Mechanisms of atrial-selective block of Na+ channels by ranolazine: I. Experimental analysis of the use-dependent block

Atrial-selective inhibition of cardiac Na+ channel current ( INa) and INa-dependent parameters has been shown to contribute to the safe and effective management of atrial fibrillation. The present study examined the basis for the atrial-selective actions of ranolazine. Whole cell INa was recorded at 15°C in canine atrial and ventricular myocytes and in human embryonic kidney (HEK)-293 cells expressing SCN5A. Tonic block was negligible at holding potentials from −140 to −100 mV, suggesting minimal drug interactions with the closed state. Trains of 40 pulses were elicited over a range of holding potentials to determine use-dependent block. Guarded receptor formalism was used to analyze the development of block during pulse trains. Use-dependent block by ranolazine increased at more depolarized holding potentials, consistent with an interaction of the drug with either preopen or inactivated states, but was unaffected by longer pulse durations between 5 and 200 ms, suggesting a weak interaction with the inactivated state. Block was significantly increased at shorter diastolic intervals between 20 and 200 ms. Responses in atrial and ventricular myocytes and in HEK-293 cells displayed a similar pattern. Ranolazine is an open state blocker that unbinds from closed Na+ channels unusually fast but is trapped in the inactivated state. Kinetic rates of ranolazine interactions with different states of atrial and ventricular Na+ channels were similar. Our data suggest that the atrial selectivity of ranolazine is due to a more negative steady-state inactivation curve, less negative resting membrane potential, and shorter diastolic intervals in atrial cells compared with ventricular cells at rapid rates.

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Mexiletine rescues the mixed phenotype in SCN5A-1795insD hiPSC-CMs

EP Europace ◽

10.1093/europace/euab116.584 ◽

2021 ◽

Vol 23 (Supplement_3) ◽

Author(s):

G Nasilli ◽

L Yiangou ◽

C Palandri ◽

AO Verkerk ◽

RP Davis ◽

...

Keyword(s):

Sodium Current ◽

Hek293 Cells ◽

Resting Membrane Potential ◽

Loss Of Function ◽

Hek 293 ◽

Hek 293 Cells ◽

Funding Sources ◽

293 Cells ◽

Induced Pluripotent ◽

Mixed Phenotype

Abstract Funding Acknowledgements Type of funding sources: None. Background The sodium channel blocker mexiletine can reduce late sodium current (INa) in patients with LQT3 syndrome, and additionally restore the decreased peak INa associated with SCN5A loss of function mutations. Purpose To investigate whether mexiletinecan rescue the mixed phenotype associated with the SCN5A-1795insD mutation in human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs). Methods and Results HEK293 cells transfected with SCN5A-1795insD and SCN5A-WT and hiPSC-CMs from a patient carrying the SCN5A-1795insD mutation were incubated with a therapeutic dose of mexiletine (10 µM) or vehicle (H2O) for 48h. Peak INa, late INa and action potential (AP) properties were assessed by patch-clamp analysis. In HEK-293 cells transfected with SCN5A-1795insD or SCN5A-WT, exposure to mexiletine caused a significant increase in peak INa, in addition to a small increase in late INa in HEK-293 cells transfected with SCN5A-1795insD. In 1795insD hiPSC-CMs, peak INa was significantly increased whereas late INa was unchanged after mexiletine treatment. Accordingly, mexiletine increased AP upstroke velocity in SCN5A-1795insD hiPSC-CMs (indicating a rescue of INa availability), while AP amplitude, resting membrane potential and AP duration were unaffected. Conclusions Chronic treatment with a therapeutic concentration of mexiletine is capable of rescuing the mixed phenotype in SCN5A-1795insD hiPSC-CMs.

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Lamin-A/C variants found in patients with cardiac conduction disease reduce sodium currents

Cardiogenetics ◽

10.4081/cardiogenetics.2018.7127 ◽

2018 ◽

Vol 8 (1) ◽

Author(s):

Michael A. Olaopa ◽

Katherine G. Spoonamore ◽

Deepak Bhakta ◽

Zhenhui Chen ◽

Patricia B.S. Celestino-Soper ◽

...

Keyword(s):

Steady State ◽

Patch Clamp ◽

Missense Variant ◽

Cardiac Conduction ◽

Lamin A ◽

Lmna Gene ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Steady State Inactivation

Variants in the LMNA gene, which encodes Lamin-A/C, have been commonly associated with cardiac conduction system diseases usually accompanying cardiomyopathy. We have seen two unrelated patients who presented with atrioventricular block (AVB) with or without cardiomyopathy. Genetic testing identified the LMNA missense variant c.1634G>A (p.R545H) and the single nucleotide deletion c.859delG (p.A287Lfs*193). The deletion leads to a shift in the reading frame and subsequent protein truncation. Since impaired Nav1.5 function has been reported to cause AVB, we sought to investigate the effects of abnormal Lamins on Nav1.5 in HEK-293 cells using patch-clamp methods. Patch-clamp studies showed that p.R545H decreased the peak INa by approximately 70%. The voltage-dependency of steady state inactivation was rightward shifted in the cells transfected with p.R545H. The p.A287Lfs*193 also decreased the peak INa by approximately 62%. The voltagedependency of steady state inactivation was rightward shifted in the cells transfected with p.A287Lfs*193. Variants of the LMNA gene caused significant reduction of the peak INa in HEK-293 cells, which may account for the patients’ AVB.

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K201 (JTV519) suppresses spontaneous Ca2+ release and [3H]ryanodine binding to RyR2 irrespective of FKBP12.6 association

Biochemical Journal ◽

10.1042/bj20070135 ◽

2007 ◽

Vol 404 (3) ◽

pp. 431-438 ◽

Cited By ~ 64

Author(s):

Donald J. Hunt ◽

Peter P. Jones ◽

Ruiwu Wang ◽

Wenqian Chen ◽

Jeff Bolstad ◽

...

Keyword(s):

Ventricular Myocytes ◽

Dependent Manner ◽

Wild Type ◽

Concentration Dependent Manner ◽

Hek 293 ◽

Human Embryonic Kidney Cells ◽

Hek 293 Cells ◽

Fk506 Binding Protein ◽

293 Cells

K201 (JTV519), a benzothiazepine derivative, has been shown to possess anti-arrhythmic and cardioprotective properties, but the mechanism of its action is both complex and controversial. It is believed to stabilize the closed state of the RyR2 (cardiac ryanodine receptor) by increasing its affinity for the FKBP12.6 (12.6 kDa FK506 binding protein) [Wehrens, Lehnart, Reiken, Deng, Vest, Cervantes, Coromilas, Landry and Marks (2004) Science 304, 292–296]. In the present study, we investigated the effect of K201 on spontaneous Ca2+ release induced by Ca2+ overload in rat ventricular myocytes and in HEK-293 cells (human embryonic kidney cells) expressing RyR2 and the role of FKBP12.6 in the action of K201. We found that K201 abolished spontaneous Ca2+ release in cardiac myocytes in a concentration-dependent manner. Treating ventricular myocytes with FK506 to dissociate FKBP12.6 from RyR2 did not affect the suppression of spontaneous Ca2+ release by K201. Similarly, K201 was able to suppress spontaneous Ca2+ release in FK506-treated HEK-293 cells co-expressing RyR2 and FKBP12.6. Furthermore, K201 suppressed spontaneous Ca2+ release in HEK-293 cells expressing RyR2 alone and in cells co-expressing RyR2 and FKBP12.6 with the same potency. In addition, K201 inhibited [3H]ryanodine binding to RyR2-wt (wild-type) and an RyR2 mutant linked to ventricular tachycardia and sudden death, N4104K, in the absence of FKBP12.6. These observations demonstrate that FKBP12.6 is not involved in the inhibitory action of K201 on spontaneous Ca2+ release. Our results also suggest that suppression of spontaneous Ca2+ release and the activity of RyR2 contributes, at least in part, to the anti-arrhythmic properties of K201.

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Lysophosphatidylcholine Augments Cav3.2 but Not Cav3.1 T-Type Ca2+ Channel Current Expressed in HEK-293 Cells

Pharmacology ◽

10.1159/000092041 ◽

2006 ◽

Vol 76 (4) ◽

pp. 192-200 ◽

Cited By ~ 8

Author(s):

Mingqi Zheng ◽

Tomoko Uchino ◽

Toshihiko Kaku ◽

Lin Kang ◽

Yan Wang ◽

...

Keyword(s):

Hek 293 ◽

Hek 293 Cells ◽

Channel Current ◽

293 Cells

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Open form of syntaxin-1A is a more potent inhibitor than wild-type syntaxin-1A of Kv2.1 channels

Biochemical Journal ◽

10.1042/bj20041625 ◽

2005 ◽

Vol 387 (1) ◽

pp. 195-202 ◽

Cited By ~ 22

Author(s):

Yuk M. LEUNG ◽

Youhou KANG ◽

Fuzhen XIA ◽

Laura SHEU ◽

Xiaodong GAO ◽

...

Keyword(s):

Current Density ◽

Open Form ◽

Wild Type ◽

Syntaxin 1A ◽

Inactivation Curve ◽

Hek 293 ◽

Channel Trafficking ◽

Hek 293 Cells ◽

Kv Channels ◽

293 Cells

We have shown that SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins not only participate directly in exocytosis, but also regulate the dominant membrane-repolarizing Kv channels (voltage-gated K+ channels), such as Kv2.1, in pancreatic β-cells. In a recent report, we demonstrated that WT (wild-type) Syn-1A (syntaxin-1A) inhibits Kv2.1 channel trafficking and gating through binding to the cytoplasmic C-terminus of Kv2.1. During β-cell exocytosis, Syn-1A converts from a closed form into an open form which reveals its active H3 domain to bind its SNARE partners SNAP-25 (synaptosome-associated protein of 25 kDa) and synaptobrevin. In the present study, we compared the effects of the WT Syn-1A and a mutant open form Syn-1A (L165A, E166A) on Kv2.1 channel trafficking and gating. When co-expressed in HEK-293 cells (human embryonic kidney-293 cells), the open form Syn-1A decreased Kv2.1 current density more than (P<0.05) the WT Syn-1A (166±35 and 371±93 pA/pF respectively; control=911±91 pA/pF). Confocal microscopy and biotinylation experiments showed that both the WT and open form Syn-1A inhibited Kv2.1 expression at the plasma membrane to a similar extent, suggesting that the stronger reduction of Kv2.1 current density by the open form compared with the WT Syn-1A is probably due to a stronger direct inhibition of channel activity. Consistently, dialysis of the recombinant open form Syn-1A protein into Kv2.1-expressing HEK-293 cells caused stronger inhibition of Kv2.1 current amplitude (P<0.05) than the WT Syn-1A protein (73±2 and 82±3% of the control respectively). We found that the H3 but not HABC domain is the putative active domain of Syn-1A, which bound to and inhibited the Kv2.1 channel. When co-expressed in HEK-293 cells, the open-form Syn-1A slowed down Kv2.1 channel activation (τ=12.3±0.8 ms) much more than (P<0.05) WT Syn-1A (τ=7.9±0.8 ms; control τ=5.5±0.6 ms). In addition, only the open form Syn-1A, but not the WT Syn-1A, caused a significant (P<0.05) left-shift in the steady-state inactivation curve (V1/2=33.1±1.3 and −29.4±1.1 mV respectively; control V1/2=−24.8±2 mV). The present study therefore indicates that the open form of Syn-1A is more potent than the WT Syn-1A in inhibiting the Kv2.1 channel. Such stronger inhibition by the open form of Syn-1A may limit K+ efflux and thus decelerate membrane repolarization during exocytosis, leading to optimization of insulin release.

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Characteristics and requirements of basal autophagy in HEK 293 cells

Autophagy ◽

10.4161/auto.25455 ◽

2013 ◽

Vol 9 (9) ◽

pp. 1407-1417 ◽

Cited By ~ 41

Author(s):

Patience Musiwaro ◽

Matthew Smith ◽

Maria Manifava ◽

Simon A. Walker ◽

Nicholas T. Ktistakis

Keyword(s):

Hek 293 ◽

Hek 293 Cells ◽

293 Cells

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Halothane Inhibition of Recombinant Cardiac L-type Ca2+ Channels Expressed in HEK-293 Cells

Anesthesiology ◽

10.1097/00000542-200512000-00009 ◽

2005 ◽

Vol 103 (6) ◽

pp. 1156-1166 ◽

Cited By ~ 7

Author(s):

Kevin J. Gingrich ◽

Son Tran ◽

Igor M. Nikonorov ◽

Thomas J. Blanck

Keyword(s):

Charge Transfer ◽

Calcium Channels ◽

Dependent Manner ◽

Current Time ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Dependent Inactivation ◽

Voltage Dependent

Background Volatile anesthetics depress cardiac contractility, which involves inhibition of cardiac L-type calcium channels. To explore the role of voltage-dependent inactivation, the authors analyzed halothane effects on recombinant cardiac L-type calcium channels (alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1), which differ by the alpha2/delta1 subunit and consequently voltage-dependent inactivation. Methods HEK-293 cells were transiently cotransfected with complementary DNAs encoding alpha1C tagged with green fluorescent protein and beta2a, with and without alpha2/delta1. Halothane effects on macroscopic barium currents were recorded using patch clamp methodology from cells expressing alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1 as identified by fluorescence microscopy. Results Halothane inhibited peak current (I(peak)) and enhanced apparent inactivation (reported by end pulse current amplitude of 300-ms depolarizations [I300]) in a concentration-dependent manner in both channel types. alpha2/delta1 coexpression shifted relations leftward as reported by the 50% inhibitory concentration of I(peak) and I300/I(peak)for alpha1Cbeta2a (1.8 and 14.5 mm, respectively) and alpha1Cbeta2aalpha2/delta1 (0.74 and 1.36 mm, respectively). Halothane reduced transmembrane charge transfer primarily through I(peak) depression and not by enhancement of macroscopic inactivation for both channels. Conclusions The results indicate that phenotypic features arising from alpha2/delta1 coexpression play a key role in halothane inhibition of cardiac L-type calcium channels. These features included marked effects on I(peak) inhibition, which is the principal determinant of charge transfer reductions. I(peak) depression arises primarily from transitions to nonactivatable states at resting membrane potentials. The findings point to the importance of halothane interactions with states present at resting membrane potential and discount the role of inactivation apparent in current time courses in determining transmembrane charge transfer.

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