Intramembrane Charge Movement Associated with Endogenous K+Channel Activity in HEK-293 Cells

2004 ◽  
Vol 24 (3) ◽  
pp. 317-330 ◽  
Author(s):  
Guillermo Avila ◽  
Alejandro Sandoval ◽  
Ricardo Felix
Keyword(s):  
K Channel ◽  
Charge Movement ◽  
Channel Activity ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Intramembrane Charge Movement

PKC-δ sensitizes Kir3.1/3.2 channels to changes in membrane phospholipid levels after M3 receptor activation in HEK-293 cells

2005 ◽  
Vol 289 (3) ◽  
pp. C543-C556 ◽  
Author(s):  
Sean G. Brown ◽  
Alison Thomas ◽  
Lodewijk V. Dekker ◽  
Andrew Tinker ◽  
Joanne L. Leaney
Keyword(s):  
Fluorescent Protein ◽  
Receptor Activation ◽  
Dominant Negative ◽  
Membrane Phospholipid ◽  
Channel Activity ◽  
Receptor Stimulation ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Channel Inhibition ◽  
293 Cells

G protein-gated inward rectifier (Kir3) channels are inhibited by activation of Gq/11-coupled receptors and this has been postulated to involve the signaling molecules protein kinase C (PKC) and/or phosphatidylinositol 4,5-bisphosphate (PIP2). Their precise roles in mediating the inhibition of this family of channels remain controversial. We examine here their relative roles in causing inhibition of Kir3.1/3.2 channels stably expressed in human embryonic kidney (HEK)-293 cells after muscarinic M3 receptor activation. In perforated patch mode, staurosporine prevented the Gq/11-mediated, M3 receptor, inhibition of channel activity. Recovery from M3-mediated inhibition was wortmannin sensitive. Whole cell currents, where the patch pipette was supplemented with PIP2, were still irreversibly inhibited by M3 receptor stimulation. When adenosine A1 receptors were co-expressed, inclusion of PIP2 rescued the A1-mediated response. Recordings from inside-out patches showed that catalytically active PKC applied directly to the intracellular membrane face inhibited the channels: a reversible effect modulated by okadaic acid. Generation of mutant heteromeric channel Kir3.1S185A/Kir3.2C-S178A, still left the channel susceptible to receptor, pharmacological, and direct kinase-mediated inhibition. Biochemically, labeled phosphate is incorporated into the channel. We suggest that PKC-δ mediates channel inhibition because recombinant PKC-δ inhibited channel activity, M3-mediated inhibition of the channel, was counteracted by overexpression of two types of dominant negative PKC-δ constructs, and, by using confocal microscopy, we have demonstrated translocation of green fluorescent protein-tagged PKC-δ to the plasma membrane on M3 receptor stimulation. Thus Kir3.1/3.2 channels are sensitive to changes in membrane phospholipid levels but this is contingent on the activity of PKC-δ after M3 receptor activation in HEK-293 cells.

Large T-antigen up-regulates Kv4.3 K+ channels through Sp1, and Kv4.3 K+ channels contribute to cell apoptosis and necrosis through activation of calcium/calmodulin-dependent protein kinase II

2012 ◽  
Vol 441 (3) ◽  
pp. 859-869 ◽  
Author(s):  
Qi Li ◽  
Ying Zhang ◽  
Yue Sheng ◽  
Rong Huo ◽  
Bo Sun ◽  
...  
Keyword(s):  
Cell Apoptosis ◽  
T Antigen ◽  
K Channel ◽  
Large T Antigen ◽  
Hek 293 ◽  
K Channels ◽  
Hek 293 Cells ◽  
Calcium Calmodulin Dependent ◽  
293 Cells ◽  
Apoptosis And Necrosis

Down-regulation of Kv4.3 K+ channels commonly occurs in multiple diseases, but the understanding of the regulation of Kv4.3 K+ channels and the role of Kv4.3 K+ channels in pathological conditions are limited. HEK (human embryonic kidney)-293T cells are derived from HEK-293 cells which are transformed by expression of the large T-antigen. In the present study, by comparing HEK-293 and HEK-293T cells, we find that HEK-293T cells express more Kv4.3 K+ channels and more transcription factor Sp1 (specificity protein 1) than HEK-293 cells. Inhibition of Sp1 with Sp1 decoy oligonucleotide reduces Kv4.3 K+ channel expression in HEK-293T cells. Transfection of pN3-Sp1FL vector increases Sp1 protein expression and results in increased Kv4.3 K+ expression in HEK-293 cells. Since the ultimate determinant of the phenotype difference between HEK-293 and HEK-293T cells is the large T-antigen, we conclude that the large T-antigen up-regulates Kv4.3 K+ channel expression through an increase in Sp1. In both HEK-293 and HEK-293T cells, inhibition of Kv4.3 K+ channels with 4-AP (4-aminopyridine) or Kv4.3 small interfering RNA induces cell apoptosis and necrosis, which are completely rescued by the specific CaMKII (calcium/calmodulin-dependent protein kinase II) inhibitor KN-93, suggesting that Kv4.3 K+ channels contribute to cell apoptosis and necrosis through CaMKII activation. In summary, we establish: (i) the HEK-293 and HEK-293T cell model for Kv4.3 K+ channel study; (ii) that large T-antigen up-regulates Kv4.3 K+ channels through increasing Sp1 levels; and (iii) that Kv4.3 K+ channels contribute to cell apoptosis and necrosis through activating CaMKII. The present study provides deep insights into the mechanism of the regulation of Kv4.3 K+ channels and the role of Kv4.3 K+ channels in cell death.

Increased inwardly rectifying potassium currents in HEK-293 cells expressing murine transient receptor potential 4

2001 ◽  
Vol 354 (3) ◽  
pp. 717-725 ◽  
Author(s):  
Zongming ZHANG ◽  
Yufang TANG ◽  
Michael Xi ZHU
Keyword(s):  
Transient Receptor Potential ◽  
Outward Current ◽  
Receptor Potential ◽  
K Channel ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Transient Receptor ◽  
Inwardly Rectifying ◽  
K Currents

Drosophila transient receptor potential (Trp) and its mammalian homologues are postulated to form capacitative Ca2+ entry or store-operated channels. Here we show that expression of murine Trp4 in HEK 293 cells also leads to an increase in inwardly rectifying K+ currents. No similar increase was found in cell lines expressing Trp1, Trp3 or Trp6. Consistent with typical characteristics of inward rectifiers, the K+ currents in Trp4-expressing cells were blocked by low millimolar concentrations of Cs+ and Ba2+, but not by 1.2mM Ca2+, and were only slightly inhibited by 5mM tetraethylammonium. Single channel recordings of excised inside-out patches revealed the presence of two conducting states of 51pS and 94pS in Trp4-expressing cells. The outward current in the excised patches was blocked by 1mM spermine, but not by 1mM Mg2+. How Trp4 expression causes the increase in the K+ currents is not known. We propose that Trp4 either participates in the formation of a novel K+ channel or up-regulates the expression or activity of endogenous inwardly rectifying K+ channels.

scholarly journals Mutation of protein kinase C phosphorylation site S1076 on α-subunits affects BKCa channel activity in HEK-293 cells

2009 ◽  
Vol 297 (4) ◽  
pp. L758-L766 ◽  
Author(s):  
Shu Zhu ◽  
Darren D. Browning ◽  
Richard E. White ◽  
David Fulton ◽  
Scott A. Barman
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Phosphorylation Site ◽  
Channel Activity ◽  
Excitatory Effect ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Smooth Muscle Function ◽  
Α Subunits

Large conductance, calcium- and voltage-activated potassium (BKCa) channels are important modulators of pulmonary vascular smooth muscle membrane potential, and phosphorylation of BKCa channels by protein kinases regulates pulmonary arterial smooth muscle function. However, little is known about the effect of phosphorylating specific channel subunits on BKCa channel activity. The present study was done to determine the effect of mutating protein kinase C (PKC) phosphorylation site serine 1076 (S1076) on transfected human BKCa channel α-subunits in human embryonic kidney (HEK-293) cells, a heterologous expression system devoid of endogenous BKCa channels. Results showed that mutating S1076 altered the effect of PKC activation on BKCa channels in HEK-293 cells. Specifically, the phospho-deficient mutation BKCa-α(S1076A)/β1 attenuated the excitatory effect of the PKC activator phorbol myristate acetate (PMA) on BKCa channels, whereas the phospho-mimetic mutation BKCa-α(S1076E)/β1 increased the excitatory effect of PMA on BKCa channels. In addition, the phospho-null mutation S1076A blocked the activating effect of cGMP-dependent protein kinase G (PKG) on BKCa channels. Collectively, these results suggest that specific putative PKC phosphorylation site(s) on human BKCa channel α-subunits influences BKCa channel activity, which may subsequently alter pulmonary vascular smooth muscle function and tone.

scholarly journals Pharmacology of human sulphonylurea receptor SUR1 and inward rectifier K+ channel Kir6.2 combination expressed in HEK-293 cells

2000 ◽  
Vol 129 (7) ◽  
pp. 1323-1332 ◽  
Author(s):  
Murali Gopalakrishnan ◽  
Eduardo J Molinari ◽  
Char-Chang Shieh ◽  
Lisa M Monteggia ◽  
Jean-Marc Roch ◽  
...  
Keyword(s):  
Inward Rectifier ◽  
K Channel ◽  
Hek 293 ◽  
Sulphonylurea Receptor ◽  
Hek 293 Cells ◽  
293 Cells

scholarly journals Expression and electrophysiology of a newly cloned splice variant of A-type K+ channel alpha subunit Kv4.3 in HEK 293 cells

1998 ◽  
Vol 76 ◽  
pp. 239
Author(s):  
Takuma Oku ◽  
Susumu Ohya ◽  
Mamiko Tanaka ◽  
Minoru Watanabe ◽  
Yuji Imaizumi
Keyword(s):  
Splice Variant ◽  
Alpha Subunit ◽  
K Channel ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Type K ◽  
293 Cells

Influence of L-type Ca channel alpha 2/delta-subunit on ionic and gating current in transiently transfected HEK 293 cells

1996 ◽  
Vol 270 (5) ◽  
pp. H1521-H1528 ◽  
Author(s):  
R. Bangalore ◽  
G. Mehrke ◽  
K. Gingrich ◽  
F. Hofmann ◽  
R. S. Kass
Keyword(s):  
Ionic Current ◽  
Charge Movement ◽  
Ca Channel ◽  
Gating Currents ◽  
Maximal Conductance ◽  
Hek 293 ◽  
Voltage Range ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Delta Subunit

We have measured ionic and gating currents in human embryonic kidney (HEK 293) cells transiently transfected with cDNAs encoding subunits of the cardiac voltage-gated L-type Ca2+ channel. Robust recombinant ionic current and associated nonlinear charge movement could be measured over a broad voltage range without contamination by endogenous channel activity. Coexpression of the alpha 2/delta-subunit along with alpha 1- and beta 2-subunits speeded activation and deactivation kinetics and significantly increased the maximal conductance of ionic current. Charge movement was measured at voltages negative to the threshold for activation of ionic current, and gating charge could be immobilized at positive holding potentials that did not inactivate ionic current. The ratio of maximal ionic conductance to maximal charge moved remained the same in the absence or presence of the alpha 2/delta-subunit. However, the maximal amount of charge moved was increased about twofold in the presence of the alpha 2/delta-subunit. These results suggest that coexpression of the alpha 2/delta-subunit enhances the expression of functional L-type channels and, in addition, provide evidence that most of the L-type channel-associated nonlinear charge movement is caused by transitions between nonconducting states of the channel protein that precede the open and inactivated states.

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.

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