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

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

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00242.2011 ◽

2011 ◽

Vol 301 (4) ◽

pp. H1606-H1614 ◽

Cited By ~ 46

Author(s):

Andrew C. Zygmunt ◽

Vladislav V. Nesterenko ◽

Sridharan Rajamani ◽

Dan Hu ◽

Hector Barajas-Martinez ◽

...

Keyword(s):

Ventricular Myocytes ◽

Selective Inhibition ◽

Resting Membrane Potential ◽

Inactivation Curve ◽

Hek 293 ◽

Hek 293 Cells ◽

Channel Current ◽

Ventricular Cells ◽

293 Cells ◽

Steady State Inactivation

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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Human α4β2 Neuronal Nicotinic Acetylcholine Receptor in HEK 293 Cells: A Patch-Clamp Study

Journal of Neuroscience ◽

10.1523/jneurosci.16-24-07880.1996 ◽

1996 ◽

Vol 16 (24) ◽

pp. 7880-7891 ◽

Cited By ~ 131

Author(s):

Bruno Buisson ◽

Murali Gopalakrishnan ◽

Stephen P. Arneric ◽

James P. Sullivan ◽

Daniel Bertrand

Keyword(s):

Patch Clamp ◽

Acetylcholine Receptor ◽

Nicotinic Acetylcholine Receptor ◽

Neuronal Nicotinic Acetylcholine Receptor ◽

Nicotinic Acetylcholine ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Clamp Study

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Temperature dependence of early and late currents in human cardiac wild-type and long Q-T ΔKPQ Na+ channels

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1998.275.6.h2016 ◽

1998 ◽

Vol 275 (6) ◽

pp. H2016-H2024 ◽

Cited By ~ 33

Author(s):

Toshihisa Nagatomo ◽

Zheng Fan ◽

Bin Ye ◽

Gayle S. Tonkovich ◽

Craig T. January ◽

...

Keyword(s):

Steady State ◽

Voltage Dependence ◽

Inactivation Kinetics ◽

Wild Type ◽

Positive Direction ◽

Hek 293 ◽

Whole Cell Patch Clamp ◽

Recovery From Inactivation ◽

293 Cells ◽

Steady State Inactivation

Na+current ( I Na) through wild-type human heart Na+channels (hH1) is important for normal cardiac excitability and conduction, and it participates in the control of repolarization and refractoriness. I Na kinetics depend strongly on temperature, but I Na for hH1 has been studied previously only at room temperature. We characterized early I Na (the peak and initial decay) and late I Na of the wild-type hH1 channel and a mutant channel (ΔKPQ) associated with congenital long Q-T syndrome. Channels were stably transfected in HEK-293 cells and studied at 23 and 33°C using whole cell patch clamp. Activation and inactivation kinetics for early I Na were twofold faster at higher temperature for both channels and shifted activation and steady-state inactivation in the positive direction, especially for ΔKPQ. For early I Na (<24 ms), ΔKPQ decayed faster than the wild type for voltages negative to −20 mV but slower for more positive voltages, suggesting a reduced voltage dependence of fast inactivation. Late I Na at 240 ms was significantly greater for ΔKPQ than for the wild type at both temperatures. The majority of late I Na for ΔKPQ was not persistent; rather, it decayed slowly, and this late component exhibited slower recovery from inactivation compared with peak I Na. Additional kinetic changes for early and peak I Na for ΔKPQ compared with the wild type at both temperatures were 1) reduced voltage dependence of steady-state inactivation with no difference in midpoint, 2) positive shift for activation kinetics, and 3) more rapid recovery from inactivation. This study represents the first description of human Na+ channel kinetics near physiological temperature and also demonstrates complex gating changes in the ΔKPQ that are present at 33°C and that may underlie the electrophysiological and clinical phenotype of congenital long Q-T Na+ channel syndromes.

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Whole-Cell Patch-Clamp Recording of Recombinant Voltage-Sensitive Ca2+ Channels Heterologously Expressed in HEK-293 Cells

Cold Spring Harbor Protocols ◽

10.1101/pdb.prot073213 ◽

2014 ◽

Vol 2014 (4) ◽

pp. pdb.prot073213 ◽

Cited By ~ 2

Author(s):

María A. Gandini ◽

Alejandro Sandoval ◽

Ricardo Felix

Keyword(s):

Patch Clamp ◽

Patch Clamp Recording ◽

Whole Cell ◽

Hek 293 ◽

Hek 293 Cells ◽

Cell Patch Clamp ◽

Whole Cell Patch Clamp ◽

293 Cells ◽

Ca2 Channels

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Electrotonic loading of anisotropic cardiac monolayers by unexcitable cells depends on connexin type and expression level

AJP Cell Physiology ◽

10.1152/ajpcell.00024.2009 ◽

2009 ◽

Vol 297 (2) ◽

pp. C339-C351 ◽

Cited By ~ 44

Author(s):

Luke C. McSpadden ◽

Robert D. Kirkton ◽

Nenad Bursac

Keyword(s):

Gap Junction ◽

Connexin 43 ◽

Cell Types ◽

Neonatal Rat ◽

Expression Level ◽

Cardiac Conduction ◽

Resting Potential ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells

Understanding how electrotonic loading of cardiomyocytes by unexcitable cells alters cardiac impulse conduction may be highly relevant to fibrotic heart disease. In this study, we optically mapped electrical propagation in confluent, aligned neonatal rat cardiac monolayers electrotonically loaded with cardiac fibroblasts, control human embryonic kidney (HEK-293) cells, or HEK-293 cells genetically engineered to overexpress the gap junction proteins connexin-43 or connexin-45. Gap junction expression and function were assessed by immunostaining, immunoblotting, and fluorescence recovery after photobleaching and were correlated with the optically mapped propagation of action potentials. We found that neonatal rat ventricular fibroblasts negative for the myofibroblast marker smooth muscle α-actin expressed connexin-45 rather than connexin-43 or connexin-40, weakly coupled to cardiomyocytes, and, without significant depolarization of cardiac resting potential, slowed cardiac conduction to 75% of control only at high (>60%) coverage densities, similar to loading effects found from HEK-293 cells expressing similar levels of connexin-45. In contrast, HEK-293 cells with connexin-43 expression similar to that of cardiomyocytes significantly decreased cardiac conduction velocity and maximum capture rate to as low as 22% and 25% of control values, respectively, while increasing cardiac action potential duration to 212% of control and cardiac resting potential from −71.6 ± 4.9 mV in controls to −65.0 ± 3.8 mV. For all unexcitable cell types and coverage densities, velocity anisotropy ratio remained unchanged. Despite the induced conduction slowing, none of the loading cell types increased the proportion of spontaneously active monolayers. These results signify connexin isoform and expression level as important contributors to potential electrical interactions between unexcitable cells and myocytes in cardiac tissue.

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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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