scholarly journals Expression and electrophysiology of a newly cloned splice variant of A-type K+ channel alpha subunit Kv4.3 in HEK 293 cells

1998 ◽  
Vol 76 ◽  
pp. 239
Author(s):  
Takuma Oku ◽  
Susumu Ohya ◽  
Mamiko Tanaka ◽  
Minoru Watanabe ◽  
Yuji Imaizumi
Keyword(s):  
Splice Variant ◽  
Alpha Subunit ◽  
K Channel ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Type K ◽  
293 Cells

Cells expressing unique Na+/Ca2+ exchange (NCX1) splice variants exhibit different susceptibilities to Ca2+ overload

2006 ◽  
Vol 290 (5) ◽  
pp. H2155-H2162 ◽  
Author(s):  
Cecilia Hurtado ◽  
Michele Prociuk ◽  
Thane G. Maddaford ◽  
Elena Dibrov ◽  
Nasrin Mesaeli ◽  
...  
Keyword(s):  
Splice Variant ◽  
Cardiac Glycosides ◽  
Splice Variants ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Neonatal Cardiomyocytes ◽  
Simulated Ischemia ◽  
293 Cells ◽  
Intracellular Ca ◽  
Specific Alternative

The Na+/Ca2+ exchanger (NCX) NCX1 exhibits tissue-specific alternative splicing. Such NCX splice variants as NCX1.1 and NCX1.3 are also differentially regulated by Na+ and Ca2+, although the physiological implications of these regulatory characteristics are unclear. On the basis of their distinct regulatory profiles, we hypothesized that cells expressing these different splice variants might exhibit unique responses to conditions promoting Ca2+ overload, such as during exposure to cardiac glycosides or simulated ischemia. NCX1.1 or NCX1.3 was expressed in human embryonic kidney (HEK)-293 cells or rat neonatal ventricular cardiomyocytes (NVC), and expression was confirmed by Western blotting and immunocytochemical analyses. HEK-293 cells lacked NCX1 protein before transfection. With use of adenoviral vectors, neonatal cardiomyocytes were induced to overexpress the NCX1.1 splice variant by nearly twofold, whereas the NCX1.3 isoform was expressed on the endogenous NCX1.1 background. Total expression was comparable for NCX1.1 and NCX1.3. Exposure of NVC to ouabain induced a significant increase in cellular Ca2+, an effect that was exaggerated in cells overexpressing NCX1.1, but not NCX1.3. The increase in intracellular Ca2+ was inhibited by 5 μM KB-R7943. Cardiomyocytes overexpressing NCX1.1 also exhibited a greater accumulation of intracellular Ca2+ in response to simulated ischemia than did cells expressing NCX1.3. Similar responses were observed in HEK-293 cells where NCX1.1 was expressed. We conclude that expression of the NCX1.3 splice variant protects against severe Ca2+ overload, whereas NCX1.1 promotes Ca2+ overload in response to cardiac glycosides and ischemic challenges. These results highlight the importance of ionic regulation in controlling NCX1 activity under conditions that promote Ca2+ overload.

scholarly journals Functional diversity of PFKFB3 splice variants in glioblastomas

2020 ◽  
Author(s):  
Ulli Heydasch ◽  
Renate Kessler ◽  
Jan-Peter Warnke ◽  
Klaus Eschrich ◽  
Nicole Scholz ◽  
...  
Keyword(s):  
Splice Variant ◽  
Splice Variants ◽  
Aerobic Glycolysis ◽  
Diagnostic Value ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Specific Effects ◽  
293 Cells ◽  
Common Notion ◽  
The Impact

AbstractTumor cells tend to metabolize glucose through aerobic glycolysis instead of oxidative phosphorylation in mitochondria. One of the rate limiting enzymes of glycolysis is 6-phosphofructo-1-kinase, which is allosterically activated by fructose 2,6-bisphosphate which in turn is produced by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2 or PFKFB). Mounting evidence suggests that cancerous tissues overexpress the PFKFB isoenzyme, PFKFB3, being causing enhanced proliferation of cancer cells.Initially, six PFKFB3 splice variants with different C-termini have been documented in humans. More recently, additional splice variants with varying N-termini were discovered the functions of which are to be uncovered.Glioblastoma is one of the deadliest forms of brain tumors. Up to now, the role of PFKFB3 splice variants in the progression and prognosis of glioblastomas is only partially understood. In this study, we first re-categorized the PFKFB3 splice variant repertoire to simplify the denomination. We investigated the impact of increased and decreased levels of PFKFB3-4 (former UBI2K4) and PFKFB3-5 (former variant 5) on the viability and proliferation rate of glioblastoma U87 and HEK-293 cells. The simultaneous knock-down of PFKFB3-4 and PFKFB3-5 led to a decrease in viability and proliferation of U87 and HEK-293 cells as well as a reduction in HEK-293 cell colony formation. Overexpression of PFKFB3-4 but not PFKFB3-5 resulted in increased cell viability and proliferation. This finding contrasts with the common notion that overexpression of PFKFB3 enhances tumor growth, but instead suggests splice variant-specific effects of PFKFB3, apparently with opposing effects on cell behaviour. Strikingly, in line with this result, we found that in human IDH-wildtype glioblastomas, the PFKFB3-4 to PFKFB3-5 ratio was significantly shifted towards PFKFB3-4 when compared to control brain samples. Our findings indicate that the expression level of distinct PFKFB3 splice variants impinges on tumorigenic properties of glioblastomas and that splice pattern may be of important diagnostic value for glioblastoma.

scholarly journals Functional diversity of PFKFB3 splice variants in glioblastomas

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0241092
Author(s):  
Ulli Heydasch ◽  
Renate Kessler ◽  
Jan-Peter Warnke ◽  
Klaus Eschrich ◽  
Nicole Scholz ◽  
...  
Keyword(s):  
Splice Variant ◽  
Splice Variants ◽  
Aerobic Glycolysis ◽  
Diagnostic Value ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Specific Effects ◽  
293 Cells ◽  
Common Notion ◽  
The Impact

Tumor cells tend to metabolize glucose through aerobic glycolysis instead of oxidative phosphorylation in mitochondria. One of the rate limiting enzymes of glycolysis is 6-phosphofructo-1-kinase, which is allosterically activated by fructose 2,6-bisphosphate which in turn is produced by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2 or PFKFB). Mounting evidence suggests that cancerous tissues overexpress the PFKFB isoenzyme, PFKFB3, being causing enhanced proliferation of cancer cells. Initially, six PFKFB3 splice variants with different C-termini have been documented in humans. More recently, additional splice variants with varying N-termini were discovered the functions of which are to be uncovered. Glioblastoma is one of the deadliest forms of brain tumors. Up to now, the role of PFKFB3 splice variants in the progression and prognosis of glioblastomas is only partially understood. In this study, we first re-categorized the PFKFB3 splice variant repertoire to simplify the denomination. We investigated the impact of increased and decreased levels of PFKFB3-4 (former UBI2K4) and PFKFB3-5 (former variant 5) on the viability and proliferation rate of glioblastoma U87 and HEK-293 cells. The simultaneous knock-down of PFKFB3-4 and PFKFB3-5 led to a decrease in viability and proliferation of U87 and HEK-293 cells as well as a reduction in HEK-293 cell colony formation. Overexpression of PFKFB3-4 but not PFKFB3-5 resulted in increased cell viability and proliferation. This finding contrasts with the common notion that overexpression of PFKFB3 enhances tumor growth, but instead suggests splice variant-specific effects of PFKFB3, apparently with opposing effects on cell behaviour. Strikingly, in line with this result, we found that in human IDH-wildtype glioblastomas, the PFKFB3-4 to PFKFB3-5 ratio was significantly shifted towards PFKFB3-4 when compared to control brain samples. Our findings indicate that the expression level of distinct PFKFB3 splice variants impinges on tumorigenic properties of glioblastomas and that splice pattern may be of important diagnostic value for glioblastoma.

Large T-antigen up-regulates Kv4.3 K+ channels through Sp1, and Kv4.3 K+ channels contribute to cell apoptosis and necrosis through activation of calcium/calmodulin-dependent protein kinase II

2012 ◽  
Vol 441 (3) ◽  
pp. 859-869 ◽  
Author(s):  
Qi Li ◽  
Ying Zhang ◽  
Yue Sheng ◽  
Rong Huo ◽  
Bo Sun ◽  
...  
Keyword(s):  
Cell Apoptosis ◽  
T Antigen ◽  
K Channel ◽  
Large T Antigen ◽  
Hek 293 ◽  
K Channels ◽  
Hek 293 Cells ◽  
Calcium Calmodulin Dependent ◽  
293 Cells ◽  
Apoptosis And Necrosis

Down-regulation of Kv4.3 K+ channels commonly occurs in multiple diseases, but the understanding of the regulation of Kv4.3 K+ channels and the role of Kv4.3 K+ channels in pathological conditions are limited. HEK (human embryonic kidney)-293T cells are derived from HEK-293 cells which are transformed by expression of the large T-antigen. In the present study, by comparing HEK-293 and HEK-293T cells, we find that HEK-293T cells express more Kv4.3 K+ channels and more transcription factor Sp1 (specificity protein 1) than HEK-293 cells. Inhibition of Sp1 with Sp1 decoy oligonucleotide reduces Kv4.3 K+ channel expression in HEK-293T cells. Transfection of pN3-Sp1FL vector increases Sp1 protein expression and results in increased Kv4.3 K+ expression in HEK-293 cells. Since the ultimate determinant of the phenotype difference between HEK-293 and HEK-293T cells is the large T-antigen, we conclude that the large T-antigen up-regulates Kv4.3 K+ channel expression through an increase in Sp1. In both HEK-293 and HEK-293T cells, inhibition of Kv4.3 K+ channels with 4-AP (4-aminopyridine) or Kv4.3 small interfering RNA induces cell apoptosis and necrosis, which are completely rescued by the specific CaMKII (calcium/calmodulin-dependent protein kinase II) inhibitor KN-93, suggesting that Kv4.3 K+ channels contribute to cell apoptosis and necrosis through CaMKII activation. In summary, we establish: (i) the HEK-293 and HEK-293T cell model for Kv4.3 K+ channel study; (ii) that large T-antigen up-regulates Kv4.3 K+ channels through increasing Sp1 levels; and (iii) that Kv4.3 K+ channels contribute to cell apoptosis and necrosis through activating CaMKII. The present study provides deep insights into the mechanism of the regulation of Kv4.3 K+ channels and the role of Kv4.3 K+ channels in cell death.

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.

scholarly journals Pharmacology of human sulphonylurea receptor SUR1 and inward rectifier K+ channel Kir6.2 combination expressed in HEK-293 cells

2000 ◽  
Vol 129 (7) ◽  
pp. 1323-1332 ◽  
Author(s):  
Murali Gopalakrishnan ◽  
Eduardo J Molinari ◽  
Char-Chang Shieh ◽  
Lisa M Monteggia ◽  
Jean-Marc Roch ◽  
...  
Keyword(s):  
Inward Rectifier ◽  
K Channel ◽  
Hek 293 ◽  
Sulphonylurea Receptor ◽  
Hek 293 Cells ◽  
293 Cells

scholarly journals Characteristics and requirements of basal autophagy in HEK 293 cells

Autophagy ◽  
2013 ◽  
Vol 9 (9) ◽  
pp. 1407-1417 ◽  
Author(s):  
Patience Musiwaro ◽  
Matthew Smith ◽  
Maria Manifava ◽  
Simon A. Walker ◽  
Nicholas T. Ktistakis
Keyword(s):  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Halothane Inhibition of Recombinant Cardiac L-type Ca2+ Channels Expressed in HEK-293 Cells

2005 ◽  
Vol 103 (6) ◽  
pp. 1156-1166 ◽  
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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