Functional Expression Of GFP-Tagged Kv1.3 And Kv1.4 Channels In HEK 293 Cells

Because HEK-293 cells are widely used for the functional expression of channels, exchangers and transporters involved in Ca2+ homoeostasis, the properties of intracellular Ca2+ stores and the methods used for measuring intracellular Ca2+ release in HEK-293 cells were evaluated. Ca2+ imaging was used to show caffeine-, carbachol- and thapsigargin-induced Ca2+ release in HEK-293 cells transfected with ryanodine receptor (RyR) cDNA, but only carbachol- and thapsigargin-induced Ca2+ release in untransfected HEK-293 cells. Intracellular Ca2+ release in untransfected HEK-293 cells was also observed if medium changes were performed by aspirating and replacing fresh medium (stop-flow), but not if medium changes were performed by a continuous over-flow procedure. Stop-flow medium-change-induced Ca2+ release in HEK-293 cells was independent of caffeine and ryanodine, demonstrating that it did not occur through RyR channels. Consistent with these observations was the observation that the level of expression of endogenous RyR proteins was below the limits of detection by Western blotting or [3H]ryanodine binding. Thus the level of endogenous expression of RyR is so low in HEK-293 cells as to provide a negligible background in relation to functional analysis of recombinant RyR molecules. These results are inconsistent with those of Querfurth et al. [Querfurth, Haughey, Greenway, Yacono, Golan and Geiger (1998) Biochem. J. 334, 79-86], who reported higher levels of endogenous RyR expression in untransfected HEK-293 cells.

Download Full-text

Cysteine Residues Are Important for Functional Expression of the Na+/Ca2+ Exchanger NCX1 in HEK 293 Cells

Annals of the New York Academy of Sciences ◽

10.1111/j.1749-6632.2002.tb04721.x ◽

2006 ◽

Vol 976 (1) ◽

pp. 89-89

Author(s):

XIAOYAN REN ◽

JUDITH KASIR ◽

HANNAH RAHAMIMOFF

Keyword(s):

Functional Expression ◽

Cysteine Residues ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells

Download Full-text

Functional expression of the human C3a receptor in HEK-293 cells by co-expression with Gα16

Molecular Immunology ◽

10.1016/s0161-5890(98)90534-x ◽

1998 ◽

Vol 35 (6-7) ◽

pp. 331

Author(s):

R.S. Ames ◽

M.A. Tornetta ◽

J.J. Foley ◽

H.M. Sarau ◽

S. Ganguly

Keyword(s):

Functional Expression ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells

Download Full-text

Leukemia inhibitory factor regulates trafficking of T-type Ca2+ channels

AJP Cell Physiology ◽

10.1152/ajpcell.00115.2010 ◽

2011 ◽

Vol 300 (3) ◽

pp. C576-C587 ◽

Cited By ~ 21

Author(s):

Deblina Dey ◽

Andrew Shepherd ◽

Judith Pachuau ◽

Miguel Martin-Caraballo

Keyword(s):

Membrane Trafficking ◽

Leukemia Inhibitory Factor ◽

Fluorescent Protein ◽

Expression System ◽

Functional Expression ◽

Dominant Negative ◽

Inhibitory Factor ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells

Neuropoietic cytokines such as ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) stimulate the functional expression of T-type Ca2+ channels in developing sensory neurons. However, the molecular and cellular mechanisms involved in the cytokine-evoked membrane expression of T-type Ca2+ channels are not fully understood. In this study we investigated the role of LIF in promoting the trafficking of T-type Ca2+ channels in a heterologous expression system. Our results demonstrate that transfection of HEK-293 cells with the rat green fluorescent protein (GFP)-tagged T-type Ca2+ channel α1H-subunit resulted in the generation of transient Ca2+ currents. Overnight treatment of α1H-GFP-transfected cells with LIF caused a significant increase in the functional expression of T-type Ca2+ channels as indicated by changes in current density. LIF also evoked a significant increase in membrane fluorescence compared with untreated cells. Disruption of the Golgi apparatus with brefeldin A inhibited the stimulatory effect of LIF, indicating that protein trafficking regulates the functional expression of T-type Ca2+ channels. Trafficking of α1H-GFP was also disrupted by cotransfection of HEK-293 cells with the dominant-negative form of ADP-ribosylation factor (ARF)1 but not ARF6, suggesting that ARF1 regulates the LIF-evoked membrane trafficking of α1H-GFP subunits. Trafficking of T-type Ca2+ channels required transient activation of the JAK and ERK signaling pathways since stimulation of HEK-293 cells with LIF evoked a considerable increase in the phosphorylation of the downstream JAK targets STAT3 and ERK. Pretreatment of HEK-293 cells with the JAK inhibitor P6 or the ERK inhibitor U0126 blocked ERK phosphorylation. Both P6 and U0126 also inhibited the stimulatory effect of LIF on T-type Ca2+ channel expression. These findings demonstrate that cytokines like LIF promote the trafficking of T-type Ca2+ channels.

Download Full-text

Phosphate transport by the human renal cotransporter NaPi-3 expressed in HEK-293 cells

AJP Cell Physiology ◽

10.1152/ajpcell.1998.274.3.c757 ◽

1998 ◽

Vol 274 (3) ◽

pp. C757-C769 ◽

Cited By ~ 27

Author(s):

Richard T. Timmer ◽

Robert B. Gunn

Keyword(s):

Renal Tubule ◽

Functional Expression ◽

Phosphate Transport ◽

Alkali Cations ◽

Hek 293 ◽

Transfected Cells ◽

Hek 293 Cells ◽

293 Cells ◽

Saturation Kinetics ◽

Phosphate Flux

The human renal Na-PO4cotransporter gene NaPi-3 was expressed in human embryonic kidney HEK-293 cells, and the transport characteristics were measured in cells transfected with a vector containing NaPi-3 or with the vector alone (sham transfected). The initial rate of32PO4influx had saturation kinetics for external Na and PO4with[Formula: see text] of 128 mM (PO4= 0.1 mM) and[Formula: see text]of 0.084 mM (extracellular Na = 143 mM) in sham- and NaPi-3-transfected cells expressing the transporter. Transfection had no effect on the Na-independent32PO4influx, but transfection increased Na-dependent32PO4influxes 2.5- to 5-fold. Of the alkali cations, only Na significantly supported PO4influx. Arsenate inhibited flux with an inhibition constant of 0.4 mM. The phosphate transport in sham- and NaPi-3-transfected cells has nearly the same temperature dependence in the absence and presence of extracellular Na. The Na-dependent phosphate flux decreased with pH in sham-transfected cells but was pH independent in transfected cells. The Na-dependent32PO4influx was inhibited by p-chloromercuriphenylsulfonate, phosphonoformate, phloretin, vanadate, and 5-( N-methyl- N-isobutyl)-amiloride but not by amiloride or other amiloride analogs. These functional characteristics are in general agreement with the known behavior of NaPi-3 homologues in the renal tubule of other species and, thus, demonstrate the fidelity of this transfection system for the study of this protein. Commensurate with the increased functional expression, there was an increase in the amount of NaPi-3 protein by Western analysis.

Download Full-text

Cloning and expression of the human transient receptor potential 4 (TRP4) gene: localization and functional expression of human TRP4 and TRP3

Biochemical Journal ◽

10.1042/bj3510735 ◽

2000 ◽

Vol 351 (3) ◽

pp. 735-746 ◽

Cited By ~ 59

Author(s):

Richard R. McKAY ◽

Caroline L. SZYMECZEK-SEAY ◽

Jean-Philippe LIEVREMONT ◽

Gary St. J. BIRD ◽

Christof ZITT ◽

...

Keyword(s):

Transient Receptor Potential ◽

Receptor Potential ◽

Functional Expression ◽

Receptor Activation ◽

Capacitative Calcium Entry ◽

Bivalent Cation ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Transient Receptor

Mammalian homologues of the Drosophila transient receptor potential (TRP) protein have been proposed to function as ion channels, and in some cases as store-operated or capacitative calcium entry channels. However, for each of the mammalian TRP proteins, different laboratories have reported distinct modes of cellular regulation. In the present study we describe the cloning and functional expression of the human form of TRP4 (hTRP4), and compare its activity with another well studied protein, hTRP3. When hTRP4 was transiently expressed in human embryonic kidney (HEK)-293 cells, basal bivalent cation permeability (barium) was increased. Whole-cell patch-clamp studies of hTRP4 expressed in Chinese hamster ovary cells revealed a constitutively active non-selective cation current which probably underlies the increased bivalent cation entry. Barium entry into hTRP4-transfected HEK-293 cells was not further increased by phospholipase C (PLC)-linked receptor activation, by intracellular calcium store depletion with thapsigargin, or by a synthetic diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol (OAG). In contrast, transient expression of hTRP3 resulted in a bivalent cation influx that was markedly increased by PLC-linked receptor activation and by OAG, but not by thapsigargin. Despite the apparent differences in regulation of these two putative channel proteins, green fluorescent protein fusions of both molecules localized similarly to the plasma-membrane, notably in discrete punctate regions suggestive of specialized signalling complexes. Our findings indicate that while both hTRP4 and hTRP3 can apparently function as cation channels, their putative roles as components of capacitative calcium entry channels are not readily demonstrable by examining their behaviour when exogenously expressed in cells.

Download Full-text

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

Download Full-text

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.

Download Full-text