scholarly journals Caveolin-1 scaffold domain interacts with TRPC1 and IP3R3 to regulate Ca2+ store release-induced Ca2+ entry in endothelial cells

2009 ◽  
Vol 296 (3) ◽  
pp. C403-C413 ◽  
Author(s):  
Premanand C. Sundivakkam ◽  
Angela M. Kwiatek ◽  
Tiffany T. Sharma ◽  
Richard D. Minshall ◽  
Asrar B. Malik ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Confocal Imaging ◽  
Microvascular Endothelial Cell ◽  
Gain Of Function ◽  
Hek 293 ◽  
Caveolin 1 ◽  
Hek 293 Cells ◽  
293 Cells

Caveolin-1 (Cav-1) regulates agonist-induced Ca2+ entry in endothelial cells; however, how Cav-1 regulates this process is poorly understood. Here, we describe that Cav-1 scaffold domain (NH2-terminal residues 82–101; CSD) interacts with transient receptor potential canonical channel 1 (TRPC1) and inositol 1,4,5-trisphosphate receptor 3 (IP3R3) to regulate Ca2+ entry. We have shown previously that the TRPC1 COOH-terminal residues 781-789 bind to CSD. In the present study, we show that the TRPC1 COOH-terminal residues 781-789 truncated (TRPC1-CΔ781-789) mutant expression abolished Ca2+ store release-induced Ca2+ influx in human dermal microvascular endothelial cell line (HMEC) and human embryonic kidney (HEK-293) cells. To understand the basis of loss of Ca2+ influx, we determined TRPC1 binding to IP3R3. We observed that the wild-type (WT)-TRPC1 but not TRPC1-CΔ781-789 effectively interacted with IP3R3. Similarly, WT-TRPC1 interacted with Cav-1, whereas TRPC1-CΔ781-789 binding to Cav-1 was markedly suppressed. We also assessed the direct binding of Cav-1 with TRPC1 and observed that the WT-Cav-1 but not the Cav-1ΔCSD effectively interacted with TRPC1. Since the interaction between TRPC1 and Cav-1ΔCSD was reduced, we measured Ca2+ store release-induced Ca2+ influx in Cav-1ΔCSD-transfected cells. Surprisingly, Cav-1ΔCSD expression showed a gain-of-function in Ca2+ entry in HMEC and HEK-293 cells. We observed a similar gain-of-function in Ca2+ entry when Cav-1ΔCSD was expressed in lung endothelial cells of Cav-1 knockout mice. Immunoprecipitation results revealed that WT-Cav-1 but not Cav-1ΔCSD interacted with IP3R3. Furthermore, we observed using confocal imaging the colocalization of IP3R3 with WT-Cav-1 but not with Cav-1ΔCSD on Ca2+ store release in endothelial cells. These findings suggest that CSD interacts with TRPC1 and IP3R3 and thereby regulates Ca2+ store release-induced Ca2+ entry in endothelial cells.

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.

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.

Canonical transient receptor potential TRPC7 can function as both a receptor- and store-operated channel in HEK-293 cells

2004 ◽  
Vol 287 (6) ◽  
pp. C1709-C1716 ◽  
Author(s):  
Jean-Philippe Lièvremont ◽  
Gary St. J. Bird ◽  
James W. Putney
Keyword(s):  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Receptor Activation ◽  
Activation Mechanism ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Canonical Transient Receptor Potential ◽  
Store Depletion ◽  
293 Cells ◽  
Transient Receptor

Previous studies on the activation mechanism of canonical transient receptor potential (TRPC) channels have often produced conflicting conclusions. All seven have been shown to be activated by phospholipase C (PLC)-coupled receptors, but TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, and TRPC7 have also been proposed to function as store-operated channels. 1 1 Although PLC activation inevitably leads to activation of store-operated channels, in this report when we refer to PLC-activated channels, we mean those channels that are specifically activated by PLC independently of store depletion. In the case of TRPC3, the expression environment and the expression level appear to determine the mode of regulation. Evidence of a close structural relative of TRPC3, TRPC7, has been presented that this channel is activated by receptor activation or by store depletion. On the basis of previous findings for TRPC3, we reasoned that subtle differences in structure or expression conditions might account for the apparent distinct gating mechanisms of TRPC7. To reexamine the mode of activation of TRPC7, we stably and transiently transfected human embryonic kidney (HEK)-293 cells with cDNA encoding for human TRPC7. We examined the ability of a PLC-activating agonist and an intracellular Ca2+ store-depleting agent to activate these channels. Our findings demonstrate that when transiently expressed in HEK-293 cells, TRPC7 forms channels that are activated by PLC-stimulating agonists, but not by Ca2+ store depletion. However, when stably expressed in HEK-293 cells, TRPC7 can be activated by either Ca2+ store depletion or PLC activation. To our knowledge, this is the first demonstration of a channel protein that can be activated by both receptor- and store-operated modes in the same cell. In addition, the results reconcile the apparently conflicting findings of other laboratories regarding TRPC7 regulation.

scholarly journals Functional significance of human trp1 and trp3 in store-operated Ca2+ entry in HEK-293 cells

2000 ◽  
Vol 278 (3) ◽  
pp. C526-C536 ◽  
Author(s):  
Xiaoyan Wu ◽  
György Babnigg ◽  
Mitchel L. Villereal
Keyword(s):  
Northern Blot Analysis ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Transient Receptor ◽  
Pcr Techniques ◽  
Antisense Construct ◽  
In Cells

The Drosophila trp (transient receptor potential) gene appears to encode the Drosophilastore-operated channel (SOC), and some mammalian trp homologues have been proposed to encode mammalian SOCs. This study provides evidence for the expression of three trp homologues (Mtrp2, Mtrp3, and Mtrp4) in fibroblasts from wild-type and src knockout mice, and four trp homologues (Htrp1, Htrp3, Htrp4, and Htrp6) in human embryonic kidney (HEK-293) cells based on RT-PCR techniques. In HEK-293 cells stably transfected with a 323-bp Htrp3 antisense construct (Htrp3AS), Northern blot analysis revealed that the expression of a 4-kb transcript was dramatically suppressed in comparison to that observed in cells stably transfected with a short Htrp3 sense construct (Htrp3S). Activity of SOCs, monitored as Ba2+ influx following Ca2+ store depletion with thapsigargin, was reduced by 32% in Htrp3AS cells in comparison with Htrp3S cells. Transient transfection of a 369-bp Htrp1 antisense construct in cells stably expressing Htrp3AS induced a higher level of inhibition (55%) of store-operated Ca2+ entry. These data suggest that Htrp1 and Htrp3 may be functional subunits of SOCs.

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.

scholarly journals Supercooling Agent Icilin Blocks a Warmth-Sensing Ion Channel TRPV3

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Muhammad Azhar Sherkheli ◽  
Guenter Gisselmann ◽  
Hanns Hatt
Keyword(s):  
Ion Channel ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Human Keratinocytes ◽  
Transient Receptor Potential Vanilloid ◽  
Neural Cells ◽  
Hek 293 ◽  
Low Concentrations ◽  
293 Cells ◽  
Transient Receptor

Transient receptor potential vanilloid subtype 3 (TRPV3) is a thermosensitive ion channel expressed in a variety of neural cells and in keratinocytes. It is activated by warmth (33–39°C), and its responsiveness is dramatically increased at nociceptive temperatures greater than 40°C. Monoterpenoids and 2-APB are chemical activators of TRPV3 channels. We found that Icilin, a known cooling substance and putative ligand of TRPM8, reversibly inhibits TRPV3 activity at nanomolar concentrations in expression systems likeXenopus laevesoocytes, HEK-293 cells, and in cultured human keratinocytes. Our data show that icilin's antagonistic effects for the warm-sensitive TRPV3 ion channel occurs at very low concentrations. Therefore, the cooling effect evoked by icilin may at least in part be due to TRPV3 inhibition in addition to TRPM8 potentiation. Blockade of TRPV3 activity by icilin at such low concentrations might have important implications for overall cooling sensations detected by keratinocytes and free nerve endings in skin. We hypothesize that blockage of TRPV3 might be a signal for cool-sensing systems (like TRPM8) to beat up the basal activity resulting in increased cold perception when warmth sensors (like TRPV3) are shut off.

Gene expression profiles in HEK-293 cells with low or high store-operated calcium entry: can regulatory as well as regulated genes be identified?

2005 ◽  
Vol 21 (1) ◽  
pp. 14-33 ◽  
Author(s):  
Tatiana K. Zagranichnaya ◽  
Xiaoyan Wu ◽  
Arpad M. Danos ◽  
Mitchel L. Villereal
Keyword(s):  
Gene Expression ◽  
Transient Receptor Potential ◽  
Expression Profiles ◽  
Physiological Role ◽  
Gene Expression Profiles ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Gene expression profiles were generated using cDNA microarray technology for clones of human embryonic kidney (HEK)-293 cells selected to have either high or low levels of store-operated Ca2+ entry (SOCE). For five high clones, three low clones, and control HEK-293 cells, duplicate Affymetrix U133A human gene arrays were run after extraction of total RNA from cells growing in the presence of serum. Of the ∼22,000 genes represented on the microarray, 58 genes had readings at least twofold higher, while 32 genes had readings at least twofold lower, in all five high SOCE clones compared with control HEK-293 cells. In the low SOCE clones, 92 genes had readings at least twofold higher, while 58 genes had readings at least twofold lower, than in HEK-293 cells. Microarray results were confirmed for 18 selected genes by real-time RT-PCR analysis; for six of those genes, predicted changes in the low SOCE clone were confirmed by an alternative method, monitoring mRNA levels in HEK-293 with SOCE decreased by expression of small interfering (si)RNA to canonical transient receptor potential protein-1. Genes regulated by SOCE are involved in signal transduction, transcription, apoptosis, metabolism, and membrane transport. These data provide insight into the physiological role of SOCE. In addition, a potential regulator of SOCE, insulin receptor substrate (IRS)-2, has been identified. A reduction of IRS-2 levels by siRNA methods in two high clones dramatically reduced SOCE, whereas overexpression of IRS-2 in a low SOCE clone elevated SOCE.

Caveolin-1 Regulates Store-Operated Ca2+ Influx by Binding of Its Scaffolding Domain to Transient Receptor Potential Channel-1 in Endothelial Cells

2006 ◽  
Vol 70 (4) ◽  
pp. 1174-1183 ◽  
Author(s):  
Angela M. Kwiatek ◽  
Richard D. Minshall ◽  
David R. Cool ◽  
Randal A. Skidgel ◽  
Asrar B. Malik ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Transient Receptor Potential Channel ◽  
Caveolin 1 ◽  
Transient Receptor

scholarly journals Mibefradil alters intracellular calcium concentration by activation of phospholipase C and IP3 receptor function

2021 ◽  
Vol 2 (1) ◽  
Author(s):  
Guilherme H. Souza Bomfim ◽  
Erna Mitaishvili ◽  
Talita Ferreira Aguiar ◽  
Rodrigo S. Lacruz
Keyword(s):  
Phospholipase C ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Ip3 Receptor ◽  
Hek 293 ◽  
Cellular Targets ◽  
293 Cells ◽  
Intracellular Ca ◽  
Transient Receptor ◽  
Orai Channels

AbstractMibefradil is a tetralol derivative originally developed as an antagonist of T-type voltage-gated calcium (Ca2+) channels to treat hypertension when used at nanomolar dosage. More recently, its therapeutic application in hypertension has declined and has been instead repurposed as a treatment of cancer cell proliferation and solid tumor growth. Beyond its function as a Cav blocker, the micromolar concentration of mibefradil can stimulate a rise in [Ca2+]cyt although the mechanism is poorly known. The chanzyme TRPM7 (transient receptor potential melastanin 7), the release of intracellular Ca2+ pools, and Ca2+ influx by ORAI channels have been associated with the increase in [Ca2+]cyt triggered by mibefradil. This study aims to investigate the cellular targets and pathways associated with mibefradil’s effect on [Ca2+]cyt. To address these questions, we monitored changes in [Ca2+]cyt in the specialized mouse epithelial cells (LS8 and ALC) and the widely used HEK-293 cells by stimulating these cells with mibefradil (0.1 μM to 100 μM). We show that mibefradil elicits an increase in [Ca2+]cyt at concentrations above 10 μM (IC50 around 50 μM) and a fast Ca2+ increase capacity at 100 μM. We found that inhibiting IP3 receptors, depleting the ER-Ca2+ stores, or blocking phospholipase C (PLC), significantly decreased the capacity of mibefradil to elevate [Ca2+]cyt. Moreover, the transient application of 100 μM mibefradil triggered Ca2+ influx by store-operated Ca2+ entry (SOCE) mediated by the ORAI channels. Our findings reveal that IP3R and PLC are potential new targets of mibefradil offering novel insights into the effects of this drug.

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