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

1998 ◽  
Vol 274 (3) ◽  
pp. C757-C769 ◽  
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.

scholarly journals Role of transient receptor potential canonical 6 (TRPC6) in non-transferrin-bound iron uptake in neuronal phenotype PC12 cells

2004 ◽  
Vol 378 (3) ◽  
pp. 975-982 ◽  
Author(s):  
James MWANJEWE ◽  
Ashok K. GROVER
Keyword(s):  
Pc12 Cells ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Neuronal Phenotype ◽  
Hek 293 ◽  
Transfected Cells ◽  
Transient Receptor Potential Canonical ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Transient Receptor

Cells take up transferrin-bound iron or NTBI (non-transferrin-bound iron). After treatment with NGF (nerve growth factor), PC12 cells exhibited a neuronal phenotype and an increase in the NTBI uptake (55Fe2+ or 55Fe3+). We loaded the cells with the dye calcein, whose fluorescence increases in the presence of Ca2+ but is quenched with Fe2+ or Fe3+. When examined using calcein fluorescence or radioactive iron, DAG (diacylglycerol)-stimulated NTBI entry was more in NGF-treated PC12 cells compared with untreated cells. All experiments were performed at 1.5 mM extracellular Ca2+. Nramp2 (natural-resistance-associated macrophage protein 2) mRNA expression did not change after the NGF treatment. Expression of the bivalent cation entry protein TRPC6 (transient receptor potential canonical 6) was detected only in the NGF-treated cells. To verify that increased NTBI uptake depended on TRPC6, we examined whether transfecting HEK-293 (human embryonic kidney 293) cells with TRPC6 also increased the NTBI (55Fe) uptake. We also cotransfected HEK-293 cells with two plasmids, one expressing TRPC6 and the other expressing the fluorescent protein DsRED2 to identify the transfected cells. Challenging the calcein-loaded HEK-293 cells (which intrinsically express the α1-adrenergic receptors) with phenylephrine or a cell-permeant DAG increased the fluorescence signal more rapidly in transfected cells compared with untransfected cells. However, when iron (Fe2+ and Fe3+) was added before adding phenylephrine or DAG, the fluorescence intensity decreased more rapidly in transfected cells compared with untransfected cells, thereby indicating a greater stimulation of the NTBI uptake in cells expressing TRPC6. We postulate that the increase in the NTBI entry into neuronal PC12 cells is through TRPC6, a pathway that is unique since it is receptor-stimulated. Since neuronal cells express TRPC6, this pathway may have a role in neurotoxicity.

scholarly journals HEK-293 cells possess a carbachol- and thapsigargin-sensitive intracellular Ca2+ store that is responsive to stop-flow medium changes and insensitive to caffeine and ryanodine

1999 ◽  
Vol 343 (1) ◽  
pp. 39-44 ◽  
Author(s):  
Jiefei TONG ◽  
Guo Guang DU ◽  
S. R. Wayne CHEN ◽  
David H. MACLENNAN
Keyword(s):  
Functional Analysis ◽  
Ryanodine Receptor ◽  
Functional Expression ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Endogenous Expression ◽  
293 Cells ◽  
Medium Change ◽  
Flow Medium ◽  
Stop Flow

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.

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

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

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

2011 ◽  
Vol 300 (3) ◽  
pp. C576-C587 ◽  
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.

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

2000 ◽  
Vol 351 (3) ◽  
pp. 735-746 ◽  
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.

NaPO4cotransport type III (PiT1) expression in human embryonic kidney cells and regulation by PTH

1999 ◽  
Vol 277 (4) ◽  
pp. F543-F551 ◽  
Author(s):  
Isabelle Fernandes ◽  
Richard Béliveau ◽  
Gérard Friedlander ◽  
Caroline Silve
Keyword(s):  
Phosphate Uptake ◽  
Phosphate Transport ◽  
Affinity Constant ◽  
Type I ◽  
Human Embryonic Kidney ◽  
Hek 293 ◽  
Phosphate Deprivation ◽  
Hek 293 Cells ◽  
Type Iii ◽  
293 Cells

The aim of the present study was to characterize the type(s) of NaPO4cotransporter expressed in the human renal cell line HEK-293 and its regulation by parathyroid hormone (PTH) in wild-type cells and in cells transfected by the PTH/PTH-related protein (PTHrP) receptor. The results showed that human embryonic kidney HEK-293 cells expressed NaPO4cotransporter type III (PiT1) mRNA and protein. In contrast, type I (NPT1) or II (NPT2) cotransporter mRNA were not expressed. Na+-dependent phosphate uptake followed a Michaelis-Menten model (apparent maximal transport rate and affinity constant: 23.32 ± 0.69 nmol PO4⋅ mg protein−1⋅ 10 min−1and 0.147 ± 0.014 mM KH2PO4, respectively), was stimulated by phosphate deprivation (maximal increase 24.5 ± 0.8%, P < 0.001, after 15 h of phosphate deprivation), and was inhibited by increasing pH (3.6 ± 0.2-fold decrease at pH 8.5, P < 0.0001). It was inhibited in a time- and concentration-dependent fashion by PTH in HEK-293 cells stably transfected by PTH/PTHrP receptors but not in parental HEK-293 cells. Maximal inhibition of Na+-dependent phosphate transport was observed at 30 min after the addition of 72 nM PTH-(1—34) (31.5 ± 2.4% inhibition, P < 0.01). PTH inhibition of phosphate transport was maintained in phosphate-deprived cells and reversed by both GF109203X (10−6M) or staurosporine (5.5 nM), two protein kinase C inhibitors. Na+-dependent phosphate uptake was also significantly inhibited by phorbol 12-myristate 13-acetate (20.9 ± 3.9% inhibition, P < 0.001) but not by dibutyril-cAMP (10−4M) or forskolin (50 μM). The physiological role played by type III NaPO4cotransport expression in the overall renal regulation of phosphate homeostasis remains to be established.

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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