Functional analysis of two isoforms of phosphatidylethanolamine N-methyltransferase

The enzyme catalysing the conversion of PE (phosphatidylethanolamine) into PC (phosphatidylcholine), PEMT (PE N-methyltransferase), exists as two isoforms, PEMT-L (longer isoform of PEMT) and PEMT-S (shorter isoform of PEMT). In the present study, to compare the functions of the two isoforms of PEMT, we established HEK (human embryonic kidney)-293 cell lines stably expressing PEMT-L and PEMT-S. Both PEMT-L and PEMT-S were localized in the ER (endoplasmic reticulum). PEMT-L, but not PEMT-S, was N-glycosylated with high-mannose oligosaccharides. The enzymatic activity of PEMT-S was much higher than that of PEMT-L. By using novel enzymatic assays for measuring PC and PE, we showed that PEMT-L and PEMT-S expression remarkably increased the cellular PC content, whereas the PE content was decreased by PEMT-S expression, but was hardly affected by PEMT-L expression. The cellular content of phosphatidylserine was also reduced by the expression of PEMT-L or PEMT-S. MS analyses demonstrated that the expression of PEMT-S led to more increases in the molecular species of PC and PC-O (ether-linked PC) with longer polyunsaturated chains than that of PEMT-L, whereas the PC-O species with shorter chains were increased more by PEMT-L expression than by PEMT-S expression, suggesting a difference in the substrate specificity of PEMT-L and PEMT-S. On the other hand, various PE and PE-O species were decreased by PEMT-S expression. In addition, PEMT-L and PEMT-S expression promoted the proliferation of HEK-293 cells. Based upon these findings, we propose a model in which the enzymatic activity and substrate specificity are regulated by the glycosylated N-terminal region of PEMT-L localized in the ER lumen.

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Bupivacaine inhibits a small conductance calcium‐activated potassium type 2 channel in human embryonic kidney 293 cells

BMC Pharmacology and Toxicology ◽

10.1186/s40360-021-00481-2 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Hongfei Chen ◽

Zhousheng Jin ◽

Fangfang Xia ◽

Zhijian Fu

Keyword(s):

Free Calcium ◽

Heart Cells ◽

Dependent Manner ◽

Human Embryonic Kidney ◽

Patch Clamp Technique ◽

Hek 293 ◽

293 Cells ◽

Ic50 Value ◽

Small Conductance

Abstract Background Bupivacaine blocks many ion channels in the heart muscle, causing severe cardiotoxicity. Small-conductance calcium-activated potassium type 2 channels (SK2 channels) are widely distributed in the heart cells and are involved in relevant physiological functions. However, whether bupivacaine can inhibit SK2 channels is still unclear. This study investigated the effect of bupivacaine on SK2 channels. Methods The SK2 channel gene was transfected into human embryonic kidney 293 cells (HEK-293 cells) with Lipofectamine 2000. The whole-cell patch-clamp technique was used to examine the effect of bupivacaine on SK2 channels. The concentration–response relationship of bupivacaine for inhibiting SK2 currents (0 mV) was fitted to a Hill equation, and the half-maximal inhibitory concentration (IC50) value was determined. Results Bupivacaine inhibited the SK2 channels reversibly in a dose-dependent manner. The IC50 value of bupivacaine, ropivacaine, and lidocaine on SK2 currents was 16.5, 46.5, and 77.8µM, respectively. The degree of SK2 current inhibition by bupivacaine depended on the intracellular concentration of free calcium. Conclusions The results of this study suggested the inhibitory effect of bupivacaine on SK2 channels. Future studies should explore the effects of SK2 on bupivacaine cardiotoxicity.

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Optimization of transfection mediated by calcium phosphate for plasmid rAAV-LacZ (recombinant adeno-associated virus–β-galactosidase reporter gene) production in suspension-cultured HEK-293 (human embryonic kidney 293) cells

Biotechnology and Applied Biochemistry ◽

10.1042/ba20060143 ◽

2007 ◽

Vol 46 (2) ◽

pp. 127 ◽

Cited By ~ 9

Author(s):

Meijin Guo ◽

Lei Feng ◽

Shuxiang Zhang ◽

Ju Chu ◽

Yingping Zhuang ◽

...

Keyword(s):

Calcium Phosphate ◽

Reporter Gene ◽

Human Embryonic Kidney ◽

Adeno Associated Virus ◽

Hek 293 ◽

293 Cells

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Effect of Curcumin on Cisplatin- and Oxaliplatin-Induced Oxidative Stress in Human Embryonic Kidney (HEK) 293 Cells

Renal Failure ◽

10.3109/0886022x.2011.577546 ◽

2011 ◽

Vol 33 (5) ◽

pp. 518-523 ◽

Cited By ~ 25

Author(s):

Mostafa I. Waly ◽

Mansour S. Al Moundhri ◽

Badreldin H. Ali

Keyword(s):

Oxidative Stress ◽

Human Embryonic Kidney ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells

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Caffeine induces Ca2+ release by reducing the threshold for luminal Ca2+ activation of the ryanodine receptor

Biochemical Journal ◽

10.1042/bj20080489 ◽

2008 ◽

Vol 414 (3) ◽

pp. 441-452 ◽

Cited By ~ 102

Author(s):

Huihui Kong ◽

Peter P. Jones ◽

Andrea Koop ◽

Lin Zhang ◽

Henry J. Duff ◽

...

Keyword(s):

Ryanodine Receptor ◽

Cardiac Arrhythmias ◽

Single Channel ◽

Kidney Cells ◽

Human Embryonic Kidney ◽

Activation Threshold ◽

Hek 293 ◽

Human Embryonic Kidney Cells ◽

293 Cells ◽

High Concentrations

Caffeine has long been used as a pharmacological probe for studying RyR (ryanodine receptor)-mediated Ca2+ release and cardiac arrhythmias. However, the precise mechanism by which caffeine activates RyRs is elusive. In the present study, we investigated the effects of caffeine on spontaneous Ca2+ release and on the response of single RyR2 (cardiac RyR) channels to luminal or cytosolic Ca2+. We found that HEK-293 cells (human embryonic kidney cells) expressing RyR2 displayed partial or ‘quantal’ Ca2+ release in response to repetitive additions of submaximal concentrations of caffeine. This quantal Ca2+ release was abolished by ryanodine. Monitoring of endoplasmic reticulum luminal Ca2+ revealed that caffeine reduced the luminal Ca2+ threshold at which spontaneous Ca2+ release occurs. Interestingly, spontaneous Ca2+ release in the form of Ca2+ oscillations persisted in the presence of 10 mM caffeine, and was diminished by ryanodine, demonstrating that unlike ryanodine, caffeine, even at high concentrations, does not hold the channel open. At the single-channel level, caffeine markedly reduced the threshold for luminal Ca2+ activation, but had little effect on the threshold for cytosolic Ca2+ activation, indicating that the major action of caffeine is to reduce the luminal, but not the cytosolic, Ca2+ activation threshold. Furthermore, as with caffeine, the clinically relevant, pro-arrhythmic methylxanthines aminophylline and theophylline potentiated luminal Ca2+ activation of RyR2, and increased the propensity for spontaneous Ca2+ release, mimicking the effects of disease-linked RyR2 mutations. Collectively, our results demonstrate that caffeine triggers Ca2+ release by reducing the threshold for luminal Ca2+ activation of RyR2, and suggest that disease-linked RyR2 mutations and RyR2-interacting pro-arrhythmic agents may share the same arrhythmogenic mechanism.

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Substrate Specificity Analysis of Endoplasmic Reticulum Glucosidase II Using Synthetic High Mannose-type Glycans

Journal of Biological Chemistry ◽

10.1074/jbc.m605457200 ◽

2006 ◽

Vol 281 (42) ◽

pp. 31502-31508 ◽

Cited By ~ 71

Author(s):

Kiichiro Totani ◽

Yoshito Ihara ◽

Ichiro Matsuo ◽

Yukishige Ito

Keyword(s):

Endoplasmic Reticulum ◽

Substrate Specificity ◽

Glucosidase Ii ◽

High Mannose

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Endogenous expression of the atypical chemokine receptor CCX-CKR (CCRL1) gene in human embryonic kidney (HEK 293) cells

Molecular and Cellular Biochemistry ◽

10.1007/s11010-015-2629-2 ◽

2015 ◽

Vol 412 (1-2) ◽

pp. 229-233 ◽

Cited By ~ 2

Author(s):

Parvin Salimi ◽

Abolghasem Esmaeili ◽

Mohammad Hashemi ◽

Mohaddeseh Behjati

Keyword(s):

Chemokine Receptor ◽

Human Embryonic Kidney ◽

Hek 293 ◽

Hek 293 Cells ◽

Endogenous Expression ◽

293 Cells

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Glutamine-181 is crucial in the enzymatic activity and substrate specificity of human endoplasmic-reticulum aminopeptidase-1

Biochemical Journal ◽

10.1042/bj20080965 ◽

2008 ◽

Vol 416 (1) ◽

pp. 109-116 ◽

Cited By ~ 21

Author(s):

Yoshikuni Goto ◽

Hiroe Tanji ◽

Akira Hattori ◽

Masafumi Tsujimoto

Keyword(s):

Endoplasmic Reticulum ◽

Amino Acid ◽

Substrate Specificity ◽

Enzymatic Activity ◽

Basic Amino Acid ◽

Hydrolytic Activity ◽

Site Directed Mutagenesis ◽

Peptide Substrates ◽

Endoplasmic Reticulum Aminopeptidase 1 ◽

Natural Peptides

ERAP-1 (endoplasmic-reticulum aminopeptidase-1) is a multifunctional enzyme with roles in the regulation of blood pressure, angiogenesis and the presentation of antigens to MHC class I molecules. Whereas the enzyme shows restricted specificity toward synthetic substrates, its substrate specificity toward natural peptides is rather broad. Because of the pathophysiological significance of ERAP-1, it is important to elucidate the molecular basis of its enzymatic action. In the present study we used site-directed mutagenesis to identify residues affecting the substrate specificity of human ERAP-1 and identified Gln181 as important for enzymatic activity and substrate specificity. Replacement of Gln181 by aspartic acid resulted in a significant change in substrate specificity, with Q181D ERAP-1 showing a preference for basic amino acids. In addition, Q181D ERAP-1 cleaved natural peptides possessing a basic amino acid at the N-terminal end more efficiently than did the wild-type enzyme, whereas its cleavage of peptides with a non-basic amino acid was significantly reduced. Another mutant enzyme, Q181E, also revealed some preference for peptides with a basic N-terminal amino acid, although it had little hydrolytic activity toward the synthetic peptides tested. Other mutant enzymes, including Q181N and Q181A ERAP-1s, revealed little enzymatic activity toward synthetic or peptide substrates. These results indicate that Gln181 is critical for the enzymatic activity and substrate specificity of ERAP-1.

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The cardiac L-type calcium channel distal carboxy terminus autoinhibition is regulated by calcium

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00396.2012 ◽

2013 ◽

Vol 304 (3) ◽

pp. H455-H464 ◽

Cited By ~ 15

Author(s):

Shawn M. Crump ◽

Douglas A. Andres ◽

Gail Sievert ◽

Jonathan Satin

Keyword(s):

Calcium Channel ◽

High Frequency ◽

Dynamic Range ◽

Physiological Function ◽

Interacting Proteins ◽

Carboxy Terminus ◽

Human Embryonic Kidney ◽

Hek 293 ◽

293 Cells ◽

New Hypothesis

The L-type calcium channel (LTCC) provides trigger Ca2+ for sarcoplasmic reticulum Ca-release, and LTCC function is influenced by interacting proteins including the LTCC distal COOH terminus (DCT) and calmodulin. DCT is proteolytically cleaved and reassociates with the LTCC complex to regulate calcium channel function. DCT reduces LTCC barium current ( IBa,L) in reconstituted channel complexes, yet the contribution of DCT to LTCC Ca2+ current ( ICa,L) in cardiomyocyte systems is unexplored. This study tests the hypothesis that DCT attenuates cardiomyocyte ICa,L. We measured LTCC current and Ca2+ transients with DCT coexpressed in murine cardiomyocytes. We also heterologously coexpressed DCT and CaV1.2 constructs with truncations corresponding to the predicted proteolytic cleavage site, CaV1.2Δ1801, and a shorter deletion corresponding to well-studied construct, CaV1.2Δ1733. DCT inhibited IBa,L in cardiomyocytes, and in human embryonic kidney (HEK) 293 cells expressing CaV1.2Δ1801 and CaV1.2Δ1733. Ca2+-CaM relieved DCT block in cardiomyocytes and HEK cells. The selective block of IBa,L combined with Ca2+-CaM effects suggested that DCT-mediated blockade may be relieved under conditions of elevated Ca2+. We therefore tested the hypothesis that DCT block is dynamic, increasing under relatively low Ca2+, and show that DCT reduced diastolic Ca2+ at low stimulation frequencies but spared high frequency Ca2+ entry. DCT reduction of diastolic Ca2+ and relief of block at high pacing frequencies and under conditions of supraphysiological bath Ca2+ suggests that a physiological function of DCT is to increase the dynamic range of Ca2+ transients in response to elevated pacing frequencies. Our data motivate the new hypothesis that DCT is a native reverse use-dependent inhibitor of LTCC current.

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