scholarly journals Adenine nucleotides inhibit recombinant N-type calcium channels via G protein-coupled mechanisms in HEK 293 cells; involvement of the P2Y13 receptor-type

2004 ◽  
Vol 141 (1) ◽  
pp. 141-151 ◽  
Author(s):  
Kerstin Wirkner ◽  
Joana Schweigel ◽  
Zoltan Gerevich ◽  
Heike Franke ◽  
Clemens Allgaier ◽  
...  
Keyword(s):  
Calcium Channels ◽  
G Protein ◽  
Adenine Nucleotides ◽  
Receptor Type ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
G Protein Coupled

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

Endocrinology ◽  
2007 ◽  
Vol 148 (7) ◽  
pp. 3236-3245 ◽  
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.

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

2007 ◽  
Vol 101 (1) ◽  
pp. 192-204 ◽  
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

Quantification of G-Protein Coupled Receptor Internalization Using G-Protein Coupled Receptor-Green Fluorescent Protein Conjugates with the ArrayScan™ High-Content Screening System

1999 ◽  
Vol 4 (2) ◽  
pp. 75-86 ◽  
Author(s):  
Bruce R. Conway ◽  
Lisa K. Minor ◽  
Jun Z. Xu ◽  
Joseph W. Gunnet ◽  
Robbin DeBiasio ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
G Protein ◽  
Fluorescent Protein ◽  
Receptor Internalization ◽  
Hek 293 ◽  
Whole Cells ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Green Fluorescent ◽  
G Protein Coupled

Many G-protein coupled receptors (GPCRs) undergo ligand-dependent homologous desensitization and internalization. Desensitization, defined as a decrease in the responsiveness to ligand, is accompanied by receptor aggregation on the cell surface and internalization via clathrin-coated pits to an intracellular endosomal compartment. In this study, we have taken advantage of the trafficking properties of GPCRs to develop a useful screening method for the identification of receptor mimetics. A series of studies were undertaken to evaluate the expression, functionality, and ligand-dependent trafficking of GPCR-green fluorescent protein (GFP) fusion conjugates stably transfected into HEK 293 cells. These GPCR-GFP expressing cells were then utilized in the validation of the ArrayScan™ (Cellomics™, Pittsburgh, PA), a microtiter plate imaging system that permits cellular and subcellular quantitation of fluorescence in whole cells. These studies demonstrated our ability to measure the internalization of a parathy-roid hormone (PTH) receptor-GFP conjugate after ligand treatment by spatially resolving internalized receptors. Internalization was time- and dose-dependent and appeared to be selective for PTH. Similar results were obtained for a β2-adrenergic receptor (β2 AR)-GFP conjugate stably expressed in HEK 293 cells. The internalized GFP-labeled receptors were visualized as numerous punctate "spots" within the cell interior. An algorithm has been developed that identifies and collects information about these spots, allowing quantification of the internalization process. Variables such as the receptor-GFP expression level, plating density, cell number per field, number of fields scanned per well, spot size, and spot intensity were evaluated during the development of this assay. The method represents a valuable tool to screen for receptor mimetics and antagonists of receptor internalization in whole cells rapidly.

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.

scholarly journals Overexpression of T-type calcium channels in HEK-293 cells increases intracellular calcium without affecting cellular proliferation

FEBS Letters ◽  
2000 ◽  
Vol 478 (1-2) ◽  
pp. 166-172 ◽  
Author(s):  
Jean Chemin ◽  
Arnaud Monteil ◽  
Christelle Briquaire ◽  
Sylvain Richard ◽  
Edward Perez-Reyes ◽  
...  
Keyword(s):  
Calcium Channels ◽  
Intracellular Calcium ◽  
Cellular Proliferation ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

scholarly journals Characterization of a human 5-hydroxytryptamine3 receptor type A (h5-HT3R-AS) subunit stably expressed in HEK 293 cells

1996 ◽  
Vol 118 (5) ◽  
pp. 1237-1245 ◽  
Author(s):  
Anthony G. Hope ◽  
John A. Peters ◽  
Angus M. Brown ◽  
Jeremy J. Lambert ◽  
Thomas P. Blackburn
Keyword(s):  
Type A ◽  
Receptor Type ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

scholarly journals BRET Analysis of GPCR Dimers in Neurons and Non-Neuronal Cells: Evidence for Inactive, Agonist, and Constitutive Conformations

2021 ◽  
Vol 22 (19) ◽  
pp. 10638
Author(s):  
Chayma El Khamlichi ◽  
Laetitia Cobret ◽  
Jean-Michel Arrang ◽  
Séverine Morisset-Lopez
Keyword(s):  
Cortical Neurons ◽  
G Protein Coupled Receptors ◽  
Constitutive Activity ◽  
Western Blots ◽  
Inverse Agonists ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Functional Consequences ◽  
G Protein Coupled

G-protein-coupled receptors (GPCRs) are dimeric proteins, but the functional consequences of the process are still debated. Active GPCR conformations are promoted either by agonists or constitutive activity. Inverse agonists decrease constitutive activity by promoting inactive conformations. The histamine H3 receptor (H3R) is the target of choice for the study of GPCRs because it displays high constitutive activity. Here, we study the dimerization of recombinant and brain H3R and explore the effects of H3R ligands of different intrinsic efficacy on dimerization. Co-immunoprecipitations and Western blots showed that H3R dimers co-exist with monomers in transfected HEK 293 cells and in rodent brains. Bioluminescence energy transfer (BRET) analysis confirmed the existence of spontaneous H3R dimers, not only in living HEK 293 cells but also in transfected cortical neurons. In both cells, agonists and constitutive activity of the H3R decreased BRET signals, whereas inverse agonists and GTPγS, which promote inactive conformations, increased BRET signals. These findings show the existence of spontaneous H3R dimers not only in heterologous systems but also in native tissues, which are able to adopt a number of allosteric conformations, from more inactive to more active states.

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