Molecular determinants of voltage-gated sodium channel regulation by the Nedd4/Nedd4-like proteins

2005 ◽  
Vol 288 (3) ◽  
pp. C692-C701 ◽  
Author(s):  
Jean-Sébastien Rougier ◽  
Miguel X. van Bemmelen ◽  
M. Christine Bruce ◽  
Thomas Jespersen ◽  
Bruno Gavillet ◽  
...  
Keyword(s):  
Cell Voltage ◽  
Tryptophan Fluorescence ◽  
General Mechanism ◽  
Hek 293 ◽  
Ww Domains ◽  
Voltage Gated ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Molecular Determinants ◽  
Py Motif

The voltage-gated Na+ channels (Nav) form a family composed of 10 genes. The COOH termini of Nav contain a cluster of amino acids that are nearly identical among 7 of the 10 members. This COOH-terminal sequence, PPSYDSV, is a PY motif known to bind to WW domains of E3 protein-ubiquitin ligases of the Nedd4 family. We recently reported that cardiac Nav1.5 is regulated by Nedd4-2. In this study, we further investigated the molecular determinants of regulation of Nav proteins. When expressed in HEK-293 cells and studied using whole cell voltage clamping, the neuronal Nav1.2 and Nav1.3 were also downregulated by Nedd4-2. Pull-down experiments using fusion proteins bearing the PY motif of Nav1.2, Nav1.3, and Nav1.5 indicated that mouse brain Nedd4-2 binds to the Nav PY motif. Using intrinsic tryptophan fluorescence imaging of WW domains, we found that Nav1.5 PY motif binds preferentially to the fourth WW domain of Nedd4-2 with a Kd of ∼55 μM. We tested the binding properties and the ability to ubiquitinate and downregulate Nav1.5 of three Nedd4-like E3s: Nedd4-1, Nedd4-2, and WWP2. Despite the fact that along with Nedd4-2, Nedd4-1 and WWP2 bind to Nav1.5 PY motif, only Nedd4-2 robustly ubiquitinated and downregulated Nav1.5. Interestingly, coexpression of WWP2 competed with the effect of Nedd4-2. Finally, using brefeldin A, we found that Nedd4-2 accelerated internalization of Nav1.5 stably expressed in HEK-293 cells. This study shows that Nedd4-dependent ubiquitination of Nav channels may represent a general mechanism regulating the excitability of neurons and myocytes via modulation of channel density at the plasma membrane.

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

2003 ◽  
Vol 285 (2) ◽  
pp. C467-C479 ◽  
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.

Changes in the Effect of Isoflurane on N -methyl-d-aspartic Acid-gated Currents in Cultured Cerebral Cortical Neurons with Time in Culture

2002 ◽  
Vol 97 (4) ◽  
pp. 856-867 ◽  
Author(s):  
Zhen Ming ◽  
Benjamin L. Griffith ◽  
George R. Breese ◽  
Robert A. Mueller ◽  
Hugh E. Criswell
Keyword(s):  
Nmda Receptors ◽  
Cortical Neurons ◽  
Splice Variants ◽  
Cell Voltage ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Cerebral Cortical Neurons ◽  
Cultured Cortical Neurons ◽  
Nr2 Subunits

Background Developmental changes in NR1 splice variants and NR2 subunits of the N-methyl-D-aspartate (NMDA) receptor have been associated with changes in the sensitivity of NMDA receptors to agonists, antagonists, and pharmacologic modulators. The authors have investigated changes in the effect of isoflurane on NMDA-gated currents from cultured cortical neurons with time in culture and related these changes to the subunit composition of the NMDA receptors. Methods N-methyl-D-aspartate-gated currents were measured using whole-cell voltage clamp recording in cortical neurons cultured for 1-4 weeks and HEK 293 cells transiently expressing NR1-1a + NR2A or NR1-1a + NR2B subunit-containing receptors. NMDA alone or NMDA with treatment agents (isoflurane or ifenprodil) was applied to cells using a U tube. Results The effect of isoflurane and the NR2B selective antagonist ifenprodil on NMDA-gated currents from cortical neurons decreased significantly with time in culture. NMDA-gated currents mediated by NR2A-containing receptors were less sensitive to isoflurane than those mediated by NR2B-containing receptors. Tachyphylaxis to repeated application of isoflurane was found in cortical neurons and HEK 293 cells with recombinant NMDA receptors. Hooked tail currents were induced by isoflurane in cultured cortical neurons and HEK 293 cells with expressed NMDA receptors. Conclusions Isoflurane inhibits NMDA-gated currents at concentrations well below 1 minimum alveolar concentration (MAC). This effect of isoflurane was subunit dependent with the NR2B-containing receptors more sensitive to isoflurane than the NR2A-containing receptors. A potent tachyphylaxis occurred after brief exposure to isoflurane.

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

2009 ◽  
Vol 296 (5) ◽  
pp. C1049-C1057 ◽  
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.

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.

Micropatterned surfaces of PDMS as growth templates for HEK 293 cells

2007 ◽  
Vol 9 (4) ◽  
pp. 475-485 ◽  
Author(s):  
R. M. Johann ◽  
Ch. Baiotto ◽  
Ph. Renaud
Keyword(s):  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Calcium Induces Expression of Cytoplasmic Gelsolin in SH-SY5Y and HEK-293 Cells

2010 ◽  
Vol 35 (7) ◽  
pp. 1075-1082 ◽  
Author(s):  
Lina Ji ◽  
Abha Chauhan ◽  
Ved Chauhan
Keyword(s):  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Cytoplasmic Gelsolin

Characteristics of rat downregulated in adenoma (rDRA) expressed in HEK 293 cells

2007 ◽  
Vol 454 (3) ◽  
pp. 441-450 ◽  
Author(s):  
Christian Barmeyer ◽  
Jeff Huaqing Ye ◽  
Shafik Sidani ◽  
John Geibel ◽  
Henry J. Binder ◽  
...  
Keyword(s):  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells
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