Functional Interaction Between Caveolin 1 and LRRC8-Mediated Volume-Regulated Anion Channel

Volume-regulated anion channel (VRAC), constituted by leucine-rich repeat-containing 8 (LRRC8) heteromers, is crucial for volume homeostasis in vertebrate cells. This widely expressed channel has been associated with membrane potential modulation, proliferation, migration, apoptosis, and glutamate release. VRAC is activated by cell swelling and by low cytoplasmic ionic strength or intracellular guanosine 5′-O-(3-thiotriphosphate) (GTP-γS) in isotonic conditions. Despite the substantial number of studies that characterized the biophysical properties of VRAC, its mechanism of activation remains a mystery. Different evidence suggests a possible effect of caveolins in modulating VRAC activity: (1) Caveolin 1 (Cav1)-deficient cells display insignificant swelling-induced Cl– currents mediated by VRAC, which can be restored by Cav1 expression; (2) Caveolin 3 (Cav3) knockout mice display reduced VRAC currents; and (3) Interaction between LRRC8A, the essential subunit for VRAC, and Cav3 has been found in transfected human embryonic kidney 293 (HEK 293) cells. In this study, we demonstrate a physical interaction between endogenous LRRC8A and Cav1 proteins, that is enhanced by hypotonic stimulation, suggesting that this will increase the availability of the channel to Cav1. In addition, LRRC8A targets plasma membrane regions outside caveolae of HEK 293 cells where it associates with non-caveolar Cav1. We propose that a rise in cell membrane tension by hypotonicity would flatten caveolae, as described previously, increasing the amount of Cav1 outside of caveolar structures interacting with VRAC. Besides, the expression of Cav1 in HEK Cav1- cells increases VRAC current density without changing the main biophysical properties of the channel. The present study provides further evidence on the relevance of Cav1 on the activation of endothelial VRAC through a functional molecular interaction.

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Functional interaction of the K-Cl cotransporter (KCC1) with the Na-K-Cl cotransporter in HEK-293 cells

AJP Cell Physiology ◽

10.1152/ajpcell.1999.276.2.c328 ◽

1999 ◽

Vol 276 (2) ◽

pp. C328-C336 ◽

Cited By ~ 89

Author(s):

Christopher M. Gillen ◽

Bliss Forbush

Keyword(s):

Cell Swelling ◽

Primary Role ◽

Functional Interaction ◽

Hek 293 ◽

Hek 293 Cells ◽

Hek Cells ◽

293 Cells ◽

Hypotonic Cell Swelling ◽

The Relationship ◽

Fold Activation

We have studied the regulation of the K-Cl cotransporter KCC1 and its functional interaction with the Na-K-Cl cotransporter. K-Cl cotransporter activity was substantially activated in HEK-293 cells overexpressing KCC1 (KCC1-HEK) by hypotonic cell swelling, 50 mM external K, and pretreatment with N-ethylmaleimide (NEM). Bumetanide inhibited 86Rb efflux in KCC1-HEK cells after cell swelling [inhibition constant ( K i) ∼190 μM] and pretreatment with NEM ( K i ∼60 μM). Thus regulation of KCC1 is consistent with properties of the red cell K-Cl cotransporter. To investigate functional interactions between K-Cl and Na-K-Cl cotransporters, we studied the relationship between Na-K-Cl cotransporter activation and intracellular Cl concentration ([Cl]i). Without stimulation, KCC1-HEK cells had greater Na-K-Cl cotransporter activity than controls. Endogenous Na-K-Cl cotransporter of KCC1-HEK cells was activated <2-fold by low-Cl hypotonic prestimulation, compared with 10-fold activation in HEK-293 cells and >20-fold activation in cells overexpressing the Na-K-Cl cotransporter (NKCC1-HEK). KCC1-HEK cells had lower resting [Cl]i than HEK-293 cells; cell volume was not different among cell lines. We found a steep relationship between [Cl]i and Na-K-Cl cotransport activity within the physiological range, supporting a primary role for [Cl]iin activation of Na-K-Cl cotransport and in apical-basolateral cross talk in ion-transporting epithelia.

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

AJP Cell Physiology ◽

10.1152/ajpcell.00470.2008 ◽

2009 ◽

Vol 296 (3) ◽

pp. C403-C413 ◽

Cited By ~ 80

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.

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Evidence for Cyclooxygenase-1 Association with Caveolin-1 and -2 in Cultured Human Embryonic Kidney (HEK 293) Cells

IUBMB Life ◽

10.1080/15216540410001699312 ◽

2004 ◽

Vol 56 (4) ◽

pp. 221-227 ◽

Cited By ~ 7

Author(s):

Nam-Hee Jung ◽

Bum-Rae Kim ◽

Hyun-Woo Kim ◽

Jin-Oh Kwak ◽

Seok Ho Cha

Keyword(s):

Human Embryonic Kidney ◽

Hek 293 ◽

Caveolin 1 ◽

Hek 293 Cells ◽

Cyclooxygenase 1 ◽

293 Cells

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Cadmium affects osmotic phase and regulatory volume decrease in cultured human embryonic kidney cells

Journal of Biological Research - Bollettino della Società Italiana di Biologia Sperimentale ◽

10.4081/jbr.2016.5650 ◽

2016 ◽

Vol 89 (1) ◽

Author(s):

Rossana Morabito ◽

Alessia Remigante ◽

Roberta Costa ◽

Silvia Dossena ◽

Giuseppa La Spada ◽

...

Keyword(s):

Cell Volume ◽

Regulatory Volume Decrease ◽

Cell Swelling ◽

Suitable Model ◽

Human Embryonic Kidney ◽

Cell Homeostasis ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Volume Decrease

The present investigation aims to verify whether cadmium (Cd2+), a metal possibly accumulated in body tissues from air and food, affects cell volume regulation capability in cultured human embryonic kidney (HEK 293 Phoenix) cells. The osmotic phase (OP), which is the expected cell swelling due to aquaporins involvement after hyposmotic challenge, and regulatory volume decrease (RVD), bringing cell volume back to control values through Ca2+-dependent ion efflux (K+ and Cl–), have been monitored in HEK 293 cells treated with Cd2+ (1-10-100 μM) for different time intervals (30 min, 3 h, overnight) and then submitted to 15 % hyposmotic shock. The results show that both 1 and 10 μM Cd2+ significantly reduced OP, whereas 100 μM impaired Cd2+ RVD mechanisms. The use of glutathione (GSH, 200 μM) confirmed that Cd2+ elicited its effect via oxidative damage, being RVD inhibition after Cd2+ treatment prevented by this antioxidant compound. Our findings show that: i) HEK 293 cells are a suitable model to assay the effect of xenobiotics on cell homeostasis; ii) Cd2+, depending on its concentration, affects cell homeostasis at different levels, i.e. water and ion permeability, responsible for, respectively, OP and RVD mechanism, adding thus more information to the knowledge of Cd2+ toxicology.

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Physical and functional interactions between the serotonin transporter and the neutral amino acid transporter ASCT2

Biochemical Journal ◽

10.1042/bcj20160315 ◽

2016 ◽

Vol 473 (13) ◽

pp. 1953-1965 ◽

Cited By ~ 7

Author(s):

Pascal Seyer ◽

Franck Vandermoere ◽

Elisabeth Cassier ◽

Joël Bockaert ◽

Philippe Marin

Keyword(s):

Plasma Membrane ◽

Molecular Mechanisms ◽

Physical Interaction ◽

Maximal Velocity ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Concomitant Reduction ◽

Solute Carrier ◽

5 Ht Uptake

The activity of serotonergic systems depends on the reuptake of extracellular serotonin via its plasma membrane serotonin [5-HT (5-hydroxytryptamine)] transporter (SERT), a member of the Na+/Cl−-dependent solute carrier 6 family. SERT is finely regulated by multiple molecular mechanisms including its physical interaction with intracellular proteins. The majority of previously identified SERT partners that control its functional activity are soluble proteins, which bind to its intracellular domains. SERT also interacts with transmembrane proteins, but its association with other plasma membrane transporters remains to be established. Using a proteomics strategy, we show that SERT associates with ASCT2 (alanine–serine–cysteine–threonine 2), a member of the solute carrier 1 family co-expressed with SERT in serotonergic neurons and involved in the transport of small neutral amino acids across the plasma membrane. Co-expression of ASCT2 with SERT in HEK (human embryonic kidney)-293 cells affects glycosylation and cell-surface localization of SERT with a concomitant reduction in its 5-HT uptake activity. Conversely, depletion of cellular ASCT2 by RNAi enhances 5-HT uptake in both HEK-293 cells and primary cultured mesencephalon neurons. Mimicking the effect of ASCT2 down-regulation, treatment of HEK-293 cells and neurons with the ASCT2 inhibitor D-threonine also increases 5-HT uptake. Moreover, D-threonine does not enhance further the maximal velocity of 5-HT uptake in cells depleted of ASCT2. Collectively, these findings provide evidence for a complex assembly involving SERT and a member of another solute carrier family, which strongly influences the subcellular distribution of SERT and the reuptake of 5-HT.

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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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