Molecular identification of a TTX-sensitive Ca2+current

The TTX-sensitive Ca2+ current [ I Ca(TTX)] observed in cardiac myocytes under Na+-free conditions was investigated using patch-clamp and Ca2+-imaging methods. Cs+ and Ca2+were found to contribute to I Ca(TTX), but TEA+ and N-methyl-d-glucamine (NMDG+) did not. HEK-293 cells transfected with cardiac Na+ channels exhibited a current that resembled I Ca(TTX) in cardiac myocytes with regard to voltage dependence, inactivation kinetics, and ion selectivity, suggesting that the cardiac Na+ channel itself gives rise to I Ca(TTX). Furthermore, repeated activation of I Ca(TTX) led to a 60% increase in intracellular Ca2+ concentration, confirming Ca2+ entry through this current. Ba2+ permeation of I Ca(TTX), reported by others, did not occur in rat myocytes or in HEK-293 cells expressing cardiac Na+channels under our experimental conditions. The report of block of I Ca(TTX) in guinea pig heart by mibefradil (10 μM) was supported in transfected HEK-293 cells, but Na+current was also blocked (half-block at 0.45 μM). We conclude that I Ca(TTX) reflects current through cardiac Na+ channels in Na+-free (or “null”) conditions. We suggest that the current be renamed I Na(null) to more accurately reflect the molecular identity of the channel and the conditions needed for its activation. The relationship between I Na(null)and Ca2+ flux through slip-mode conductance of cardiac Na+ channels is discussed in the context of ion channel biophysics and “permeation plasticity.”

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

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00958.2005 ◽

2006 ◽

Vol 290 (5) ◽

pp. H2155-H2162 ◽

Cited By ~ 27

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.

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Interaction between bradykinin and natriuretic peptides via RGS protein activation in HEK-293 cells

AJP Cell Physiology ◽

10.1152/ajpcell.00033.2012 ◽

2012 ◽

Vol 303 (12) ◽

pp. C1260-C1268 ◽

Cited By ~ 6

Author(s):

Marina Dobrivojević ◽

Aleksandra Sinđić ◽

Bayram Edemir ◽

Stefanie Kalweit ◽

Wolf-Georg Forssmann ◽

...

Keyword(s):

Signaling Pathway ◽

Natriuretic Peptide ◽

Natriuretic Peptides ◽

Specific Inhibitor ◽

Rgs Proteins ◽

Patch Clamp Technique ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Intracellular Ca

In this study, the interaction of natriuretic peptides (NP) and bradykinin (BK) signaling pathways was identified by measuring membrane potential ( Vm) and intracellular Ca2+ using the patch-clamp technique and flow cytometry in HEK-293 cells. BK and NP receptor mRNA was identified using RT-PCR. BK (100 nM) depolarized cells activating bradykinin receptor type 2 (B2R) and Ca2+-dependent Cl− channels inhibitable by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 10 μM). The BK-induced Ca2+ signal was blocked by the B2R inhibitor HOE 140. [Des-Arg9]-bradykinin, an activator of B1R, had no effect on intracellular Ca2+. NP [atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and urodilatin] depolarized HEK-293 cells inhibiting K+ channels. ANP, urodilatin, BNP [binding to natriuretic peptide receptor (NPR)-A] and 8-bromo-(8-Br)-cGMP inhibited the BK-induced depolarization while CNP (binding to NPR-Bi) failed to do so. The inhibitory effect on BK-triggered depolarization could be reversed by blocking PKG using the specific inhibitor KT 5823. BK-stimulated depolarization as well as Ca2+ signaling was completely blocked by the phospholipase C (PLC) inhibitor U-73122 (10 nM). The inositol 1,4,5-trisphosphate receptor blocker 2-aminoethoxydiphenyl borate (2-APB; 50 μM) completely inhibited the BK-induced Ca2+ signaling. UTP, another activator of the PLC-mediated Ca2+ signaling pathway, was blocked by U-73122 as well but not by 8-Br-cGMP, indicating an intermediate regulatory step for NP via PKG in BK signaling such as regulators of G-protein signaling (RGS) proteins. When RGS proteins were inhibited by CCG-63802 in the presence of BK and 8-Br-cGMP, cells started to depolarize again. In conclusion, as natural antagonists of the B2R signaling pathway, NP may also positively interact in pathological conditions caused by BK.

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Intracellular calcium measurements as a method in studies on activity of purinergic P2X receptor channels

AJP Cell Physiology ◽

10.1152/ajpcell.00042.2003 ◽

2003 ◽

Vol 285 (2) ◽

pp. C467-C479 ◽

Cited By ~ 38

Author(s):

Mu-Lan He ◽

Hana Zemkova ◽

Taka-aki Koshimizu ◽

Melanija Tomić ◽

Stanko S. Stojilkovic

Keyword(s):

Purinergic Receptors ◽

Host Cells ◽

P2x Receptor ◽

Wild Type ◽

Hek 293 ◽

Human Embryonic Kidney Cells ◽

Voltage Gated ◽

Hek 293 Cells ◽

293 Cells ◽

Intracellular Ca

Extracellular nucleotide-activated purinergic receptors (P2XRs) are a family of cation-permeable channels that conduct small cations, including Ca2+, leading to the depolarization of cells and subsequent stimulation of voltage-gated Ca2+ influx in excitable cells. Here, we studied the spatiotemporal characteristics of intracellular Ca2+ signaling and its dependence on current signaling in excitable mouse immortalized gonadotropin-releasing hormone-secreting cells (GT1) and nonexcitable human embryonic kidney cells (HEK-293) cells expressing wild-type and chimeric P2XRs. In both cell types, P2XR generated depolarizing currents during the sustained ATP stimulation, which desensitized in order (from rapidly desensitizing to nondesensitizing): P2X3R > P2X2b + X4R > P2X2bR > P2X2a + X4R > P2X4R > P2X2aR > P2X7R. HEK-293 cells were not suitable for studies on P2XR-mediated Ca2+ influx because of the coactivation of endogenously expressed Ca2+-mobilizing purinergic P2Y receptors. However, when expressed in GT1 cells, all wild-type and chimeric P2XRs responded to agonist binding with global Ca2+ signals, which desensitized in the same order as current signals but in a significantly slower manner. The global distribution of Ca2+ signals was present independently of the rate of current desensitization. The temporal characteristics of Ca2+ signals were not affected by voltage-gated Ca2+ influx and removal of extracellular sodium. Ca2+ signals reflected well the receptor-specific EC50 values for ATP and the extracellular Zn2+ and pH sensitivities of P2XRs. These results indicate that intracellular Ca2+ measurements are useful for characterizing the pharmacological properties and messenger functions of P2XRs, as well as the kinetics of channel activity, when the host cells do not express other members of purinergic receptors.

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Thiamin uptake by the human-derived renal epithelial (HEK-293) cells: cellular and molecular mechanisms

AJP Renal Physiology ◽

10.1152/ajprenal.00078.2006 ◽

2006 ◽

Vol 291 (4) ◽

pp. F796-F805 ◽

Cited By ~ 34

Author(s):

Balasubramaniem Ashokkumar ◽

Nosratola D. Vaziri ◽

Hamid M. Said

Keyword(s):

Molecular Mechanisms ◽

Critical Role ◽

Mrna Levels ◽

Cellular Functions ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Glomerular Filtrate ◽

Intracellular Ca ◽

Ph Dependent

Thiamin (vitamin B1) is essential for normal cellular functions. The kidneys play a critical role in regulating body thiamin homeostasis, by salvaging the vitamin via reabsorption from the glomerular filtrate, but little is known about the mechanism(s) and regulation of thiamin transport in the human renal epithelia at cellular and molecular levels. Using the human-derived renal epithelial HEK-293 cells as a model, we have addressed these issues. Our results showed [3H]thiamin uptake to be 1) temperature and energy dependent but Na+ independent, 2) pH dependent with higher uptake at alkaline/neutral buffer pH compared with acidic pH, 3) saturable as a function of concentration over the nanomolar (apparent Km = 70.0 ± 18.4 nM) and micromolar (apparent Km = 2.66 ± 0.18 μM) ranges, 4) cis-inhibited by unlabeled thiamin and its structural analogs but not by unrelated organic cations, 5) trans-stimulated by unlabeled thiamin, and 6) competitively inhibited by amiloride with an apparent Ki of 0.6 mM. Using a gene-specific small-interference RNAs (siRNAs) approach, human thiamin transporters 1 and 2 (hTHTR-1 and hTHTR-2) were both found to be expressed and contributed toward total carrier-mediated thiamin uptake. Maintaining the cells in thiamin-deficient medium led to a significant ( P < 0.01) and specific upregulation in [3H]thiamin uptake, which was associated with an increase in hTHTR-1 and hTHTR-2 protein and mRNA levels as well as promoter activities. Uptake of thiamin by HEK-293 cells also appeared to be under the regulation of an intracellular Ca2+/calmodulin-mediated pathway. These studies demonstrate for the first time that thiamin uptake by HEK-293 cells is mediated via a specific pH-dependent process, which involves both the hTHTR-1 and hTHTR-2. In addition, the uptake process appears to be under the regulation of an intracellular Ca2+/CaM-mediated pathway and also adaptively upregulated in thiamin deficiency via transcriptional regulatory mechanism(s) that involves both the hTHTR-1 and hTHTR-2.

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Voltage-gated sodium channel modulation by σ-receptors in cardiac myocytes and heterologous systems

AJP Cell Physiology ◽

10.1152/ajpcell.00431.2008 ◽

2009 ◽

Vol 296 (5) ◽

pp. C1049-C1057 ◽

Cited By ~ 73

Author(s):

Molly Johannessen ◽

Subramaniam Ramachandran ◽

Logan Riemer ◽

Andrea Ramos-Serrano ◽

Arnold E. Ruoho ◽

...

Keyword(s):

Cardiac Myocytes ◽

Integral Membrane Protein ◽

Receptor Ligands ◽

Pipette Solution ◽

Hek 293 ◽

Voltage Gated ◽

Hek 293 Cells ◽

Novel Structure ◽

293 Cells ◽

Voltage Gated Ion Channels

The σ-receptor, a broadly distributed integral membrane protein with a novel structure, is known to modulate various voltage-gated K+ and Ca2+ channels through a mechanism that involves neither G proteins nor phosphorylation. The present study investigated the modulation of the heart voltage-gated Na+ channel (Nav1.5) by σ-receptors. The σ1-receptor ligands [SKF-10047 and (+)-pentazocine] and σ1/σ2-receptor ligands (haloperidol and ditolylguanidine) all reversibly inhibited Nav1.5 channels to varying degrees in human embryonic kidney 293 (HEK-293) cells and COS-7 cells, but the σ1-receptor ligands were less effective in COS-7 cells. The same four ligands also inhibited Na+ current in neonatal mouse cardiac myocytes. In σ1-receptor knockout myocytes, the σ1-receptor-specific ligands were far less effective in modulating Na+ current, but the σ1/σ2-receptor ligands modulated Na+ channels as well as in wild type. Photolabeling with the σ1-receptor photoprobe [125I]-iodoazidococaine demonstrated that σ1-receptors were abundant in heart and HEK-293 cells, but scarce in COS-7 cells. This difference was consistent with the greater efficacy of σ1-receptor-specific ligands in HEK-293 cells than in COS-7 cells. σ-Receptors modulated Na+ channels despite the omission of GTP and ATP from the patch pipette solution. σ-Receptor-mediated inhibition of Na+ current had little if any voltage dependence and produced no change in channel kinetics. Na+ channels represent a new addition to the large number of voltage-gated ion channels modulated by σ-receptors. The modulation of Nav1.5 channels by σ-receptors in the heart suggests an important pathway by which drugs can alter cardiac excitability and rhythmicity.

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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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