Inhibitory effect of protopine on KATP channel subunits expressed in HEK-293 cells

Background and Objective. ATP-sensitive potassium (KATP) channel couples cell metabolism to excitability. To explore role of KATP channels in cellular photobiomodulation, we designed experiment to study effect of low intensity 808 nm laser irradiation on the activity of membrane KATP channel.Study Design/Materials and Methods. Plasmids encoding Kir6.2 was constructed and heterologously expressed in cultured mammalian HEK-293 cells. The patch-clamp and data acquisition systems were used to record KATP channel current before and after irradiation. A laser beam of Ga-As 808 nm at 5 mW/cm2was used in experiments. A one-way ANOVA test followed by apost hocStudent-Newman-Keuls test was used to assess the statistical differences between data groups.Results. Obvious openings of KATP channels of Kir6.2-transfected HEK-293 cells and excised patches were recorded during and after low intensity 808 nm laser irradiation. Compared with the channels that did not undergo irradiation, open probability, current amplitude, and dwell time of KATP channels after irradiation improved.Conclusions. Low intensity 808 nm laser irradiation may activate membrane KATP channels of Kir6.2-transfected HEK-293 cells and in excised patches.

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Calcium-sensing receptors control CYP27B1-luciferase expression. Transcriptional and post-transcriptional mechanisms

Journal of the Endocrine Society ◽

10.1210/jendso/bvab057 ◽

2021 ◽

Author(s):

Alice Huang ◽

Lenah Binmahfouz ◽

Dale P Hancock ◽

Paul H Anderson ◽

Donald T Ward ◽

...

Keyword(s):

Fold Increase ◽

Firefly Luciferase ◽

Inhibitory Effect ◽

Renilla Luciferase ◽

Luciferase Expression ◽

Mrna Levels ◽

Hek 293 ◽

Hek 293 Cells ◽

Calcium Sensing ◽

293 Cells

Abstract 25-hydroxyvitamin D 1α-hydroxylase (encoded by CYP27B1), which catalyses the synthesis of 1,25-dihydroxyvitamin D3, is subject to negative or positive modulation by extracellular Ca 2+ (Ca 2+o) depending on the tissue. However, the Ca 2+ sensors and underlying mechanisms are unidentified. We tested whether calcium-sensing receptors (CaSRs) mediate Ca 2+o-dependent control of 1α-hydroxylase using HEK-293 cells stably expressing the CaSR (HEK-CaSR cells). In HEK-CaSR cells, but not control HEK-293 cells, co-transfected with reporter genes for CYP27B1-Photinus pyralis (firefly) luciferase and control Renilla luciferase, an increase in Ca 2+o from 0.5 to 3.0 mM induced a 2-3 fold increase in firefly-luciferase activity as well as mRNA and protein levels. Surprisingly, firefly-luciferase was specifically suppressed at Ca 2+o ≥ 5.0 mM, demonstrating biphasic Ca 2+o control. Both phases were mediated by CaSRs as revealed by positive and negative modulators. However, Ca 2+o induced simple monotonic increases in firefly-luciferase and endogenous CYP27B1 mRNA levels, indicating that the inhibitory effect of high Ca 2+o was post-transcriptional. Studies with inhibitors and the CaSR C-terminal mutant T888A identified roles for PKC, phosphorylation of T888, and ERK1/2 in high Ca 2+o-dependent suppression of firefly-luciferase. Blockade of both PKC and ERK1/2 abolished Ca 2+o-stimulated firefly-luciferase, demonstrating that either PKC or ERK1/2 is sufficient to stimulate the CYP27B1 promoter. A key CCAAT box (–74 bp to –68 bp), which is regulated downstream of PKC and ERK1/2 was required for both basal transcription and Ca 2+o-mediated transcriptional upregulation. The CaSR mediates Ca 2+o-dependent transcriptional upregulation of 1α-hydroxylase and an additional CaSR-mediated mechanism is identified by which Ca 2+o can promote luciferase and possibly 1α-hydroxylase breakdown.

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The inhibitory effect of diphenyltin on gap junctional intercellular communication in HEK-293 cells is reduced by thioredoxin reductase 1

Toxicology Letters ◽

10.1016/j.toxlet.2008.09.012 ◽

2008 ◽

Vol 183 (1-3) ◽

pp. 45-51 ◽

Cited By ~ 4

Author(s):

J SROKA ◽

J CZYZ ◽

M WOJEWODA ◽

Z MADEJA

Keyword(s):

Intercellular Communication ◽

Thioredoxin Reductase ◽

Inhibitory Effect ◽

Gap Junctional Intercellular Communication ◽

Hek 293 ◽

Hek 293 Cells ◽

Thioredoxin Reductase 1 ◽

293 Cells ◽

Gap Junctional

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Proximal C-terminal domain of sulphonylurea receptor 2A interacts with pore-forming Kir6 subunits in KATP channels

Biochemical Journal ◽

10.1042/bj20031087 ◽

2004 ◽

Vol 379 (1) ◽

pp. 173-181 ◽

Cited By ~ 14

Author(s):

Richard D. RAINBOW ◽

Marian JAMES ◽

Diane HUDMAN ◽

Mohammed AL JOHI ◽

Harprit SINGH ◽

...

Keyword(s):

Cell Membrane ◽

Katp Channel ◽

Direct Interaction ◽

Katp Channels ◽

Full Length ◽

Hek 293 ◽

Sulphonylurea Receptor ◽

Hek 293 Cells ◽

Terminal Domain ◽

293 Cells

Functional KATP (ATP-sensitive potassium) channels are hetero-octamers of four Kir6 (inwardly rectifying potassium) channel subunits and four SUR (sulphonylurea receptor) subunits. Possible interactions between the C-terminal domain of SUR2A and Kir6.2 were investigated by co-immunoprecipitation of rat SUR2A C-terminal fragments with full-length Kir6.2 and by analysis of cloned KATP channel function and distribution in HEK-293 cells (human embryonic kidney 293 cells) in the presence of competing rSUR2A fragments. Three maltose-binding protein–SUR2A fusions, rSUR2A-CTA (rSUR2A residues 1254–1545), rSUR2A-CTB (residues 1254–1403) and rSUR2A-CTC (residues 1294–1403), were co-immunoprecipitated with full-length Kir6.2 using a polyclonal anti-Kir6.2 antiserum. A fourth C-terminal domain fragment, rSUR2A-CTD (residues 1358–1545) did not co-immunoprecipitate with Kir6.2 under the same conditions, indicating a direct interaction between Kir6.2 and a 65-amino-acid section of the cytoplasmic C-terminal region of rSUR2A between residues 1294 and 1358. ATP- and glibenclamide-sensitive K+ currents were decreased in HEK-293 cells expressing full-length Kir6 and SUR2 subunits that were transiently transfected with fragments rSUR2A-CTA, rSUR2A-CTC and rSUR2A-CTE (residues 1294–1359) compared with fragment rSUR2A-CTD or mock-transfected cells, suggesting either channel inhibition or a reduction in the number of functional KATP channels at the cell surface. Anti-KATP channel subunit-associated fluorescence in the cell membrane was substantially lower and intracellular fluorescence increased in rSUR2A-CTE expressing cells; thus, SUR2A fragments containing residues 1294–1358 reduce current by decreasing the number of channel subunits in the cell membrane. These results identify a site in the C-terminal domain of rSUR2A, between residues 1294 and 1358, whose direct interaction with full-length Kir6.2 is crucial for the assembly of functional KATP channels.

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Bupivacaine inhibits a small conductance calcium-activated potassium type 2 channel (SK2) in HEK 293 cells

10.21203/rs.2.338/v1 ◽

2019 ◽

Author(s):

Hongfei Chen ◽

Fangfang Xia ◽

Zhousheng Jin ◽

Yuting He ◽

Zhengjie Chen ◽

...

Keyword(s):

Inhibitory Effect ◽

Heart Cells ◽

Dependent Manner ◽

Patch Clamp Technique ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Ic50 Value ◽

Small Conductance

Abstract Background: Bupivacaine blocks many ion channels in the heart muscle, which could cause severe cardiotoxicity. Small conductance calcium-activated potassium type 2 channels (SK2 channels) are widely distributed in the heart cells and are involved in relevant physiological functions. However, whether bupivacaine can inhibit SK2 channels is still unknown. This study investigated the effect of bupivacaine on SK2 channels. Methods: The SK2 channel gene was transfected into human embryonic kidney 293 cells (HEK-293 cells) with Lipofectamine 2000. The whole-cell patch clamp technique was used to study the effect of bupivacaine on SK2 channels. The inhibitory effect of various concentrations of bupivacaine on SK2 currents exhibited a non-linear relation, and the half-maximal inhibitory concentration (IC50) value was determined. Results: Bupivacaine inhibited the SK2 channels reversibly in a dose-dependent manner. The IC50 value of bupivacaine, ropivacaine and lidocaine on the SK2 current was 133.7, 189.3, and 885.8 µM, respectively. The degree of SK2 current inhibition by bupivacaine was dependent on the intracellular concentration of free calcium. Conclusions: The results of this study suggested a new inhibitory effect of bupivacaine on SK2 channels. Future studies should be concerned with the effects of SK2 on bupivacaine cardiotoxicity. Keywords: Bupivacaine, SK2 channel, inhibition, cardiotoxicity, HEK 293.

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Inhibition of Small-Conductance, Ca2+-Activated K+ Current by Ondansetron

Frontiers in Pharmacology ◽

10.3389/fphar.2021.651267 ◽

2021 ◽

Vol 12 ◽

Author(s):

Shuai Guo ◽

Zhenhui Chen ◽

Peng-Sheng Chen ◽

Michael Rubart

Keyword(s):

Inhibitory Effect ◽

Sk Channels ◽

Patch Clamp Technique ◽

Whole Cell ◽

Hek 293 ◽

Hek 293 Cells ◽

Whole Cell Patch Clamp ◽

293 Cells ◽

Inwardly Rectifying ◽

Small Conductance

Background: Small-conductance Ca2+-activated K+ channels (SK channels) have been proposed as antiarrhythmic targets for the treatment of atrial fibrillation. We previously demonstrated that the 5-HT3 receptor antagonist ondansetron inhibits heterologously expressed, human SK2 (hSK2) currents as well as native cardiac SK currents in a physiological extra-/intracellular [K+] gradient at therapeutic (i.e., sub-micromolar) concentrations. A recent study, using symmetrical [K+] conditions, challenged this result. The goal of the present study was to revisit the inhibitory effect of ondansetron on hSK2-mediated currents in symmetrical [K+] conditions.Experimental Approach: The whole-cell patch clamp technique was used to investigate the effects of ondansetron and apamin on hSK2-mediated currents expressed in HEK 293 cells. Currents were measured in symmetrical [K+] conditions in the presence of 100 nM [Ca2+]o.Results: Expression of hSK2 produced inwardly rectifying whole-cell currents in the presence of 400 nM free cytosolic Ca2+. Ondansetron inhibited whole-cell hSK2 currents with IC50 values of 154 and 113 nM at −80 and 40 mV, respectively. Macroscopic current inhibited by ondansetron and current inhibited by apamin exhibited inwardly rectifying current-voltage relationships with similar reversal potentials (apamin, ∼5 mV and ondansetron, ∼2 mV). Ondansetron (1 μM) in the continuing presence of apamin (100 nM) had no effect on hSK2-mediated whole-cell currents. Wild-type HEK 293 cells did not express ondansetron- or apamin-sensitive currents.Conclusion: Ondansetron in sub-micromolar concentrations inhibits hSK2 currents even under altered ionic conditions.

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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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