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.

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 Sodium Channel Nav1.3 Is Expressed by Polymorphonuclear Neutrophils during Mouse Heart and Kidney Ischemia InVivo and Regulates Adhesion, Transmigration, and Chemotaxis of Human and Mouse Neutrophils In Vitro

2018 ◽  
Vol 128 (6) ◽  
pp. 1151-1166 ◽  
Author(s):  
Marit Poffers ◽  
Nathalie Bühne ◽  
Christine Herzog ◽  
Anja Thorenz ◽  
Rongjun Chen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Sodium Channel ◽  
Sodium Channels ◽  
Polymorphonuclear Neutrophils ◽  
Human Neutrophils ◽  
Mouse Heart ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels

Abstract Background Voltage-gated sodium channels generate action potentials in excitable cells, but they have also been attributed noncanonical roles in nonexcitable cells. We hypothesize that voltage-gated sodium channels play a functional role during extravasation of neutrophils. Methods Expression of voltage-gated sodium channels was analyzed by polymerase chain reaction. Distribution of Nav1.3 was determined by immunofluorescence and flow cytometry in mouse models of ischemic heart and kidney injury. Adhesion, transmigration, and chemotaxis of neutrophils to endothelial cells and collagen were investigated with voltage-gated sodium channel inhibitors and lidocaine in vitro. Sodium currents were examined with a whole cell patch clamp. Results Mouse and human neutrophils express multiple voltage-gated sodium channels. Only Nav1.3 was detected in neutrophils recruited to ischemic mouse heart (25 ± 7%, n = 14) and kidney (19 ± 2%, n = 6) in vivo. Endothelial adhesion of mouse neutrophils was reduced by tetrodotoxin (56 ± 9%, unselective Nav-inhibitor), ICA121431 (53 ± 10%), and Pterinotoxin-2 (55 ± 9%; preferential inhibitors of Nav1.3, n = 10). Tetrodotoxin (56 ± 19%), ICA121431 (62 ± 22%), and Pterinotoxin-2 (59 ± 22%) reduced transmigration of human neutrophils through endothelial cells, and also prevented chemotactic migration (n = 60, 3 × 20 cells). Lidocaine reduced neutrophil adhesion to 60 ± 9% (n = 10) and transmigration to 54 ± 8% (n = 9). The effect of lidocaine was not increased by ICA121431 or Pterinotoxin-2. Conclusions Nav1.3 is expressed in neutrophils in vivo; regulates attachment, transmigration, and chemotaxis in vitro; and may serve as a relevant target for antiinflammatory effects of lidocaine.

scholarly journals Axons provide the secretory machinery for trafficking of voltage-gated sodium channels in peripheral nerve

2016 ◽  
Vol 113 (7) ◽  
pp. 1823-1828 ◽  
Author(s):  
Carolina González ◽  
José Cánovas ◽  
Javiera Fresno ◽  
Eduardo Couve ◽  
Felipe A. Court ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Sodium Channel ◽  
Sodium Channels ◽  
Neural Function ◽  
Axonal Membrane ◽  
Local Synthesis ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels

The regulation of the axonal proteome is key to generate and maintain neural function. Fast and slow axoplasmic waves have been known for decades, but alternative mechanisms to control the abundance of axonal proteins based on local synthesis have also been identified. The presence of the endoplasmic reticulum has been documented in peripheral axons, but it is still unknown whether this localized organelle participates in the delivery of axonal membrane proteins. Voltage-gated sodium channels are responsible for action potentials and are mostly concentrated in the axon initial segment and nodes of Ranvier. Despite their fundamental role, little is known about the intracellular trafficking mechanisms that govern their availability in mature axons. Here we describe the secretory machinery in axons and its contribution to plasma membrane delivery of sodium channels. The distribution of axonal secretory components was evaluated in axons of the sciatic nerve and in spinal nerve axons after in vivo electroporation. Intracellular protein trafficking was pharmacologically blocked in vivo and in vitro. Axonal voltage-gated sodium channel mRNA and local trafficking were examined by RT-PCR and a retention-release methodology. We demonstrate that mature axons contain components of the endoplasmic reticulum and other biosynthetic organelles. Axonal organelles and sodium channel localization are sensitive to local blockade of the endoplasmic reticulum to Golgi transport. More importantly, secretory organelles are capable of delivering sodium channels to the plasma membrane in isolated axons, demonstrating an intrinsic capacity of the axonal biosynthetic route in regulating the axonal proteome in mammalian axons.

scholarly journals Stereoselectivity of Propafenone RyR2 Channel Block and Prevention of Ventricular Arrhythmia In Vivo

2011 ◽  
Vol 100 (3) ◽  
pp. 180a
Author(s):  
Michela Faggione ◽  
Hyun Seok Hwang ◽  
Bjorn C. Knollmann
Keyword(s):  
Ventricular Arrhythmia ◽  
Channel Block ◽  
Ryr2 Channel

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.

Antiepileptic Drug Cellular Mechanisms of Action: Where Does Lamotrigine Fit In?

1997 ◽  
Vol 12 (1_suppl) ◽  
pp. S2-S9 ◽  
Author(s):  
Douglas A. Coulter
Keyword(s):  
Sodium Channel ◽  
Antiepileptic Drugs ◽  
Sodium Channels ◽  
Clinical Efficacy ◽  
Calcium Currents ◽  
Channel Block ◽  
Cellular Mechanisms ◽  
Absence Seizures ◽  
Gaba A ◽  
Low Threshold

Current frontline antiepileptic drugs tend to fall into several cellular mechanistic categories, and these categories often correlate with the clinical spectrum of action of the various antiepileptic drugs. Many antiepileptic drugs effective in control of partial and generalized tonic-clonic seizures are use- and voltage-dependent blockers of sodium channels. This mechanism selectively dampens pathologic activation of sodium channels, without interacting with normal sodium channel function. Examples include phenytoin, carbamazepine, valproic acid, and lamotrigine. Many antiepileptic drugs effective in control of generalized absence seizures block low threshold calcium currents. Low threshold calcium channels are present in high densities in thalamic neurons, and these channels trigger regenerative bursts that drive normal and pathologic thalamocortical rhythms, including the spike wave discharges of absence seizures. Examples include ethosuximide, trimethadione, and methsuximide. Several antiepileptic drugs that have varying clinical actions interact with the γ-aminobutyric acid (GABA)ergic system. Diazepam and clonazepam selectively augment function of a subset of GABA A receptors, and these drugs are broad-spectrum antiepileptic drugs. In contrast, barbiturates augment function of all types of GABAA receptors, and are ineffective in control of generalized absence seizures, but effective in control of many other seizure types. Tiagabine and vigabatrin enhance cerebrospinal levels of GABA by interfering with reuptake and degradation of GABA, respectively. These antiepileptic drugs are effective in partial seizures. Lamotrigine is effective against both partial and generalized seizures, including generalized absence seizures. Its sole documented cellular mechanism of action is sodium channel block, a mechanism shared by phenytoin and carbamazepine. These drugs are ineffective against absence seizures. Consequently, unless there are unique aspects to the sodium channel block by lamotrigine, it seems unlikely that this mechanism alone could explain its broad clinical efficacy. Therefore, lamotrigine may have as yet uncharacterized cellular actions, which could combine with its sodium channel blocking actions, to account for its broad clinical efficacy. (J Child Neurol 1997;12(Suppl 1):S2-S9).

scholarly journals Modified kinetics and selectivity of sodium channels in frog skeletal muscle fibers treated with aconitine.

1982 ◽  
Vol 80 (5) ◽  
pp. 713-731 ◽  
Author(s):  
D T Campbell
Keyword(s):  
Skeletal Muscle ◽  
Sodium Channel ◽  
Sodium Channels ◽  
State Level ◽  
Steady State Level ◽  
Channel Block ◽  
Frog Skeletal Muscle ◽  
Channel Kinetics ◽  
Ionic Selectivity ◽  
Channel Selectivity

The effect of the plant alkaloid aconitine on sodium channel kinetics, ionic selectivity, and blockage by protons and tetrodotoxin (TTX) has been studied in frog skeletal muscle. Treatment with 0.25 or 0.3 mM aconitine alters sodium channels so that the threshold of activation is shifted 40-50 mV in the hyperpolarized direction. In contrast to previous results in frog nerve, inactivation is complete for depolarizations beyond about -60 mV. After aconitine treatment, the steady state level of inactivation is shifted approximately 20 mV in the hyperpolarizing direction. Concomitant with changes in channel kinetics, the relative permeability of the sodium channel to NH4,K, and Cs is increased. This altered selectivity is not accompanied by altered block by protons or TTX. The results suggest that sites other than those involved in channel block by protons and TTX are important in determining sodium channel selectivity.

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 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.

scholarly journals Sodium channel activation underlies transfluthrin repellency in Aedes aegypti

2021 ◽  
Vol 15 (7) ◽  
pp. e0009546
Author(s):  
Felipe Andreazza ◽  
Wilson R. Valbon ◽  
Qiang Wang ◽  
Feng Liu ◽  
Peng Xu ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Sodium Channel ◽  
Sodium Channels ◽  
Human Disease ◽  
Disease Vectors ◽  
Channel Activation ◽  
Principal Mechanism ◽  
Pyrethroid Insecticides ◽  
Voltage Gated Sodium Channels ◽  
Sodium Channel Activation

Background Volatile pyrethroid insecticides, such as transfluthrin, have received increasing attention for their potent repellent activities in recent years for controlling human disease vectors. It has been long understood that pyrethroids kill insects by promoting activation and inhibiting inactivation of voltage-gated sodium channels. However, the mechanism of pyrethroid repellency remains poorly understood and controversial. Methodology/Principal findings Here, we show that transfluthrin repels Aedes aegypti in a hand-in-cage assay at nonlethal concentrations as low as 1 ppm. Contrary to a previous report, transfluthrin does not elicit any electroantennogram (EAG) responses, indicating that it does not activate olfactory receptor neurons (ORNs). The 1S-cis isomer of transfluthrin, which does not activate sodium channels, does not elicit repellency. Mutations in the sodium channel gene that reduce the potency of transfluthrin on sodium channels decrease transfluthrin repellency but do not affect repellency by DEET. Furthermore, transfluthrin enhances DEET repellency. Conclusions/Significance These results provide a surprising example that sodium channel activation alone is sufficient to potently repel mosquitoes. Our findings of sodium channel activation as the principal mechanism of transfluthrin repellency and potentiation of DEET repellency have broad implications in future development of a new generation of dual-target repellent formulations to more effectively repel a variety of human disease vectors.

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