Inhibitory effects of hesperetin on Nav1.5 channels stably expressed in HEK 293 cells and on the voltage-gated cardiac sodium current in human atrial myocytes

Abstract Background: Propofol is widely used clinically for the induction and maintenance of anesthesia. Clinical case reports have shown that propofol has an antiatrial tachycardia/fibrillation effect; however, the related ionic mechanisms are not fully understood. The current study investigates the effects of propofol on human cardiac potassium channels. Methods: The whole cell patch voltage clamp technique was used to record transient outward potassium current (Ito) and ultrarapidly activating delayed rectifier potassium current (IKur) in human atrial myocytes and hKv1.5, human ether-à-go-go-related gene (hERG), and hKCNQ1/hKCNE1 channels stably expressed in HEK 293 cells. Current clamp mode was used to record action potentials in human atrial myocytes. Results: In human atrial myocytes, propofol inhibited Ito in a concentration-dependent manner (IC50 = 33.5 ± 2.0 μM for peak current, n = 6) by blocking open channels without affecting the voltage-dependent kinetics or the recovery time constant; propofol decreased IKur (IC50 = 35.3 ± 1.9 μM, n = 6) in human atrial myocytes and inhibited hKv1.5 current expressed in HEK 293 cells by preferentially binding to the open channels. Action potential duration at 90% repolarization was slightly prolonged by 30 μM propofol in human atrial myocytes. In addition, propofol also suppressed hERG and hKCNQ1/hKCNE1 channels expressed in HEK 293 cells. Conclusion: Propofol inhibits multiple human cardiac potassium channels, including human atrial Ito and IKur, as well as hKv1.5, hERG, and hKCNQ1/hKCNE1 channels stably expressed in HEK 293 cells, and slightly prolongs human atrial action potential duration, which may contribute to the antiatrial tachycardia/fibrillation effects observed in patients who receive propofol.

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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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Molecular determinants of voltage-gated sodium channel regulation by the Nedd4/Nedd4-like proteins

AJP Cell Physiology ◽

10.1152/ajpcell.00460.2004 ◽

2005 ◽

Vol 288 (3) ◽

pp. C692-C701 ◽

Cited By ~ 87

Author(s):

Jean-Sébastien Rougier ◽

Miguel X. van Bemmelen ◽

M. Christine Bruce ◽

Thomas Jespersen ◽

Bruno Gavillet ◽

...

Keyword(s):

Cell Voltage ◽

Tryptophan Fluorescence ◽

General Mechanism ◽

Hek 293 ◽

Ww Domains ◽

Voltage Gated ◽

Hek 293 Cells ◽

293 Cells ◽

Molecular Determinants ◽

Py Motif

The voltage-gated Na+ channels (Nav) form a family composed of 10 genes. The COOH termini of Nav contain a cluster of amino acids that are nearly identical among 7 of the 10 members. This COOH-terminal sequence, PPSYDSV, is a PY motif known to bind to WW domains of E3 protein-ubiquitin ligases of the Nedd4 family. We recently reported that cardiac Nav1.5 is regulated by Nedd4-2. In this study, we further investigated the molecular determinants of regulation of Nav proteins. When expressed in HEK-293 cells and studied using whole cell voltage clamping, the neuronal Nav1.2 and Nav1.3 were also downregulated by Nedd4-2. Pull-down experiments using fusion proteins bearing the PY motif of Nav1.2, Nav1.3, and Nav1.5 indicated that mouse brain Nedd4-2 binds to the Nav PY motif. Using intrinsic tryptophan fluorescence imaging of WW domains, we found that Nav1.5 PY motif binds preferentially to the fourth WW domain of Nedd4-2 with a Kd of ∼55 μM. We tested the binding properties and the ability to ubiquitinate and downregulate Nav1.5 of three Nedd4-like E3s: Nedd4-1, Nedd4-2, and WWP2. Despite the fact that along with Nedd4-2, Nedd4-1 and WWP2 bind to Nav1.5 PY motif, only Nedd4-2 robustly ubiquitinated and downregulated Nav1.5. Interestingly, coexpression of WWP2 competed with the effect of Nedd4-2. Finally, using brefeldin A, we found that Nedd4-2 accelerated internalization of Nav1.5 stably expressed in HEK-293 cells. This study shows that Nedd4-dependent ubiquitination of Nav channels may represent a general mechanism regulating the excitability of neurons and myocytes via modulation of channel density at the plasma membrane.

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Inhibitory effects of flavonoids on TNF-α-induced IL-8 gene expression in HEK 293 cells

BMB Reports ◽

10.5483/bmbrep.2009.42.5.265 ◽

2009 ◽

Vol 42 (5) ◽

pp. 265-270 ◽

Cited By ~ 43

Author(s):

Soo-Hyoung Lee ◽

Young-Jin Kim ◽

Sang-Hoon Kwon ◽

Young-Hee Lee ◽

Soo-Young Choi ◽

...

Keyword(s):

Gene Expression ◽

Inhibitory Effects ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Tnf Α

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Intracellular calcium measurements as a method in studies on activity of purinergic P2X receptor channels

AJP Cell Physiology ◽

10.1152/ajpcell.00042.2003 ◽

2003 ◽

Vol 285 (2) ◽

pp. C467-C479 ◽

Cited By ~ 38

Author(s):

Mu-Lan He ◽

Hana Zemkova ◽

Taka-aki Koshimizu ◽

Melanija Tomić ◽

Stanko S. Stojilkovic

Keyword(s):

Purinergic Receptors ◽

Host Cells ◽

P2x Receptor ◽

Wild Type ◽

Hek 293 ◽

Human Embryonic Kidney Cells ◽

Voltage Gated ◽

Hek 293 Cells ◽

293 Cells ◽

Intracellular Ca

Extracellular nucleotide-activated purinergic receptors (P2XRs) are a family of cation-permeable channels that conduct small cations, including Ca2+, leading to the depolarization of cells and subsequent stimulation of voltage-gated Ca2+ influx in excitable cells. Here, we studied the spatiotemporal characteristics of intracellular Ca2+ signaling and its dependence on current signaling in excitable mouse immortalized gonadotropin-releasing hormone-secreting cells (GT1) and nonexcitable human embryonic kidney cells (HEK-293) cells expressing wild-type and chimeric P2XRs. In both cell types, P2XR generated depolarizing currents during the sustained ATP stimulation, which desensitized in order (from rapidly desensitizing to nondesensitizing): P2X3R > P2X2b + X4R > P2X2bR > P2X2a + X4R > P2X4R > P2X2aR > P2X7R. HEK-293 cells were not suitable for studies on P2XR-mediated Ca2+ influx because of the coactivation of endogenously expressed Ca2+-mobilizing purinergic P2Y receptors. However, when expressed in GT1 cells, all wild-type and chimeric P2XRs responded to agonist binding with global Ca2+ signals, which desensitized in the same order as current signals but in a significantly slower manner. The global distribution of Ca2+ signals was present independently of the rate of current desensitization. The temporal characteristics of Ca2+ signals were not affected by voltage-gated Ca2+ influx and removal of extracellular sodium. Ca2+ signals reflected well the receptor-specific EC50 values for ATP and the extracellular Zn2+ and pH sensitivities of P2XRs. These results indicate that intracellular Ca2+ measurements are useful for characterizing the pharmacological properties and messenger functions of P2XRs, as well as the kinetics of channel activity, when the host cells do not express other members of purinergic receptors.

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Voltage-gated sodium channel modulation by σ-receptors in cardiac myocytes and heterologous systems

AJP Cell Physiology ◽

10.1152/ajpcell.00431.2008 ◽

2009 ◽

Vol 296 (5) ◽

pp. C1049-C1057 ◽

Cited By ~ 73

Author(s):

Molly Johannessen ◽

Subramaniam Ramachandran ◽

Logan Riemer ◽

Andrea Ramos-Serrano ◽

Arnold E. Ruoho ◽

...

Keyword(s):

Cardiac Myocytes ◽

Integral Membrane Protein ◽

Receptor Ligands ◽

Pipette Solution ◽

Hek 293 ◽

Voltage Gated ◽

Hek 293 Cells ◽

Novel Structure ◽

293 Cells ◽

Voltage Gated Ion Channels

The σ-receptor, a broadly distributed integral membrane protein with a novel structure, is known to modulate various voltage-gated K+ and Ca2+ channels through a mechanism that involves neither G proteins nor phosphorylation. The present study investigated the modulation of the heart voltage-gated Na+ channel (Nav1.5) by σ-receptors. The σ1-receptor ligands [SKF-10047 and (+)-pentazocine] and σ1/σ2-receptor ligands (haloperidol and ditolylguanidine) all reversibly inhibited Nav1.5 channels to varying degrees in human embryonic kidney 293 (HEK-293) cells and COS-7 cells, but the σ1-receptor ligands were less effective in COS-7 cells. The same four ligands also inhibited Na+ current in neonatal mouse cardiac myocytes. In σ1-receptor knockout myocytes, the σ1-receptor-specific ligands were far less effective in modulating Na+ current, but the σ1/σ2-receptor ligands modulated Na+ channels as well as in wild type. Photolabeling with the σ1-receptor photoprobe [125I]-iodoazidococaine demonstrated that σ1-receptors were abundant in heart and HEK-293 cells, but scarce in COS-7 cells. This difference was consistent with the greater efficacy of σ1-receptor-specific ligands in HEK-293 cells than in COS-7 cells. σ-Receptors modulated Na+ channels despite the omission of GTP and ATP from the patch pipette solution. σ-Receptor-mediated inhibition of Na+ current had little if any voltage dependence and produced no change in channel kinetics. Na+ channels represent a new addition to the large number of voltage-gated ion channels modulated by σ-receptors. The modulation of Nav1.5 channels by σ-receptors in the heart suggests an important pathway by which drugs can alter cardiac excitability and rhythmicity.

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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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Micropatterned surfaces of PDMS as growth templates for HEK 293 cells

Biomedical Microdevices ◽

10.1007/s10544-007-9054-6 ◽

2007 ◽

Vol 9 (4) ◽

pp. 475-485 ◽

Cited By ~ 13

Author(s):

R. M. Johann ◽

Ch. Baiotto ◽

Ph. Renaud

Keyword(s):

Hek 293 ◽

Hek 293 Cells ◽

293 Cells

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