Bursts of action potential waveforms relieve G-protein inhibition of recombinant P/Q-type Ca2+ channels in HEK 293 cells.

The activity of native L-type Ca channels can be facilitated by strong depolarizations. The cardiac Ca channel α1C-subunit was transiently expressed in human embryonic kidney (HEK-293) cells, but these channels did not exhibit voltage-dependent facilitation. Coexpression of the Ca channel β1a- or β2a-subunit with the α1C-subunit enabled voltage-dependent facilitation in 40% of cells tested. The onset of facilitation in α1C + β1a-expressing HEK-293 cells was rapid after a depolarization to +100 mV (τ = 7.0 ms). The kinetic features of the facilitated currents were comparable to those observed for voltage-dependent relief of G protein inhibition demonstrated for many neuronal Ca channels; however, intracellular dialysis with guanosine 5′- O-(2-thiodiphosphate) and guanosine 5′- O-(3-thiotriphosphate) in the patch pipette had no effect on facilitation. Stimulation of G protein-coupled receptors, either endogenous (somatostatin receptors) or coexpressed (adenosine A1receptors), did not affect voltage-dependent facilitation. These results indicate that the cardiac Ca channel α1C-subunit can exhibit voltage-dependent facilitation in HEK-293 cells only when coexpressed with an auxiliary β-subunit and that this facilitation is independent of G protein pathways.

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

Circulation ◽

10.1161/circ.118.suppl_18.s_525-c ◽

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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Activation of the Novel Estrogen Receptor G Protein-Coupled Receptor 30 (GPR30) at the Plasma Membrane

Endocrinology ◽

10.1210/en.2006-1605 ◽

2007 ◽

Vol 148 (7) ◽

pp. 3236-3245 ◽

Cited By ~ 290

Author(s):

E. Filardo ◽

J. Quinn ◽

Y. Pang ◽

C. Graeber ◽

S. Shaw ◽

...

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

G Protein ◽

Intracellular Camp ◽

Calcium Stores ◽

G Protein Coupled Receptor ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

G Protein Coupled

G protein-coupled receptor 30 (GPR30), a seven-transmembrane receptor (7TMR), is associated with rapid estrogen-dependent, G protein signaling and specific estrogen binding. At present, the subcellular site of GPR30 action is unclear. Previous studies using antibodies and fluorochrome-labeled estradiol (E2) have failed to detect GPR30 on the cell surface, suggesting that GPR30 may function uniquely among 7TMRs as an intracellular receptor. Here, we show that detectable expression of GPR30 on the surface of transfected HEK-293 cells can be selected by fluorescence-activated cell sorting. Expression of GPR30 on the cell surface was confirmed by confocal microscopy using the lectin concanavalin A as a plasma membrane marker. Stimulation of GPR30-expressing HEK-293 cells with 17β-E2 caused sequestration of GPR30 from the cell surface and resulted in its codistribution with clathrin and mobilization of intracellular calcium stores. Evidence that GPR30 signals from the cell surface was obtained from experiments demonstrating that the cell-impermeable E2-protein conjugates E2-BSA and E2-horseradish peroxidase promote GPR30-dependent elevation of intracellular cAMP concentrations. Subcellular fractionation studies further support the plasma membrane as a site of GPR30 action with specific [3H]17β-E2 binding and G protein activation associated with plasma membrane but not microsomal, or other fractions, prepared from HEK-293 or SKBR3 breast cancer cells. These results suggest that GPR30, like other 7TMRs, functions as a plasma membrane receptor.

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Activation of ERK, JNK, Akt, and G-protein coupled signaling by hybrid angiotensin II AT1/bradykinin B2 receptors expressed in HEK-293 cells

Journal of Cellular Biochemistry ◽

10.1002/jcb.21161 ◽

2007 ◽

Vol 101 (1) ◽

pp. 192-204 ◽

Cited By ~ 9

Author(s):

Jun Yu ◽

David Lubinsky ◽

Natia Tsomaia ◽

Zhenhua Huang ◽

Linda Taylor ◽

...

Keyword(s):

Angiotensin Ii ◽

G Protein ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

B2 Receptors ◽

G Protein Coupled

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Intravenous Anesthetic Propofol Inhibits Multiple Human Cardiac Potassium Channels

Anesthesiology ◽

10.1097/aln.0000000000000495 ◽

2015 ◽

Vol 122 (3) ◽

pp. 571-584 ◽

Cited By ~ 19

Author(s):

Lei Yang ◽

Hui Liu ◽

Hai-Ying Sun ◽

Gui-Rong Li

Keyword(s):

Potassium Channels ◽

Action Potential ◽

Action Potential Duration ◽

Potassium Current ◽

Open Channels ◽

Atrial Myocytes ◽

Hek 293 ◽

Human Atrial Myocytes ◽

Hek 293 Cells ◽

293 Cells

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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