The EGFP/hERG fusion protein alter the electrophysiological properties of hERG channels in HEK293 cells

Cisapride, a gastrointestinal prokinetic agent, is known to cause long Q-T syndrome and ventricular arrhythmias. The cellular mechanism is not known. The human ether-á-go-go-related gene ( HERG), which encodes the rapidly activating delayed rectifier K+current and is important in cardiac repolarization, may serve as a target for the action of cisapride. We tested the hypothesis that cisapride blocks HERG. The whole cell patch-clamp recording technique was used to study HERG channels stably expressed heterologously in HEK293 cells. Under voltage-clamp conditions, cisapride block of HERG is dose dependent with a half-maximal inhibitory concentration of 6.5 nM at 22°C ( n = 25 cells). Currents rapidly recovered with drug washout. The onset of block by cisapride required channel activation indicative of open or inactivated state blockage. Block of HERG with cisapride after channel activation was voltage dependent. At −20 mV, 10 nM cisapride reduced HERG tail-current amplitude by 5%, whereas, at +20 mV, the tail-current amplitude was reduced by 45% ( n = 4 cells). At −20 and +20 mV, 100 nM cisapride reduced tail-current amplitude by 66 and 90%, respectively. We conclude that cisapride is a potent blocker of HERG channels expressed in HEK293 cells. This effect may account for the clinical occurrence of Q-T prolongation and ventricular arrhythmias observed with cisapride.

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Mechanisms of IhERG/IKr Modulation by α1-Adrenoceptors in HEK293 Cells and Cardiac Myocytes

Cellular Physiology and Biochemistry ◽

10.1159/000453180 ◽

2016 ◽

Vol 40 (6) ◽

pp. 1261-1273 ◽

Cited By ~ 3

Author(s):

Janire Urrutia ◽

Aintzane Alday ◽

Mónica Gallego ◽

L. Layse Malagueta-Vieira ◽

Ivan Arael Aréchiga-Figueroa ◽

...

Keyword(s):

Ventricular Fibrillation ◽

Cardiac Myocytes ◽

Torsades De Pointes ◽

Hek293 Cells ◽

Herg Channel ◽

Delayed Rectifier ◽

Patch Clamp Technique ◽

K Current ◽

Herg Channels ◽

Intracellular Pathway

Background: The rapid delayed rectifier K+ current (IKr), carried by the hERG protein, is one of the main repolarising currents in the human heart and a reduction of this current increases the risk of ventricular fibrillation. α1-adrenoceptors (α1-AR) activation reduces IKr but, despite the clear relationship between an increase in the sympathetic tone and arrhythmias, the mechanisms underlying the α1-AR regulation of the hERG channel are controversial. Thus, we aimed to investigate the mechanisms by which α1-AR stimulation regulates IKr. Methods: α1-adrenoceptors, hERG channels, auxiliary subunits minK and MIRP1, the non PIP2-interacting mutant D-hERG (with a deletion of the 883-894 amino acids) in the C-terminal and the non PKC-phosphorylable mutant N-terminal truncated-hERG (NTK-hERG) were transfected in HEK293 cells. Cell membranes were extracted by centrifugation and the different proteins were visualized by Western blot. Potassium currents were recorded by the patch-clamp technique. IKr was recorded in isolated feline cardiac myocytes. Results: Activation of the α1-AR reduces the amplitude of IhERG and IKr through a positive shift in the activation half voltage, which reduces the channel availability at physiological membrane potentials. The intracellular pathway connecting the α1-AR to the hERG channel in HEK293 cells includes activation of the Gαq protein, PLC activation and PIP2 hydrolysis, activation of PKC and direct phosphorylation of the hERG channel N-terminal. The PKC-mediated IKr channel phosphorylation and subsequent IKr reduction after α1-AR stimulation was corroborated in feline cardiac myocytes. Conclusions: These findings clarify the link between sympathetic nervous system hyperactivity and IKr reduction, one of the best characterized causes of torsades de pointes and ventricular fibrillation.

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Dimethyl sulfoxide effects on hERG channels expressed in HEK293 cells

Journal of Pharmacological and Toxicological Methods ◽

10.1016/j.vascn.2006.03.002 ◽

2006 ◽

Vol 54 (2) ◽

pp. 164-172 ◽

Cited By ~ 25

Author(s):

Xiaoyi Du ◽

Daniel Lu ◽

Eric D. Daharsh ◽

Aizhen Yao ◽

Rebecca Dewoody ◽

...

Keyword(s):

Dimethyl Sulfoxide ◽

Hek293 Cells ◽

Herg Channels

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Construction of a HERG mutant L539fs/47-*558W pEGFP vector and the expression of the fusion protein in HEK293 cells

Journal of Medical Colleges of PLA ◽

10.1016/s1000-1948(13)60034-9 ◽

2013 ◽

Vol 28 (4) ◽

pp. 193-205

Author(s):

Junbo ZHANG ◽

Ying LÜ ◽

Aifeng ZHANG ◽

Chaofeng SUN ◽

Wenqi HAN ◽

...

Keyword(s):

Fusion Protein ◽

Hek293 Cells

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Establishing the Role of EPS15, DVL2 and Cortactin in CALM-AF10 Leukemogenesis

Blood ◽

10.1182/blood-2021-152644 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3312-3312

Author(s):

Rafi Kazi ◽

Waitman Kurt Aumann ◽

Pritha Bagchi ◽

Donald Tope ◽

Daniel S. Wechsler

Keyword(s):

Fusion Protein ◽

Cell Line ◽

Nuclear Pore Complex ◽

Nuclear Export ◽

Hek293 Cells ◽

Leukemia Cells ◽

Nuclear Pore ◽

U937 Cells ◽

Biotin Ligase ◽

Hoxa Genes

Abstract Background: Leukemia is the most common type of childhood cancer. Although the prognosis for many pediatric leukemias has improved, leukemias associated with the t(10;11) CALM-AF10 translocation remain difficult to treat. CALM-AF10 leukemias account for ~5-10% of childhood T-cell acute lymphoblastic leukemia (T-ALL)as well as a subset of acute myeloid leukemia (AML). CALM-AF10 leukemias exhibit increased expression of proleukemic HOXA genes, but relatively little is known about the cellular mechanisms that drive CALM-AF10 leukemogenesis. Our laboratory has demonstrated that the CALM protein contains a nuclear export signal (NES) that is critical for CALM-AF10-dependent leukemogenesis. The NES interacts with the CRM1/XPO1 nuclear export receptor, which shuttles proteins from the nucleus to the cytoplasm through the nuclear pore complex. We have shown that transcriptional activation of HOXA genes by CALM-AF10 is dependent on its interaction with CRM1. Importantly, CRM1 does not contain a recognized DNA binding domain, and it is not currently understood how the CALM-AF10/CRM1 complex interacts with regulatory regions of HOXA genes. To identify proteins that mediate the interaction between the CALM-AF10/CRM1 complex and DNA, we took advantage of a proximity-based labeling approach using BioID2, a second-generation biotin ligase. When fused to a protein of interest and in the presence of biotin, BioID2 biotinylates proteins in close proximity to the ligase. These biotinylated proteins can then be identified by mass spectrometry (MS). Methods: We prepared an expression plasmid in which BioID2 was cloned in-frame with CALM-AF10. Human Embryonic Kidney 293 (HEK293) cells were transiently transfected with BioID2-CALM-AF10 and grown in the presence or absence of biotin. MS was performed to identify candidate interacting proteins. We validated direct interactions of candidate proteins with CALM-AF10 using co-immunoprecipitation experiments in HEK293 cells transfected with a CALM-AF10 plasmid. We confirmed that candidate proteins are present in murine CALM-AF10 leukemia cells via Western blotting. In order to efficiently knockout (KO) candidate proteins, we have generated a human U937 cell line (which harbors a t(10;11) CALM-AF10 translocation) with a stable incorporated Cas9. To assess whether KO of EPS15, DVL2 or CTTN affects HOXA5 expression, we performed RT-qPCR in U937-Cas9 cells lines with confirmed KO. Results: We carried out three independent transfections/MS experiments, which identified 71, 95 and 61 proteins, respectively. Of the proteins identified, 12 candidates were common to all three experiments . Importantly, we identified Disruptor Of Telomeric silencing 1-Like (DOT1L), a protein known to interact with AF10, and Nuclear pore complex protein 214 (NUP214), a protein that interacts with CRM1 and that is involved in leukemogenic translocations. We chose EPS15, DVL2 and CTTN for further study, as each of these proteins plays a role in leukemogenesis. We performed initial validation of direct interactions via co-immunoprecipitation and found that all three proteins co-precipitate with CALM-AF10. Western blotting showed that all three proteins are expressed in a murine CALM-AF10 leukemia cell line. We effectively knocked out EPS15 protein expression in U937 cells, and showed that HOXA5 expression is reduced in the setting of EPS15 knockout. Conclusion: We used biotin ligase-dependent proximity-based labeling to identify candidate proteins that potentially interact with the CALM-AF10 fusion protein. Our identification of DOT1L validates the approach, since DOT1L is known to interact with CALM-AF10. We have started to investigate three candidate proteins - EPS15, DVL2 and CTTN - all of which are involved in leukemogenic transformation. We have shown that EPS15, DVL2 and CTTN are expressed in murine CALM-AF10 leukemia cells and directly interact with the CALM-AF10 fusion protein. Knockout of EPS15 in U937 cells results in decreased HOXA5 expression, suggesting the importance of EPS15 in CALM-AF10 leukemogenesis. Evaluation of the roles of these proteins in leukemogenesis may lead to identification of novel pathways involved in CALM-AF10 leukemogenesis. Disclosures No relevant conflicts of interest to declare.

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Alternative splicing of Cav1.2 channel exons in smooth muscle cells of resistance-size arteries generates currents with unique electrophysiological properties

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00109.2009 ◽

2009 ◽

Vol 297 (2) ◽

pp. H680-H688 ◽

Cited By ~ 28

Author(s):

Xiaoyang Cheng ◽

Judith Pachuau ◽

Eva Blaskova ◽

Maria Asuncion-Chin ◽

Jianxi Liu ◽

...

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Muscle Cells ◽

Full Length ◽

Hek293 Cells ◽

Electrophysiological Properties ◽

Splice Variation ◽

Voltage Dependent ◽

Exon 9 ◽

Hyperpolarizing Shift

Voltage-dependent calcium (Ca2+, CaV1.2) channels are the primary Ca2+ entry pathway in smooth muscle cells of resistance-size (myogenic) arteries, but their molecular identity remains unclear. Here we identified and quantified CaV1.2 α1-subunit splice variation in myocytes of rat resistance-size (100–200 μm diameter) cerebral arteries. Full-length clones containing either exon 1b or the recently identified exon 1c exhibited additional primary splice variation at exons 9*, 21/22, 31/32, and ± 33. Real-time PCR confirmed the findings from full-length clones and indicated that the major CaV1.2 variant contained exons 1c, 8, 21, and 32+33, with ∼57% containing 9*. Exon 9* was more prevalent in clones containing 1c (72%) than in those containing 1b (33%), suggesting exon-selective combinatorial splicing. To examine the functional significance of this splicing profile, membrane currents produced by each of the four exon 1b/c/ ± 9* variants were characterized following transfection in HEK293 cells. Exon 1c and 9* caused similar hyperpolarizing shifts in both current-voltage relationships and voltage-dependent activation of currents. Furthermore, exon 9* induced a hyperpolarizing shift only in the voltage-dependent activation of channels containing exon 1b, but not in those containing exon 1c. In contrast, exon 1b, 1c, or +9* did not alter voltage-dependent inactivation. In summary, we have identified the CaV1.2 α1-subunit splice variant population that is expressed in myocytes of resistance-size arteries and the unique electrophysiological properties of recombinant channels formed by exon 1 and 9* variation. The predominance of exon 1c and 9* in smooth muscle cell CaV1.2 channels causes a hyperpolarizing shift in the voltage sensitivity of currents toward the physiological arterial voltage range.

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Abstract 2736: Mutations In The Nonpore Region of The HERG Gene Are at Risk of Acquired Long QT Syndrome

Circulation ◽

10.1161/circ.116.suppl_16.ii_607-a ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Yuichiro Sakamoto ◽

Hidekazu Ino ◽

Noboru Fujino ◽

Katsuharu Uchiyama ◽

Kenshi Hayashi ◽

...

Keyword(s):

Clinical Characteristics ◽

Electrophysiological Study ◽

Herg Channel ◽

Cardiac Events ◽

Electrophysiological Properties ◽

Acquired Long Qt Syndrome ◽

Increased Risk ◽

Increase Risk ◽

Few Data ◽

Herg Channels

Introduction : Mutations involving the KCNH2 gene in HERG channel are responsible for type2 LQTS. Previous study showed patients with mutations in the pore region of the HERG gene are markedly increased risk for arrhythmia-related cardiac events compared with patients with nonpore mutations. However, a few data exist regarding mode of onset and frequency in arrhythmic events of mutations in the nonpore regions of the HERG channel. Hypothesis :We hypothesized that patients with mutations in the nonpore regions of the HERG gene increase risk for cardiac events under a specific condition. Methods: We examined 104 unrelated subjects with LQTS. Genetic analysis of KCNH2 gene was performed using standard genetic tests for probands and families of genotyped ones.Between subjects with and without pore mutations,difference in clinical characteristics such as QTc intervals, LQT score and major cardiac events are evaluated by standard statistical methods. Result: We identified 7 LQT2 patients with 3 pore mutations and 17 patients with 7 nonpore mutation, and evaluated clinical characteristics and characterized the electrophysiological properties of these mutations. The baseline QTc intervals were significantly longer with pore than with nonpore mutations (513 ± 60ms vs. 479 ± 21ms, p<0.01). The LQTS scores determined by Schwartz’s criteria were also significantly higher with pore mutations (4.5 ± 1.2 vs. 2.9 ± 1.2, p<0.01). However the frequency of LQTS-related cardiac events with nonpore mutaion was as high as with pore mutations (57 % vs. 30 %, N.S). Significantly 4 of 5 cardiac events with nonpore mutations occurred under medications (Probucol) or conditions (Hypokalemia) known to favor QT prolongation, although only 1 of 4 cardiac events with pore mutations did. Electrophysiological study revealed that the tail current amplitude through one nonpore mutant channel(M124T)was significantly decreased as compared that of WT HERG channel and Probucol significantly inhibited M124T HERG channels. Conclusions: These data demonstrate that LQT2 patients with nonpore mutations are also at high risk of arrhythmia-related events in the presence of drugs or other environmental stressors.

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A Model to Study Platelet Integrin Variants Using Transfected Human Embryonic Kidney-293 Cells Expressing Fluorescent αIIbβ3-Fusion Proteins

Blood ◽

10.1182/blood.v112.11.5364.5364 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5364-5364

Author(s):

Volker Rainer Stoldt ◽

Abdelouahid El Khattouti ◽

Rudiger E. Scharf

Keyword(s):

Fusion Protein ◽

G Protein ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Laser Scanning Microscopy ◽

Hek293 Cells ◽

Confocal Laser ◽

Chimeric Antibody ◽

Human Embryonic Kidney ◽

G Protein Coupled

Abstract The HPA-1 polymorphism of platelet integrin αIIbβ3 (GPIIb-IIIa) arises from a thymidine to cytosine transition in position 1565 of the ITGB3 gene. This transition leads to an amino acid exchange at residue 33 of the mature β3 subunit. The resulting isoforms are HPA-1a (Leu33) or HPA-1b (Pro33). We have shown that the HPA-1b variant of αIIbβ3 is associated with premature manifestation of myocardial infarction in patients suffering from coronary artery disease (Zotz et al. J Thromb Haemost 2005). This observation has lead to the hypothesis that the HPA-1b variant of αIIbβ3 may increase platelet thrombogenicity. Recently, we have also demonstrated that HPA-1b/1b platelets adhering onto fibrinogen are more resistant than HPA-1a/1a platelets when exposed to arterial shear rates of 1000 to 1500 sec-1 (Loncar et al. Thromb J 2007). To explore the molecular nature of the postulated prothrombotic phenotype of HPA-1b in further detail, we have now overexpressed the yellow fluorescent protein (YFP) or the cyan fluorescent protein (CFP) fused to the cytoplasmic tails of the αIIb or β3 subunit of both αIIbβ3 variants in human embryonic kidney-293 (HEK293) cells. Clones were screened for their cyan and yellow fluorescence. Ten positive clones of each αIIbβ3 variant were detected and characterized by Western blotting identifying the 140 kD αIIb-CFP fusion protein and the 113 kD β3-YFP fusion protein with appropriate antibodies directed against the αIIb subunit, the β3 subunit or the fluorescent proteins. Stable HEK293 clones expressing the HPA-1 receptor isoforms on the cell surface were functionally analyzed by flow cytometry (Becton Dickinson), confocal laser scanning microscopy (Zeiss), and a fluorescence imager (Thermo). We used fluorophore (PE)-conjugated complex-specific (PM6/248) and activation-dependent (PAC-1) antibodies and fluorescently tagged fibrinogen (Alexa647- Fg) in combination with phorbol-12-myristate-13-acetate (PMA) or organic acid (1-stearoyl-2-arachidonoyl-sn-glycerol, SAG). Corresponding controls were performed with the chimeric antibody abciximab (ReoPro) to block fibrinogen binding to αIIbβ3 or with pertussis toxin (PTX) to inhibit G-protein-coupled inside-out signal transduction. Functional integrity of the integrin variants (HPA-1a and HPA-1b) was demonstrated by intact activation through G-protein-coupled receptors with SAG and by specific binding of Alexa647 fibrinogen to αIIbβ3 indicating proper insertion of the receptor complex into the plasma membrane of transfected cells. In the presence of PTX or abciximab, activation and ligand binding function of αIIbβ3 were completely (>95%) inhibited in both isoforms of HPA-1. Cytoplasmic conformational changes upon integrin activation using either PMA or SAG were followed by fluorescence resonance energy transfer (FRET) and quantified by FRET signal disappearance due to allosteric changes of the cytoplasmic domains. Stimulation with PMA and SAG caused FRET signal disappearance to same extent in each HPA-1 variant. However, the dynamics of signal disappearance appeared to be faster in the HPA-1b than in the HPA-1a variant of the cell clones studied so far. This observation corresponds to the prothrombotic phenotype of HPA-1b. In conclusion, our results demonstrate that we have generated a cellular model which can be useful to study molecular properties of αIIbβ3 variants and to explore the nature of the prothrombotic HPA-1b phenotype in further detail.

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