Extracellular Ca2+-sensing receptor is a promiscuous divalent cation sensor that responds to lead

The extracellular Ca2+-sensing receptor (CaR) responds to polycations, including Ca2+ and neomycin. This receptor is a physiological regulator of systemic Ca2+ metabolism and may also mediate the toxic effects of hypercalcemia. A number of divalent cations, including Pb2+, Co2+, Cd2+, and Fe2+, are toxic to the kidney, brain, and other tissues where the CaR is expressed. To determine which divalent cations can activate the CaR, we expressed the human CaR in HEK-293 cells and measured activation of phospholipase A2(PLA2) and the mitogen-activated protein kinase p42ERK in response to potential agonists for the receptor. HEK-293 cells expressing the nonfunctional mutant CaR R796W served as controls. Extracellular Ca2+, Ba2+, Cd2+, Co2+, Fe2+, Gd3+, Ni2+, Pb2+, and neomycin activated the CaR, but Hg2+and Fe3+ did not. We analyzed the kinetics of activation of p42ERK and PLA2 by the CaR in response to Ca2+, Co2+, and Pb2+. The EC50 values ranged from ∼0.1 mM for Pb2+ to ∼4.0 mM for Ca2+. The Hill coefficients were >3, indicating multiple cooperative ligand binding sites or subunits. Submaximal concentrations of Ca2+ and Pb2+ were additive for activation of the CaR. The EC50 for Ca2+ or Pb2+ was reduced four- to fivefold by the presence of the other ion. These divalent cations also activated PLA2 via the CaR in Madin-Darby canine kidney cells that stably express the CaR. We conclude that many divalent cations activate the CaR and that their effects are additive. The facts that the CaR is a promiscuous polycation sensor and that the effects of these ions are additive to activate it suggest that the CaR may contribute to the toxicity of some heavy metals such as Pb2+, Cd2+, Co2+, and Fe2+ for the kidney and other tissues where it is expressed.

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Involvement of Mitogen-Activated Protein Kinase in Agonist-Induced Phosphorylation of the μ-Opioid Receptor in HEK 293 Cells

Journal of Neurochemistry ◽

10.1046/j.1471-4159.2000.0740414.x ◽

2001 ◽

Vol 74 (1) ◽

pp. 414-422 ◽

Cited By ~ 69

Author(s):

Harald Schmidt ◽

Stefan Schulz ◽

Marcus Klutzny ◽

Thomas Koch ◽

Manuela Händel ◽

...

Keyword(s):

Protein Kinase ◽

Opioid Receptor ◽

Mitogen Activated Protein Kinase ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Mitogen Activated Protein ◽

Μ Opioid Receptor

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Regulation of apoptosis signal-regulating kinase 1 by protein phosphatase 2Cϵ

Biochemical Journal ◽

10.1042/bj20070231 ◽

2007 ◽

Vol 405 (3) ◽

pp. 591-596 ◽

Cited By ~ 31

Author(s):

Jun-ichi Saito ◽

Shinnosuke Toriumi ◽

Kenjiro Awano ◽

Hidenori Ichijo ◽

Keiichi Sasaki ◽

...

Keyword(s):

Protein Phosphatase ◽

Feedback Regulation ◽

Dominant Negative ◽

Mitogen Activated Protein Kinase ◽

Regulation Mechanism ◽

H2o2 Treatment ◽

Hek 293 ◽

Human Embryonic Kidney Cells ◽

Hek 293 Cells ◽

293 Cells

ASK1 (apoptosis signal-regulating kinase 1), a MKKK (mitogen-activated protein kinase kinase kinase), is activated in response to cytotoxic stresses, such as H2O2 and TNFα (tumour necrosis factor α). ASK1 induction initiates a signalling cascade leading to apoptosis. After exposure of cells to H2O2, ASK1 is transiently activated by autophosphorylation at Thr845. The protein then associates with PP5 (protein serine/threonine phosphatase 5), which inactivates ASK1 by dephosphorylation of Thr845. Although this feedback regulation mechanism has been elucidated, it remains unclear how ASK1 is maintained in the dephosphorylated state under non-stressed conditions. In the present study, we have examined the possible role of PP2Cϵ (protein phosphatase 2Cϵ), a member of PP2C family, in the regulation of ASK1 signalling. Following expression in HEK-293 cells (human embryonic kidney cells), wild-type PP2Cϵ inhibited ASK1-induced activation of an AP-1 (activator protein 1) reporter gene. Conversely, a dominant-negative PP2Cϵ mutant enhanced AP-1 activity. Exogenous PP2Cϵ associated with exogenous ASK1 in HEK-293 cells under non-stressed conditions, inactivating ASK1 by decreasing Thr845 phosphorylation. The association of endogenous PP2Cϵ and ASK1 was also observed in mouse brain extracts. PP2Cϵ directly dephosphorylated ASK1 at Thr845in vitro. In contrast with PP5, PP2Cϵ transiently dissociated from ASK1 within cells upon H2O2 treatment. These results suggest that PP2Cϵ maintains ASK1 in an inactive state by dephosphorylation in quiescent cells, supporting the possibility that PP2Cϵ and PP5 play different roles in H2O2-induced regulation of ASK1 activity.

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Infection kinetics of human adenovirus serotype 41 in HEK 293 cells

Memórias do Instituto Oswaldo Cruz ◽

10.1590/s0074-02762009000500013 ◽

2009 ◽

Vol 104 (5) ◽

pp. 736-744 ◽

Cited By ~ 11

Author(s):

Joselma Siqueira-Silva ◽

Fernanda Perez Yeda ◽

Anne-Laure Favier ◽

Paulette Mezin ◽

Misael Leonardo Silva ◽

...

Keyword(s):

Human Adenovirus ◽

Adenovirus Serotype ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Kinetics Of

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CNNM proteins selectively bind to the TRPM7 channel to stimulate divalent cation entry into cells

PLoS Biology ◽

10.1371/journal.pbio.3001496 ◽

2021 ◽

Vol 19 (12) ◽

pp. e3001496

Author(s):

Zhiyong Bai ◽

Jianlin Feng ◽

Gijs A. C. Franken ◽

Namariq Al’Saadi ◽

Na Cai ◽

...

Keyword(s):

Divalent Cation ◽

Divalent Cations ◽

Hek 293 ◽

Hek 293 Cells ◽

Cellular Life ◽

Binding Partners ◽

Cation Uptake ◽

Electrophysiological Recordings ◽

293 Cells ◽

Inactivating Mutation

Magnesium is essential for cellular life, but how it is homeostatically controlled still remains poorly understood. Here, we report that members of CNNM family, which have been controversially implicated in both cellular Mg2+ influx and efflux, selectively bind to the TRPM7 channel to stimulate divalent cation entry into cells. Coexpression of CNNMs with the channel markedly increased uptake of divalent cations, which is prevented by an inactivating mutation to the channel’s pore. Knockout (KO) of Trpm7 in cells or application of the TRPM7 channel inhibitor NS8593 also interfered with CNNM-stimulated divalent cation uptake. Conversely, KO of CNNM3 and CNNM4 in HEK-293 cells significantly reduced TRPM7-mediated divalent cation entry, without affecting TRPM7 protein expression or its cell surface levels. Furthermore, we found that cellular overexpression of phosphatases of regenerating liver (PRLs), known CNNMs binding partners, stimulated TRPM7-dependent divalent cation entry and that CNNMs were required for this activity. Whole-cell electrophysiological recordings demonstrated that deletion of CNNM3 and CNNM4 from HEK-293 cells interfered with heterologously expressed and native TRPM7 channel function. We conclude that CNNMs employ the TRPM7 channel to mediate divalent cation influx and that CNNMs also possess separate TRPM7-independent Mg2+ efflux activities that contribute to CNNMs’ control of cellular Mg2+ homeostasis.

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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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