of voltage-gated Sodium (NaV) Channel activation and fast inactivation. A complete description of NaV Channel proton modulation, however, has not been reported. The majority of NaV Channel proton modulation studies have been completed in intact tissue. Additionally, several NaV Channel isoforms are expressed in cardiac tissue. Characterizing the proton modulation of the cardiac NaV Channel, Nav1.5, will thus help define its contribution to ischemic arrhythmogenesis, where extracellular pH drops from pH 7.4 to as low as pH 6.0 within ∼10 min of its onset. We expressed the human variant of Nav1.5 with and without the modulating β1 subunit in Xenopus oocytes. Lowering extracellular pH from 7.4 to 6.0 affected a range of biophysical gating properties heretofore unreported. Specifically, acidic pH destabilized the fast-inactivated and slow-inactivated states, and elevated persistent INa. These data were incorporated into a ventricular action potential model that displayed a reduced maximum rate of depolarization as well as disparate increases in epicardial, mid-myocardial, and endocardial action potential durations, indicative of an increased heterogeneity of repolarization. Portions of these data were previously reported in abstract form.
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pH Modulation of the Cardiac Voltage Gated Sodium Channel, Nav1.5
Biophysical Journal, 2010Co-Authors: David K. Jones, Thomas W. Claydon, Peter C. RubenAbstract:Alterations in the function of the cardiac voltage-gated Sodium Channel (Nav1.5) are a known cause of cardiac disease and arrhythmia. Elevated concentrations of protons decrease conductance and depolarize the voltage dependence of activation and steady-state fast inactivation (SSFI) of Nav1.5 Channels (Zhang & Siegelbaum, 1991, Khan et al., 2006). A complete analysis of the effects of low pH on Nav1.5 Channel kinetics has not previously been reported. We sought to characterize the effects of low pH on Nav1.5 kinetics. Nav1.5 was co-expressed in Xenopus laevis oocytes with the β1 subunit, and currents were recorded at 20 °C using the cut-open voltage clamp technique with the extracellular solution titrated to either pH 7.4 (control) or pH 6.0. Application of solution at pH 6.0 significantly depolarized the voltage dependence of activation and SSFI; −34.4 ± 0.3mV to −25.2 ± 0.2mV and −76.4 ± 0.1mV to −72.7 ± 0.2mV, respectively. The apparent valences of activation and SSFI were significantly decreased; from 3.4 ± 0.12e to 2.5 ± 0.04e, and from −4.6 ± 0.07e to −4.1 ± 0.09e, respectively. At pH 6.0, the fast time constant of use-dependent inactivation was significantly increased and the use dependent current reduction was decreased from 40.6 ± 0.12% to 34.8 ± 0.05%. The rates of open-state fast inactivation onset were significantly decreased at potentials between −30mV and +30mV, and the rates of recovery at −90mV and −80mV were significantly increased. There was also a visible increase in window current. All effects were reversible upon reperfusion of solution at pH 7.4. Taken together, these data suggest that lowering extracellular pH from 7.4 to 6.0 destabilizes the fast-inactivated state of Nav1.5 Channels, an effect that could act as an arrhythmogenic trigger during ischemic events.
Jean-sébastien Rougier - One of the best experts on this subject based on the ideXlab platform.
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Single-Molecule Localization of the Cardiac Voltage-Gated Sodium Channel Reveals Different Modes of Reorganization at Cardiomyocyte Membrane Domains.
Circulation. Arrhythmia and electrophysiology, 2020Co-Authors: Sarah H. Vermij, Mario Delmar, Jean-sébastien Rougier, Esperanza Agullo-pascual, Eli Rothenberg, Hugues AbrielAbstract:Background: Mutations in the gene encoding the cardiac voltage-gated Sodium Channel Nav1.5 cause various cardiac arrhythmias. This variety may arise from different determinants of Nav1.5 expression...
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Calmodulin binds to the N-terminal domain of the cardiac Sodium Channel Nav1.5
bioRxiv, 2020Co-Authors: Zizun Wang, Jean-sébastien Rougier, Sarah H. Vermij, Valentin Sottas, Anna Shestak, Daniela Ross-kaschitza, E. V. Zaklyazminskaya, Andy Hudmon, Hugues AbrielAbstract:ABSTRACT The cardiac voltage-gated Sodium Channel Nav1.5 conducts the rapid inward Sodium current crucial for cardiomyocyte excitability. Loss-of-function mutations in its gene SCN5A are linked to cardiac arrhythmias such as Brugada Syndrome (BrS). Several BrS-associated mutations in the Nav1.5 N-terminal domain exert a dominant-negative effect (DNE) on wild-type Channel function, for which mechanisms remain poorly understood. We aim to contribute to the understanding of BrS pathophysiology by characterizing three mutations in the Nav1.5 N-terminal domain (NTD): Y87C–here newly identified–, R104W and R121W. In addition, we hypothesize that the calcium sensor protein calmodulin is a new NTD binding partner. Recordings of whole-cell Sodium currents in TsA-201 cells expressing WT and variant Nav1.5 showed that Y87C and R104W but not R121W exert a DNE on WT Channels. Biotinylation assays revealed reduction in fully glycosylated Nav1.5 at the cell surface and in whole-cell lysates. Localization of Nav1.5 WT Channel with the ER however did not change in the presence of variants, shown by transfected and stained rat neonatal cardiomyocytes. We next demonstrated that calmodulin binds Nav1.5 N-terminus using in silico modeling, SPOTS, pull-down and proximity ligation assays. This binding is impaired in the R121W variant and in a Nav1.5 construct missing residues 80-105, a predicted calmodulin binding site. In conclusion, we present the first evidence that calmodulin binds to the Nav1.5 NTD, which seems to be a determinant for the DNE.
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Calmodulin binds to the N-terminal domain of the cardiac Sodium Channel Nav1.5.
Channels, 2020Co-Authors: Zizun Wang, Jean-sébastien Rougier, Sarah H. Vermij, Valentin Sottas, Anna Shestak, Daniela Ross-kaschitza, E. V. Zaklyazminskaya, Andy Hudmon, Hugues AbrielAbstract:The cardiac voltage-gated Sodium Channel Nav1.5 conducts the rapid inward Sodium current crucial for cardiomyocyte excitability. Loss-of-function mutations in its gene SCN5A are linked to cardiac arrhythmias such as Brugada Syndrome (BrS). Several BrS-associated mutations in the Nav1.5 N-terminal domain (NTD) exert a dominant-negative effect (DNE) on wild-type Channel function, for which mechanisms remain poorly understood. We aim to contribute to the understanding of BrS pathophysiology by characterizing three mutations in the Nav1.5 NTD: Y87C-here newly identified-, R104W, and R121W. In addition, we hypothesize that the calcium sensor protein calmodulin is a new NTD binding partner. Recordings of whole-cell Sodium currents in TsA-201 cells expressing WT and variant Nav1.5 showed that Y87C and R104W but not R121W exert a DNE on WT Channels. Biotinylation assays revealed reduction in fully glycosylated Nav1.5 at the cell surface and in whole-cell lysates. Localization of Nav1.5 WT Channel with the ER did not change in the presence of variants, as shown by transfected and stained rat neonatal cardiomyocytes. We demonstrated that calmodulin binds the Nav1.5 NTD using in silico modeling, SPOTS, pull-down, and proximity ligation assays. Calmodulin binding to the R121W variant and to a Nav1.5 construct missing residues 80-105, a predicted calmodulin-binding site, is impaired. In conclusion, we describe the new natural BrS Nav1.5 variant Y87C and present first evidence that calmodulin binds to the Nav1.5 NTD, which seems to be a determinant for the DNE.
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Calmodulin binds to the N-terminal domain of the cardiac Sodium Channel Nav1.5
Channels (Austin Tex.), 2020Co-Authors: Zizun Wang, Geoffrey S. Pitt, Jean-sébastien Rougier, Sarah H. Vermij, Valentin Sottas, Anna Shestak, Daniela Ross-kaschitza, E. V. Zaklyazminskaya, Andy Hudmon, Hugues AbrielAbstract:The cardiac voltage-gated Sodium Channel Nav1.5 conducts the rapid inward Sodium current crucial for cardiomyocyte excitability. Loss-of-function mutations in its gene SCN5A are linked to cardiac a...
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pdz domain binding motif regulates cardiomyocyte compartment specific Nav1.5 Channel expression and function
Circulation, 2014Co-Authors: Ludovic Gillet, Jean-sébastien Rougier, Maxime Albesa, Jakob Ogrodnik, Arie O Verkerk, Rianne Wolswinkel, Julien Barc, Maria C Essers, Ninda Syam, Roos F MarsmanAbstract:Background—Sodium Channel Nav1.5 underlies cardiac excitability and conduction. The last 3 residues of Nav1.5 (Ser-Ile-Val) 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 Nav1.5 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 (ΔSIV). ΔSIV mice displayed reduced Nav1.5 expression and Sodium current (INa), specifically at the lateral myocyte membrane, whereas Nav1.5 expression and INa at the intercalated disks were unaffected. Optical mapping of ΔSIV hearts revealed that ventricular conduction velocity was preferentially decreased in the transversal direction to myocardial fiber orientation, leading to increased anisotropy of ventricula...
Walter J. Chazin - One of the best experts on this subject based on the ideXlab platform.
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Solution NMR Structure of Apo-Calmodulin in Complex with the IQ Motif of Human Cardiac Sodium Channel Nav1.5
Journal of Molecular Biology, 2011Co-Authors: Benjamin Chagot, Walter J. ChazinAbstract:The function of the human voltage-gated Sodium Channel Na V 1.5 is regulated in part by intracellular calcium signals. The ubiquitous calcium sensor protein calmodulin (CaM) is an important part of the complex calcium-sensing apparatus in Na V 1.5. CaM interacts with an IQ motif in the large intracellular C-terminal domain of the Channel. Using co-expression and co-purification, we have been able to isolate a CaM-IQ motif complex and to determine its high-resolution structure in absence of calcium using multi-dimensional solution NMR. Under these conditions, the Na V 1.5 IQ motif interacts with the C-terminal domain (C-lobe) of CaM, with the N-terminal domain remaining free in solution. The structure reveals that the C-lobe adopts a semi-open conformation with the IQ motif bound in a narrow hydrophobic groove. Sequence similarities between voltage-gated Sodium Channels and voltage-gated calcium Channels suggest that the structure of the CaM-Na V 1.5 IQ motif complex can serve as a general model for the interaction between CaM and ion Channels IQ motifs under conditions of low calcium. The structure also provides insight into the biochemical basis for disease-associated mutations that map to the IQ motif in Na V 1.5.
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Solution NMR Structure of Apo-Calmodulin in Complex with the IQ Motif of Human Cardiac Sodium Channel Nav1.5
Journal of Molecular Biology, 2010Co-Authors: Benjamin Chagot, Walter J. ChazinAbstract:The function of the human voltage-gated Sodium Channel Nav1.5 is regulated in part by intracellular calcium signals. The ubiquitous calcium sensor protein calmodulin (CaM) is an important part of the complex calcium-sensing apparatus in Nav1.5. CaM interacts with an IQ (isoleucine–glutamine) motif in the large intracellular C-terminal domain of the Channel. Using co-expression and co-purification, we have been able to isolate a CaM–IQ motif complex and to determine its high-resolution structure in absence of calcium using multi-dimensional solution NMR. Under these conditions, the Nav1.5 IQ motif interacts with the C-terminal domain (C-lobe) of CaM, with the N-terminal domain remaining free in solution. The structure reveals that the C-lobe adopts a semi-open conformation with the IQ motif bound in a narrow hydrophobic groove. Sequence similarities between voltage-gated Sodium Channels and voltage-gated calcium Channels suggest that the structure of the CaM–Nav1.5 IQ motif complex can serve as a general model for the interaction between CaM and ion Channel IQ motifs under low-calcium conditions. The structure also provides insight into the biochemical basis for disease-associated mutations that map to the IQ motif in Nav1.5.
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Solution NMR structure of the C-terminal EF-hand domain of human cardiac Sodium Channel Nav1.5
Journal of Biological Chemistry, 2008Co-Authors: Benjamin Chagot, Franck Potet, Jeffrey R. Balser, Walter J. ChazinAbstract:Abstract The voltage-gated Sodium Channel Nav1.5 is responsible for the initial upstroke of the action potential in cardiac tissue. Levels of intracellular calcium modulate inactivation gating of Nav1.5, in part through a C-terminal EF-hand calcium binding domain. The significance of this structure is underscored by the fact that mutations within this domain are associated with specific cardiac arrhythmia syndromes. In an effort to elucidate the molecular basis for calcium regulation of Channel function, we have determined the solution structure of the C-terminal EF-hand domain using multidimensional heteronuclear NMR. The structure confirms the existence of the four-helix bundle common to EF-hand domain proteins. However, the location of this domain is shifted with respect to that predicted on the basis of a consensus 12-residue EF-hand calcium binding loop in the sequence. This finding is consistent with the weak calcium affinity reported for the isolated EF-hand domain; high affinity binding is observed only in a construct with an additional 60 residues C-terminal to the EF-hand domain, including the IQ motif that is central to the calcium regulatory apparatus. The binding of an IQ motif peptide to the EF-hand domain was characterized by isothermal titration calorimetry and nuclear magnetic resonance spectroscopy. The peptide binds between helices I and IV in the EF-hand domain, similar to the binding of target peptides to other EF-hand calcium-binding proteins. These results suggest a molecular basis for the coupling of the intrinsic (EF-hand domain) and extrinsic (calmodulin) components of the calcium-sensing apparatus of Nav1.5.
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Calcium-dependent regulation of the voltage-gated Sodium Channel hH1: intrinsic and extrinsic sensors use a common molecular switch.
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Vikas N. Shah, Jeffrey R. Balser, Tammy L Wingo, Kevin L. Weiss, Christina K. Williams, Walter J. ChazinAbstract:The function of the human cardiac voltage-gated Sodium Channel Nav1.5 (hH1) is regulated in part by binding of calcium to an EF hand in the C-terminal cytoplasmic domain. hH1 is also regulated via an extrinsic calcium-sensing pathway mediated by calmodulin (CaM) via binding to an IQ motif immediately adjacent to the EF-hand domain. The intrinsic EF-hand domain is shown here to interact with the IQ motif, which controls calcium affinity. Remarkably, mutation of the IQ residues has only a minor effect on CaM affinity but drastically reduces calcium affinity of the EF-hand domain, whereas the Brugada mutation A1924T significantly reduces CaM affinity but has no effect on calcium affinity of the EF-hand domain. Moreover, the differences in the biochemical effects of the mutations directly correlate with contrasting effects on Channel electrophysiology. A comprehensive model is proposed in which the hH1 IQ motif serves as a molecular switch, coupling the intrinsic and extrinsic calcium sensors.
David K. Jones - One of the best experts on this subject based on the ideXlab platform.
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Proton modulation of cardiac I Na: a potential arrhythmogenic trigger.
Handbook of experimental pharmacology, 2014Co-Authors: David K. Jones, Peter C. RubenAbstract:Voltage-gated Sodium (NaV) Channels generate the upstroke and mediate duration of the ventricular action potential, thus they play a critical role in mediating cardiac excitability. Cardiac ischemia triggers extracellular pH to drop as low as pH 6.0, within just 10 min of its onset. Heightened proton concentrations reduce Sodium conductance and alter the gating parameters of the cardiac-specific voltage-gated Sodium Channel, Nav1.5. Most notably, acidosis destabilizes fast inactivation, which plays a critical role in regulating action potential duration. The changes in Nav1.5 Channel gating contribute to cardiac dysfunction during ischemia that can cause syncope, cardiac arrhythmia, and even sudden cardiac death. Understanding NaV Channel modulation by protons is paramount to treatment and prevention of the deleterious effects of cardiac ischemia and other triggers of cardiac acidosis.
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Proton Modulation of Gating Currents in the Cardiac Voltage-Gated Sodium Channel, Nav1.5
Biophysical Journal, 2012Co-Authors: David K. Jones, Thomas W. Claydon, Peter C. RubenAbstract:Low pH reduces single Channel conductance and destabilizes the fast-inactivated state of the cardiac voltage-gated Sodium Channel (Nav1.5) by increasing both window and persistent currents (Jones et al., 2011, Biophys. J. 101(7)). Since fast inactivation is tightly coupled to NaV Channel voltage sensor activation, we hypothesized that alterations in the kinetics of voltage sensor movement may underlie the destabilization of the fast-inactivated state in Nav1.5. To test this, we expressed Nav1.5 Channels in Xenopus oocytes and recorded gating currents using a cut-open voltage clamp with extracellular solution titrated to either pH 7.4 (control) or pH 6.0. At pH 6.0, compared to pH 7.4, the V1/2 of the Q(V) curve was significantly depolarized (from −57.8±4.3 mV to −40.8±5.1 mV). Additionally, the slow time constant of charge recovery was significantly reduced from 16.1±5.0 ms at pH 7.4 to 9.7±4.2 ms at pH 6.0. These data suggest a molecular basis for the increased persistent and window currents previously shown in Nav1.5 Channels at reduced extracellular pH. Specifically, protons may electrostatically affect the rate of voltage sensor movement, either by directly binding to extracellular residues (e.g. H880) or indirectly by binding to carboxylates in the pore domain (Kahn et al., J Physiol. 2002, 543). (Supported by an NSERC Discovery Grant to PCR, a CFI Infrastructure grant to PCR and TC, and a CIHR Vanier Scholarship to DKJ.)
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Extracellular proton modulation of the cardiac voltage-gated Sodium Channel, Nav1.5.
Biophysical Journal, 2011Co-Authors: David K. Jones, Colin H. Peters, Thomas W. Claydon, S.a. Tolhurst, Peter C. RubenAbstract:Low pH depolarizes the voltage dependence of voltage-gated Sodium (NaV) Channel activation and fast inactivation. A complete description of NaV Channel proton modulation, however, has not been reported. The majority of NaV Channel proton modulation studies have been completed in intact tissue. Additionally, several NaV Channel isoforms are expressed in cardiac tissue. Characterizing the proton modulation of the cardiac NaV Channel, Nav1.5, will thus help define its contribution to ischemic arrhythmogenesis, where extracellular pH drops from pH 7.4 to as low as pH 6.0 within ∼10 min of its onset. We expressed the human variant of Nav1.5 with and without the modulating β1 subunit in Xenopus oocytes. Lowering extracellular pH from 7.4 to 6.0 affected a range of biophysical gating properties heretofore unreported. Specifically, acidic pH destabilized the fast-inactivated and slow-inactivated states, and elevated persistent INa. These data were incorporated into a ventricular action potential model that displayed a reduced maximum rate of depolarization as well as disparate increases in epicardial, mid-myocardial, and endocardial action potential durations, indicative of an increased heterogeneity of repolarization. Portions of these data were previously reported in abstract form.
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pH Modulation of the Cardiac Voltage Gated Sodium Channel, Nav1.5
Biophysical Journal, 2010Co-Authors: David K. Jones, Thomas W. Claydon, Peter C. RubenAbstract:Alterations in the function of the cardiac voltage-gated Sodium Channel (Nav1.5) are a known cause of cardiac disease and arrhythmia. Elevated concentrations of protons decrease conductance and depolarize the voltage dependence of activation and steady-state fast inactivation (SSFI) of Nav1.5 Channels (Zhang & Siegelbaum, 1991, Khan et al., 2006). A complete analysis of the effects of low pH on Nav1.5 Channel kinetics has not previously been reported. We sought to characterize the effects of low pH on Nav1.5 kinetics. Nav1.5 was co-expressed in Xenopus laevis oocytes with the β1 subunit, and currents were recorded at 20 °C using the cut-open voltage clamp technique with the extracellular solution titrated to either pH 7.4 (control) or pH 6.0. Application of solution at pH 6.0 significantly depolarized the voltage dependence of activation and SSFI; −34.4 ± 0.3mV to −25.2 ± 0.2mV and −76.4 ± 0.1mV to −72.7 ± 0.2mV, respectively. The apparent valences of activation and SSFI were significantly decreased; from 3.4 ± 0.12e to 2.5 ± 0.04e, and from −4.6 ± 0.07e to −4.1 ± 0.09e, respectively. At pH 6.0, the fast time constant of use-dependent inactivation was significantly increased and the use dependent current reduction was decreased from 40.6 ± 0.12% to 34.8 ± 0.05%. The rates of open-state fast inactivation onset were significantly decreased at potentials between −30mV and +30mV, and the rates of recovery at −90mV and −80mV were significantly increased. There was also a visible increase in window current. All effects were reversible upon reperfusion of solution at pH 7.4. Taken together, these data suggest that lowering extracellular pH from 7.4 to 6.0 destabilizes the fast-inactivated state of Nav1.5 Channels, an effect that could act as an arrhythmogenic trigger during ischemic events.