scholarly journals Measurement of Resting Cytosolic Ca2+Concentrations and Ca2+Store Size in HEK-293 Cells Transfected with Malignant Hyperthermia or Central Core Disease Mutant Ca2+Release Channels

1999 ◽  
Vol 274 (2) ◽  
pp. 693-702 ◽  
Author(s):  
Jiefei Tong ◽  
Tommie V. McCarthy ◽  
David H. MacLennan
Keyword(s):  
Malignant Hyperthermia ◽  
Central Core ◽  
Central Core Disease ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

scholarly journals Functional Studies of RYR1  Mutations in the Skeletal Muscle Ryanodine Receptor Using Human RYR1  Complementary DNA

2010 ◽  
Vol 112 (6) ◽  
pp. 1350-1354 ◽  
Author(s):  
Keisaku Sato ◽  
Neil Pollock ◽  
Kathryn M. Stowell
Keyword(s):  
Skeletal Muscle ◽  
Malignant Hyperthermia ◽  
Calcium Channel ◽  
Ryanodine Receptor ◽  
Human Skeletal Muscle ◽  
Binding Assays ◽  
Complementary Dna ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Background Malignant hyperthermia is associated with mutations within the gene encoding the skeletal muscle ryanodine receptor, the calcium channel that releases Ca from sarcoplasmic reticulum stores triggering muscle contraction, and other metabolic activities. More than 200 variants have been identified in the ryanodine receptor, but only some of these have been shown to functionally affect the calcium channel. To implement genetic testing for malignant hyperthermia, variants must be shown to alter the function of the channel. A number of different ex vivo methods can be used to demonstrate functionality, as long as cells from human patients can be obtained and cultured from at least two unrelated families. Because malignant hyperthermia is an uncommon disorder and many variants seem to be private, including the newly identified H4833Y mutation, these approaches are limited. Methods The authors cloned the human skeletal muscle ryanodine receptor complementary DNA and expressed both normal and mutated forms in HEK-293 cells and carried out functional analysis using ryanodine binding assays in the presence of a specific agonist, 4-chloro-m-cresol, and the antagonist Mg. Results Transiently expressed human ryanodine receptor proteins colocalized with an endoplasmic reticulum marker in HEK-293 cells. Ryanodine binding assays confirmed that mutations causing malignant hyperthermia resulted in a hypersensitive channel, while those causing central core disease resulted in a hyposensitive channel. Conclusions The functional assays validate recombinant human skeletal muscle ryanodine receptor for analysis of variants and add an additional mutation (H4833Y) to the repertoire of mutations that can be used for the genetic diagnosis of malignant hyperthermia.

scholarly journals Central core disease mutations R4892W, I4897T and G4898E in the ryanodine receptor isoform 1 reduce the Ca2+ sensitivity and amplitude of Ca2+-dependent Ca2+ release

2004 ◽  
Vol 382 (2) ◽  
pp. 557-564 ◽  
Author(s):  
Guo Guang DU ◽  
Vijay K. KHANNA ◽  
Xinghua GUO ◽  
David H. MacLENNAN
Keyword(s):  
Ryanodine Receptor ◽  
Central Core ◽  
Central Core Disease ◽  
Peak Amplitude ◽  
Wild Type ◽  
Release Channel ◽  
Hek 293 ◽  
Disease Mutations ◽  
293 Cells ◽  
Ryanodine Receptor 1

Three CCD (central core disease) mutants, R4892W (Arg4892→Trp), I4897T and G4898E, in the pore region of the skeletal-muscle Ca2+-release channel RyR1 (ryanodine receptor 1) were characterized using a newly developed assay that monitored Ca2+ release in the presence of Ca2+ uptake in microsomes isolated from HEK-293 cells (human embryonic kidney 293 cells), co-expressing each of the three mutants together with SERCA1a (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase 1a). Both Ca2+ sensitivity and peak amplitude of Ca2+ release were either absent from or sharply decreased in homotetrameric mutants. Co-expression of wild-type RyR1 with mutant RyR1 (heterotetrameric mutants) restored Ca2+ sensitivity partially, in the ratio 1:2, or fully, in the ratio 1:1. Peak amplitude was restored only partially in the ratio 1:2 or 1:1. Reduced amplitude was not correlated with maximum Ca2+ loading or the amount of expressed RyR1 protein. High-affinity [3H]ryanodine binding and caffeine-induced Ca2+ release were also absent from the three homotetrameric mutants. These results indicate that decreased Ca2+ sensitivity is one of the serious defects in these three excitation–contraction uncoupling CCD mutations. In CCD skeletal muscles, where a mixture of wild-type and mutant RyR1 is expressed, these defects are expected to decrease Ca2+-induced Ca2+ release, as well as orthograde Ca2+ release, in response to transverse tubular membrane depolarization.

scholarly journals Characteristics and requirements of basal autophagy in HEK 293 cells

Autophagy ◽  
2013 ◽  
Vol 9 (9) ◽  
pp. 1407-1417 ◽  
Author(s):  
Patience Musiwaro ◽  
Matthew Smith ◽  
Maria Manifava ◽  
Simon A. Walker ◽  
Nicholas T. Ktistakis
Keyword(s):  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

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

2005 ◽  
Vol 103 (6) ◽  
pp. 1156-1166 ◽  
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.

Micropatterned surfaces of PDMS as growth templates for HEK 293 cells

2007 ◽  
Vol 9 (4) ◽  
pp. 475-485 ◽  
Author(s):  
R. M. Johann ◽  
Ch. Baiotto ◽  
Ph. Renaud
Keyword(s):  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Calcium Induces Expression of Cytoplasmic Gelsolin in SH-SY5Y and HEK-293 Cells

2010 ◽  
Vol 35 (7) ◽  
pp. 1075-1082 ◽  
Author(s):  
Lina Ji ◽  
Abha Chauhan ◽  
Ved Chauhan
Keyword(s):  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Cytoplasmic Gelsolin

Characteristics of rat downregulated in adenoma (rDRA) expressed in HEK 293 cells

2007 ◽  
Vol 454 (3) ◽  
pp. 441-450 ◽  
Author(s):  
Christian Barmeyer ◽  
Jeff Huaqing Ye ◽  
Shafik Sidani ◽  
John Geibel ◽  
Henry J. Binder ◽  
...  
Keyword(s):  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Abstract 5316: hERG 1b as a Potential Target for Inherited and Acquired Long QT Syndrome

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Gail A Robertson ◽  
Harinath Sale ◽  
David Tester ◽  
Thomas J O’Hara ◽  
Pallavi Phartiyal ◽  
...  
Keyword(s):  
Action Potential ◽  
Long Qt Syndrome ◽  
Time Course ◽  
Drug Sensitivity ◽  
Long Qt ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Acquired Long Qt Syndrome ◽  
293 Cells ◽  
Qt Syndrome

Cardiac I Kr is a critical repolarizing current in the heart and a target for inherited and acquired long QT syndrome. Biochemical studies show that native I Kr channels are heteromers composed of both hERG 1a and 1b subunits, yet our current understanding of I Kr functional properties derives primarily from studies of homo-oligomers of the original hERG 1a isolate. The hERG 1a and 1b subunits are identical except at the amino (NH2) terminus, which in hERG 1b is much shorter and has a unique primary sequence. We compared the biophysical properties of currents produced by hERG 1a and 1a/1b channels expressed in HEK-293 cells at near-physiological temperatures. We found that heteromeric hERG 1a/1b currents are much larger than hERG 1a currents and conduct 80% more charge during an action potential. This surprising difference corresponds to a two-fold increase in the apparent rates of activation and recovery from inactivation, which reduces rectification and facilitates current rebound during repolarization. Kinetic modeling shows these gating differences account quantitatively for the differences in current amplitude between the two channel types. Depending on the action potential model used, loss of 1b predicts an increase in action potential duration of 27 ms (7%) or 41 ms (17%), respectively. Drug sensitivity was also different. Compared to homomeric 1a channels, heteromeric 1a/1b channels were inhibited by E-4031 with a slower time course and a corresponding four-fold positive shift in the IC 50 . Differences in current kinetics and drug sensitivity were modeled by “NH2 mode” gating with conformational states bound by the amino terminus in hERG 1a homomers but not 1a/1b heteromers. The importance of hERG 1b in vivo is supported by the identification of a 1b-specific A8V missense mutation in 1/269 unrelated genotype-negative LQTS patients and absent in 400 control alleles. Mutant 1bA8V expressed alone or with hERG 1a in HEK-293 cells nearly eliminated 1b protein. Thus, mutations specifically disrupting hERG 1b function are expected to reduce cardiac I Kr , prolong QT interval and enhance drug sensitivity, thus representing a potential mechanism underlying inherited or acquired LQTS.

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