scholarly journals Human geneSLC41A1encodes for the Na+/Mg2+exchanger

2012 ◽  
Vol 302 (1) ◽  
pp. C318-C326 ◽  
Author(s):  
Martin Kolisek ◽  
Axel Nestler ◽  
Jürgen Vormann ◽  
Monika Schweigel-Röntgen
Keyword(s):  
Mammalian Cells ◽  
Molecular Level ◽  
Transport Activity ◽  
Human Embryonic Kidney ◽  
Efflux System ◽  
Dependent Protein Kinase ◽  
Complete Replacement ◽  
Hek 293 ◽  
293 Cells ◽  
Dependent Protein

Magnesium (Mg2+), the second most abundant divalent intracellular cation, is involved in the vast majority of intracellular processes, including the synthesis of nucleic acids, proteins, and energy metabolism. The concentration of intracellular free Mg2+([Mg2+]i) in mammalian cells is therefore tightly regulated to its optimum, mainly by an exchange of intracellular Mg2+for extracellular Na+. Despite the importance of this process for cellular Mg2+homeostasis, the gene(s) encoding for the functional Na+/Mg2+exchanger is (are) still unknown. Here, using the fluorescent probe mag-fura 2 to measure [Mg2+]ichanges, we examine Mg2+extrusion from hSLC41A1-overexpressing human embryonic kidney (HEK)-293 cells. A three- to fourfold elevation of [Mg2+]iwas accompanied by a five- to ninefold increase of Mg2+efflux. The latter was strictly dependent on extracellular Na+and reduced by 91% after complete replacement of Na+with N-methyl-d-glucamine. Imipramine and quinidine, known unspecific Na+/Mg2+exchanger inhibitors, led to a strong 88% to 100% inhibition of hSLC41A1-related Mg2+extrusion. In addition, our data show regulation of the transport activity via phosphorylation by cAMP-dependent protein kinase A. As these are the typical characteristics of a Na+/Mg2+exchanger, we conclude that the human SLC41A1 gene encodes for the Na+/Mg2+exchanger, the predominant Mg2+efflux system. Based on this finding, the analysis of Na+/Mg2+exchanger regulation and its involvement in the pathogenesis of diseases such as Parkinson's disease and hypertension at the molecular level should now be possible.

scholarly journals Molecular mechanism of TMEM16A regulation: role of CaMKII and PP1/PP2A

2019 ◽  
Vol 317 (6) ◽  
pp. C1093-C1106 ◽  
Author(s):  
Ramon J. Ayon ◽  
Matthew B. Hawn ◽  
Joydeep Aoun ◽  
Michael Wiwchar ◽  
Abigail S. Forrest ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Okadaic Acid ◽  
Inhibitory Peptide ◽  
Dependent Protein Kinase ◽  
Initial Current ◽  
Pipette Solution ◽  
Hek 293 ◽  
293 Cells ◽  
Dependent Protein

This study explored the mechanism by which Ca2+-activated Cl− channels (CaCCs) encoded by the Tmem16a gene are regulated by calmodulin-dependent protein kinase II (CaMKII) and protein phosphatases 1 (PP1) and 2A (PP2A). Ca2+-activated Cl− currents ( IClCa) were recorded from HEK-293 cells expressing mouse TMEM16A. IClCa were evoked using a pipette solution in which free Ca2+ concentration was clamped to 500 nM, in the presence (5 mM) or absence of ATP. With 5 mM ATP, IClCa decayed to <50% of the initial current magnitude within 10 min after seal rupture. IClCa rundown seen with ATP-containing pipette solution was greatly diminished by omitting ATP. IClCa recorded after 20 min of cell dialysis with 0 ATP were more than twofold larger than those recorded with 5 mM ATP. Intracellular application of autocamtide-2-related inhibitory peptide (5 µM) or KN-93 (10 µM), two specific CaMKII inhibitors, produced a similar attenuation of TMEM16A rundown. In contrast, internal application of okadaic acid (30 nM) or cantharidin (100 nM), two nonselective PP1 and PP2A blockers, promoted the rundown of TMEM16A in cells dialyzed with 0 ATP. Mutating serine 528 of TMEM16A to an alanine led to a similar inhibition of TMEM16A rundown to that exerted by either one of the two CaMKII inhibitors tested, which was not observed for three putative CaMKII consensus sites for phosphorylation (T273, T622, and S730). Our results suggest that TMEM16A-mediated CaCCs are regulated by CaMKII and PP1/PP2A. Our data also suggest that serine 528 of TMEM16A is an important contributor to the regulation of IClCa by CaMKII.

scholarly journals Depletion of the polyamines spermidine and spermine by overexpression of spermidine/spermine N1-acetyltransferase 1 (SAT1) leads to mitochondria-mediated apoptosis in mammalian cells

2015 ◽  
Vol 468 (3) ◽  
pp. 435-447 ◽  
Author(s):  
Swati Mandal ◽  
Ajeet Mandal ◽  
Myung Hee Park
Keyword(s):  
Mammalian Cells ◽  
Growth Arrest ◽  
Mitochondrial Pathway ◽  
Human Embryonic Kidney ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Catabolic Enzyme

Adenoviral overexpression of a polyamine catabolic enzyme spermidine (SSAT1)/spermine N1-acetyltransferase 1 (SAT1) in human embryonic kidney (HEK) 293 cells leads to a rapid depletion of spermidine and spermine, growth arrest and apoptosis through the intrinsic mitochondrial pathway.

scholarly journals Interaction of Styrylpyridinium Compound with Pathogenic Candida albicans Yeasts and Human Embryonic Kidney HEK-293 Cells

2020 ◽  
Vol 9 (1) ◽  
pp. 48
Author(s):  
Simona Vaitkienė ◽  
Laura Bekere ◽  
Gunars Duburs ◽  
Rimantas Daugelavičius
Keyword(s):  
Candida Albicans ◽  
Mammalian Cells ◽  
Efflux Pump ◽  
Efflux Transporter ◽  
Human Embryonic Kidney ◽  
Abcb1 Gene ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Lipophilic Cation

Candida albicans-caused local and systemic diseases are a serious health issue worldwide, leading to high mycosis-associated morbidity and mortality. Efficient combinations of novel compounds with commonly used antifungals could be an important tool for fighting infections. The aim of this study was to evaluate the interaction of synthesized 4-(4-cyanostyryl)-1-dodecylpyridin-1-ium (CSDP+) bromide alone or in combination with fluconazole with yeast and mammalian cells. We investigated cytotoxicity of the tested agents to mammalian HEK-293 cells and the influence of CSDP+ on the ability of C. albicans wt and a clinical isolate to adhere to HEK-293. Accumulation of lipophilic cation ethidium (Et+) was used to monitor the activity of efflux pumps in HEK-293 cells. The effect of CSDP+ on the expression of the main efflux transporter genes and transcription factors in C.albicans cells as well as HEK-293 efflux pump gene ABCB1 was determined. The study showed that CSDP+ alone and in combination with fluconazole was nontoxic to HEK-293 cells and was able to reduce C.albicans adhesion. The treatment of C.albicans cells with CSDP+ in combination with fluconazole resulted in a considerable overexpression of the MDR1 and MRR1 genes. The findings suggest that these genes could be associated with efflux-related resistance to fluconazole. Measurements of Et+ fluorescence and analysis of ABCB1 gene expression demonstrated that mammalian cells were not sensitive to concentrations of CSDP+ affecting C. albicans.

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

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.

Cyclic AMP-dependent protein kinase regulates sensitivity of cells to multiple drugs

1987 ◽  
Vol 7 (9) ◽  
pp. 3098-3106
Author(s):  
I Abraham ◽  
R J Hunter ◽  
K E Sampson ◽  
S Smith ◽  
M M Gottesman ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Mammalian Cells ◽  
Multiple Drug Resistance ◽  
Regulatory Subunit ◽  
Parent Cell ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Antimitotic Drug ◽  
Multiple Drugs

The isolation of mutant cell lines affecting the activity of cyclic AMP (cAMP)-dependent protein kinase (PK-A) has made it possible to determine the function of this kinase in mammalian cells. We found that both a CHO cell mutant with a defective regulatory subunit (RI) for PK-A and a transfectant cell line expressing the same mutant kinase were sensitive to multiple drugs, including puromycin, adriamycin, actinomycin D, and some antimitotic drugs. The mutant and transfectant cells, after treatment with a concentration of the antimitotic drug colcemid that had no marked effect on the wild-type parent cell, had a severely disrupted microtubule network. The phenotype of hypersensitivity to the antimitotic drug colcemid was used to select revertants of the transfectant and the original mutant. These revertants simultaneously regained normal multiple drug resistance and cAMP sensitivity, thus establishing that the characteristics of colcemid sensitivity and cAMP resistance are linked. Four revertants of the transfectant reverted because of loss or rearrangement of the transfected mutant RI gene. These revertants, as well as one revertant selected from the original mutant, had PK-A activities equal to or higher than that of the parent. In these genetic studies, in which linkage of expression of a PK-A mutation with drug sensitivity is demonstrated, it was established that the PK-A system is involved in regulating resistance of mammalian cells to multiple drugs.

GFP Fusions for Fluorescence Detection of Ca2+ and Ca2+-Calmodulin in Living Cells

1998 ◽  
Vol 4 (S2) ◽  
pp. 1008-1009
Author(s):  
Anthony Persechini
Keyword(s):  
Mammalian Cells ◽  
Dissociation Constants ◽  
Hek 293 ◽  
Fluorescent Indicators ◽  
Calmodulin Binding ◽  
Binding Domains ◽  
293 Cells ◽  
20 Nm ◽  
Binding Sequence ◽  
Expression In Mammalian Cells

We have previously described fluorescent indicators for Ca2+ (FIP-CAs) and (Ca2+)4-calmodulin (FIP-CBs) whose responses are based on a ligand-dependent decrease in fluorescence energy transfer (FRET) between GFP variants. The indicators for (Ca2+)4-calmodulin contain calmodulin-binding domains, those for Ca2+ also contain an integral calmodulin (CaM) domain. We have developed new versions of these indicators constructed with enhanced blue- and red-shifted GFPs suitable for stable and transient expression in mammalian cells, and have begun to use them to investigate the relationships between the free intracellular concentrations of Ca2+ ([Ca2+]i) and (Ca2+)4-CaM ([(Ca2+)4-CaM]i). When the blue-shifted fluorophore is excited at 380 nm these constructs exhibit an emission peak at 505 nm due to FRET to the red-shifted fluorophore.We have made FIP-CBs with dissociation constants for (Ca2+)4-CaM of 0.5 nM, 20 nM, 300 nM and > 20 μM by introducing R →Q substitutions in the CaM-binding sequence, and have stablyexpressed them in HEK-293 cells (Fig. 1).

scholarly journals Cyclic AMP-dependent protein kinase regulates sensitivity of cells to multiple drugs.

1987 ◽  
Vol 7 (9) ◽  
pp. 3098-3106 ◽  
Author(s):  
I Abraham ◽  
R J Hunter ◽  
K E Sampson ◽  
S Smith ◽  
M M Gottesman ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Mammalian Cells ◽  
Multiple Drug Resistance ◽  
Regulatory Subunit ◽  
Parent Cell ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Antimitotic Drug ◽  
Multiple Drugs

The isolation of mutant cell lines affecting the activity of cyclic AMP (cAMP)-dependent protein kinase (PK-A) has made it possible to determine the function of this kinase in mammalian cells. We found that both a CHO cell mutant with a defective regulatory subunit (RI) for PK-A and a transfectant cell line expressing the same mutant kinase were sensitive to multiple drugs, including puromycin, adriamycin, actinomycin D, and some antimitotic drugs. The mutant and transfectant cells, after treatment with a concentration of the antimitotic drug colcemid that had no marked effect on the wild-type parent cell, had a severely disrupted microtubule network. The phenotype of hypersensitivity to the antimitotic drug colcemid was used to select revertants of the transfectant and the original mutant. These revertants simultaneously regained normal multiple drug resistance and cAMP sensitivity, thus establishing that the characteristics of colcemid sensitivity and cAMP resistance are linked. Four revertants of the transfectant reverted because of loss or rearrangement of the transfected mutant RI gene. These revertants, as well as one revertant selected from the original mutant, had PK-A activities equal to or higher than that of the parent. In these genetic studies, in which linkage of expression of a PK-A mutation with drug sensitivity is demonstrated, it was established that the PK-A system is involved in regulating resistance of mammalian cells to multiple drugs.

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