Abstract P368: Caveolin-3 Prevents Swelling-induced Cell Lysis Via Regulation Of
            I
            Cl,swell
            Expression And Activity

Background: Caveolae membrane structures harbor mechanosensitive chloride channels (MCCs) which form a swelling-activated chloride current ( I Cl,swell ) and play an important role in cell volume regulation and mechano-electrical signal transduction. However, the role of muscle-specific caveolar scaffolding protein caveolin-3 (Cav3) in regulation of MCCs expression, activity, and contribution to cell viability in response to mechanical stress remains unclear. We hypothesized that Cav3-based mechano-protection is enabled by complimentary expression of MCCs. Methods and Results: Experiments were performed on native (Cav3-) and Cav3-transfected (Cav3+) HEK-293 cells. Cell stretch was mimicked by light (220 mOsM) or extreme hypoosmotic swelling (<20mOsM). Cav3+ HEK-293 cells were significantly resistant to extreme hypotonic solutions (15 minute incubation) compared to Cav3- HEK-293 cells, and this mechano-protection was significantly reduced when exposed to I Cl,swell selective inhibitor DCPIB (1 μM). We found that three MCCs (ClC-2, ClC-3, and SWELL1, also known as LRRC8A) contain caveolin-binding motifs in their structure, indicating their possible localization in caveolae structures. Co-IP analysis confirmed association of SWELL1 with Cav3. Interestingly, Cav3+ HEK-293 cells showed a significant (by 2-fold) increase of SWELL1 protein level, while ClC-2/3 protein levels remained unchanged. This was accompanied by a 2-fold increase of I Cl,swell , but no change in mRNA expression levels. FRET analysis showed a <10 nm membrane and intracellular association between Cav3 and tested MCCs. Furthermore, Cav3/SWELL1 membrane FRET efficiency was halved in light hypoosmotic solution, as well as after disruption of caveolae structures via cholesterol depletion by 1-hour treatment with 10 μM methyl-β-cyclodextrin. Cav3/ClC-2/3 average membrane FRET efficiency remained unchanged in hypotonic solution. Conclusions: We concluded that of MCCs tested, SWELL1 abundance and activity is regulated by Cav3 and that their association relies on membrane tension and caveolae integrity. The present study highlights the mechano-protective properties of Cav3 which are partially facilitated by complimentary SWELL1 expression and activity.

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Abstract 423: Caveolin-3 Enriches and Dynamically Interacts With Swell1

Circulation Research ◽

10.1161/res.127.suppl_1.423 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Daniel G Turner ◽

Leonid Tyan ◽

Sami Stroebel ◽

Frank Deguire ◽

Di Lang ◽

...

Keyword(s):

Protein Expression ◽

Hek293 Cells ◽

Scaffolding Protein ◽

Cell Swelling ◽

Hypotonic Solution ◽

Hek 293 ◽

Dynamic Relationship ◽

293 Cells ◽

Fret Efficiency ◽

Oxide Synthase

Caveolae are small (50-100nm) membrane invaginations formed by caveolin proteins enriched with cholesterol and lipids. Caveolae play a crucial role in mechanoprotection and mechano-electrical transduction by buffering membrane tension and facilitating activation of mechanosensitive ion channels, including a recently discovered swelling-activated chloride channel SWELL1 (also known as LRRC8A). However, the dynamic relationship between the muscle-specific caveolar scaffolding protein caveolin-3 (Cav3) and SWELL1 is poorly understood. The objective of this study was to determine how Cav3 interacts with SWELL1 channels and modulates their activity during mechano-electrical transduction. In HEK 293 cells transfected with Cav3, co-immunoprecipitation analysis recapitulated cardiac data showing association between SWELL1 and Cav3. Using transiently expressed Cav3-GFP and SWELL1-mCherry fusion proteins in HEK293 cells, we observed a high FRET efficiency between the two proteins in an isotonic (1T) solution, confirming their close (<5nm) proximity. In a hypotonic solution (0.7T, mimicking cell stretch), FRET efficiency decreased two-fold. Furthermore, FRET efficiency decreased two-fold to control levels when incubated with methyl-beta cyclodextrin, a cholesterol solubilizer. These data suggest that the relationship between Cav3 and SWELL1 is dependent on membrane mechanical tension and caveolae lipid raft integrity. Interestingly, in transfected cells, SWELL1 protein expression and whole cell swelling-activated chloride current ( I Cl,swell ) were increased four-fold and two-fold, respectively, while mRNA expression was reduced two-fold. This may indicate that caveolae formed by Cav3 expression enrich for SWELL1 and increase their half-life, thus requiring lower mRNA availability despite higher protein expression. Our findings indicate a close dynamic interplay between Cav3 and SWELL1, with a strong regulatory action of Cav3 on I Cl,swell activity. Given that I Cl,swell increases and interprotein FRET efficiency decreases in hypotonic solution, it is likely that Cav3 inhibits the activation of SWELL1 similarly to its known inhibition of nitric oxide synthase.

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Optimal expression of cloned NMDAR1/NMDAR2A heteromeric glutamate receptors: a biochemical characterization

Biochemical Journal ◽

10.1042/bj2960877 ◽

1993 ◽

Vol 296 (3) ◽

pp. 877-883 ◽

Cited By ~ 77

Author(s):

M Cik ◽

P L Chazot ◽

F A Stephenson

Keyword(s):

Rat Brain ◽

Biochemical Characterization ◽

Radioligand Binding ◽

Fold Increase ◽

Binding Activity ◽

Immunological Methods ◽

Hek 293 ◽

Hek 293 Cells ◽

Adult Rat ◽

293 Cells

The N-methyl-D-aspartate R1 (NMDAR1) and NMDAR2A subunits were expressed transiently either alone or in combination in human embryonic kidney (HEK) 293 cells. The biochemical and pharmacological properties of the cloned receptors were compared with those of adult rat brain NMDA receptors using both immunological methods with a newly developed anti-NMDAR2A-(1435-1445) antibody and [3H]MK801 radioligand binding activity. Anti-NMDAR2A-(1435-1445) antibodies recognized specifically four immunoreactive species with M(r)s of 180,000, 122,000, 97,000 and 54,000 in rat brain, but only a single band of M(r) 180,000 in HEK 293 cells singly transfected with plasmid pCISNMDAR2A. N-deglycosylation of HEK cell membranes yielded a 165,000-M(r) immunoreactive species, which is in agreement with the size predicted from the cDNA sequence for the mature NMDAR2A subunit. Co-expression of NMDAR1 and NMDAR2A subunits in HEK 293 cells resulted in cell death. Thus conditions were established for the optimum expression of heteromeric receptors in viable cells, including a requirement for DL-2-amino-5-phosphonopentanoic acid (AP5) in the culture medium post-transfection. Cells transfected with pCISNMDAR1 and pCISNMDAR2A combined yielded a 10-fold increase in the number of [3H]MK801 binding sites compared with single subunit expression. MK801 had similar affinity for the expressed receptors as for those found in adult rat and mouse brain. These results demonstrate that the NMDAR1 and NMDAR2A receptor subunits co-assemble to form a heteromeric complex with properties similar to those of the native receptors of adult mammalian forebrain. Furthermore, the conditions reported for maximal transient expression provide a basis for further structure-activity studies.

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Calcium-sensing receptors control CYP27B1-luciferase expression. Transcriptional and post-transcriptional mechanisms

Journal of the Endocrine Society ◽

10.1210/jendso/bvab057 ◽

2021 ◽

Author(s):

Alice Huang ◽

Lenah Binmahfouz ◽

Dale P Hancock ◽

Paul H Anderson ◽

Donald T Ward ◽

...

Keyword(s):

Fold Increase ◽

Firefly Luciferase ◽

Inhibitory Effect ◽

Renilla Luciferase ◽

Luciferase Expression ◽

Mrna Levels ◽

Hek 293 ◽

Hek 293 Cells ◽

Calcium Sensing ◽

293 Cells

Abstract 25-hydroxyvitamin D 1α-hydroxylase (encoded by CYP27B1), which catalyses the synthesis of 1,25-dihydroxyvitamin D3, is subject to negative or positive modulation by extracellular Ca 2+ (Ca 2+o) depending on the tissue. However, the Ca 2+ sensors and underlying mechanisms are unidentified. We tested whether calcium-sensing receptors (CaSRs) mediate Ca 2+o-dependent control of 1α-hydroxylase using HEK-293 cells stably expressing the CaSR (HEK-CaSR cells). In HEK-CaSR cells, but not control HEK-293 cells, co-transfected with reporter genes for CYP27B1-Photinus pyralis (firefly) luciferase and control Renilla luciferase, an increase in Ca 2+o from 0.5 to 3.0 mM induced a 2-3 fold increase in firefly-luciferase activity as well as mRNA and protein levels. Surprisingly, firefly-luciferase was specifically suppressed at Ca 2+o ≥ 5.0 mM, demonstrating biphasic Ca 2+o control. Both phases were mediated by CaSRs as revealed by positive and negative modulators. However, Ca 2+o induced simple monotonic increases in firefly-luciferase and endogenous CYP27B1 mRNA levels, indicating that the inhibitory effect of high Ca 2+o was post-transcriptional. Studies with inhibitors and the CaSR C-terminal mutant T888A identified roles for PKC, phosphorylation of T888, and ERK1/2 in high Ca 2+o-dependent suppression of firefly-luciferase. Blockade of both PKC and ERK1/2 abolished Ca 2+o-stimulated firefly-luciferase, demonstrating that either PKC or ERK1/2 is sufficient to stimulate the CYP27B1 promoter. A key CCAAT box (–74 bp to –68 bp), which is regulated downstream of PKC and ERK1/2 was required for both basal transcription and Ca 2+o-mediated transcriptional upregulation. The CaSR mediates Ca 2+o-dependent transcriptional upregulation of 1α-hydroxylase and an additional CaSR-mediated mechanism is identified by which Ca 2+o can promote luciferase and possibly 1α-hydroxylase breakdown.

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Myristoylated rhinovirus VP4 protein activates TLR2-dependent proinflammatory gene expression

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00365.2018 ◽

2019 ◽

Vol 317 (1) ◽

pp. L57-L70 ◽

Cited By ~ 4

Author(s):

J. Kelley Bentley ◽

Mingyuan Han ◽

Suraj Jaipalli ◽

Joanna L. Hinde ◽

Jing Lei ◽

...

Keyword(s):

Affinity Chromatography ◽

Mrna Expression ◽

Hela Cells ◽

Membrane Surface ◽

Airway Epithelial Cells ◽

Mouse Bone Marrow ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Fret Efficiency

Asthma exacerbations are often caused by rhinovirus (RV). We and others have shown that Toll-like receptor 2 (TLR2), a membrane surface receptor that recognizes bacterial lipopeptides and lipoteichoic acid, is required and sufficient for RV-induced proinflammatory responses in vitro and in vivo. We hypothesized that viral protein-4 (VP4), an internal capsid protein that is myristoylated upon viral replication and externalized upon viral binding, is a ligand for TLR2. Recombinant VP4 and myristoylated VP4 (MyrVP4) were purified by Ni-affinity chromatography. MyrVP4 was also purified from RV-A1B-infected HeLa cells by urea solubilization and anti-VP4 affinity chromatography. Finally, synthetic MyrVP4 was produced by chemical peptide synthesis. MyrVP4-TLR2 interactions were assessed by confocal fluorescence microscopy, fluorescence resonance energy transfer (FRET), and monitoring VP4-induced cytokine mRNA expression in the presence of anti-TLR2 and anti-VP4. MyrVP4 and TLR2 colocalized in TLR2-expressing HEK-293 cells, mouse bone marrow-derived macrophages, human bronchoalveolar macrophages, and human airway epithelial cells. Colocalization was absent in TLR2-null HEK-293 cells and blocked by anti-TLR2 and anti-VP4. Cy3-labeled MyrVP4 and Cy5-labeled anti-TLR2 showed an average fractional FRET efficiency of 0.24 ± 0.05, and Cy5-labeled anti-TLR2 increased and unlabeled MyrVP4 decreased FRET efficiency. MyrVP4-induced chemokine mRNA expression was higher than that elicited by VP4 alone and was attenuated by anti-TLR2 and anti-VP4. Cytokine expression was similarly increased by MyrVP4 purified from RV-infected HeLa cells and synthetic MyrVP4. We conclude that, during RV infection, MyrVP4 and TLR2 interact to generate a proinflammatory response.

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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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