Tyrosine Phosphorylation Modulates Current Amplitude and Kinetics of a Neuronal Voltage-Gated Potassium Channel

1997 ◽  
Vol 78 (3) ◽  
pp. 1563-1573 ◽  
Author(s):  
Debra A. Fadool ◽  
Todd C. Holmes ◽  
Kevin Berman ◽  
Daniel Dagan ◽  
Irwin B. Levitan
Keyword(s):  
Tyrosine Kinase ◽  
Potassium Channel ◽  
Tyrosine Phosphorylation ◽  
Current Amplitude ◽  
Hek 293 ◽  
Voltage Gated ◽  
Tyrosine Residues ◽  
Channel Protein ◽  
293 Cells ◽  
Kinetics Of

Fadool, Debra A., Todd C. Holmes, Kevin Berman, Daniel Dagan, and Irwin B. Levitan. Tyrosine phosphorylation modulates current amplitude and kinetics of a neuronal voltage-gated potassium channel. J. Neurophysiol. 78: 1563–1573, 1997. The modulation of the Kv1.3 potassium channel by tyrosine phosphorylation was studied. Kv1.3 was expressed in human embryonic kidney (HEK 293) cells, and its activity was measured by cell-attached patch recording. The amplitude of the characteristic C-type inactivating Kv1.3 current is reduced by >95%, in all cells tested, when the channel is co-expressed with the constitutively active nonreceptor tyrosine kinase, v-Src. This v-Src–induced suppression of current is accompanied by a robust tyrosine phosphorylation of the channel protein. No suppression of current or tyrosine phosphorylation of Kv1.3 protein is observed when the channel is co-expressed with R385A v-Src, a mutant with severely impaired tyrosine kinase activity. v-Src–induced suppression of Kv1.3 current is relieved by pretreatment of the HEK 293 cells with two structurally different tyrosine kinase inhibitors, herbimycin A and genistein. Furthermore, Kv1.3 channel protein is processed properly and targeted to the plasma membrane in v-Src cotransfected cells, as demonstrated by confocal microscopy using an antibody directed against an extracellular epitope on the channel. Thus v-Src–induced suppression of Kv1.3 current is not mediated through decreased channel protein expression or interference with its targeting to the plasma membrane. v-Src co-expression also slows the C-type inactivation and speeds the deactivation of the residual Kv1.3 current. Mutational analysis demonstrates that each of these modulatory changes, in current amplitude and kinetics, requires the phosphorylation of Kv1.3 at multiple tyrosine residues. Furthermore, a different combination of tyrosine residues is involved in each of the modulatory changes. These results emphasize the complexity of signal integration at the level of a single ion channel.

scholarly journals Modulation of the Kv1.3 Potassium Channel by Receptor Tyrosine Kinases

1997 ◽  
Vol 110 (5) ◽  
pp. 601-610 ◽  
Author(s):  
Mark R. Bowlby ◽  
Debra A. Fadool ◽  
Todd C. Holmes ◽  
Irwin B. Levitan
Keyword(s):  
Tyrosine Kinase ◽  
Potassium Channel ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Mutational Analysis ◽  
Inactivation Kinetics ◽  
Hek 293 ◽  
Voltage Dependent

The voltage-dependent potassium channel, Kv1.3, is modulated by the epidermal growth factor receptor (EGFr) and the insulin receptor tyrosine kinases. When the EGFr and Kv1.3 are coexpressed in HEK 293 cells, acute treatment of the cells with EGF during a patch recording can suppress the Kv1.3 current within tens of minutes. This effect appears to be due to tyrosine phosphorylation of the channel, as it is blocked by treatment with the tyrosine kinase inhibitor erbstatin, or by mutation of the tyrosine at channel amino acid position 479 to phenylalanine. Previous work has shown that there is a large increase in the tyrosine phosphorylation of Kv1.3 when it is coexpressed with the EGFr. Pretreatment of EGFr and Kv1.3 cotransfected cells with EGF before patch recording also results in a decrease in peak Kv1.3 current. Furthermore, pretreatment of cotransfected cells with an antibody to the EGFr ligand binding domain (α-EGFr), which blocks receptor dimerization and tyrosine kinase activation, blocks the EGFr-mediated suppression of Kv1.3 current. Insulin treatment during patch recording also causes an inhibition of Kv1.3 current after tens of minutes, while pretreatment for 18 h produces almost total suppression of current. In addition to depressing peak Kv1.3 current, EGF treatment produces a speeding of C-type inactivation, while pretreatment with the α-EGFr slows C-type inactivation. In contrast, insulin does not influence C-type inactivation kinetics. Mutational analysis indicates that the EGF-induced modulation of the inactivation rate occurs by a mechanism different from that of the EGF-induced decrease in peak current. Thus, receptor tyrosine kinases differentially modulate the current magnitude and kinetics of a voltage-dependent potassium channel.

scholarly journals Tyrosine-phosphorylation-dependent and Rho-protein-mediated control of cellular phosphatidylinositol 4,5-bisphosphate levels

1998 ◽  
Vol 334 (3) ◽  
pp. 625-631 ◽  
Author(s):  
Ulrich RÜMENAPP ◽  
Martina SCHMIDT ◽  
Simone OLESCH ◽  
Sabine OTT ◽  
Christoph von EICHEL-STREIBER ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Cellular Level ◽  
Tyrosine Phosphatases ◽  
Rho Proteins ◽  
Toxin B ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Tyrphostin 23

The polyphosphoinositide PtdIns(4,5)P2, best known as a substrate for phospholipase C isozymes, has recently been recognized to be involved in a variety of other cellular processes. The aim of this study was to examine whether the cellular levels of this versatile phospholipid are controlled by tyrosine phosphorylation. The studies were performed in human embryonic kidney (HEK)-293 cells stably expressing the M3 muscarinic acetylcholine receptor. Inhibition of tyrosine phosphatases by pervanadate induced an up-to-approx.-2.5-fold increase in the total cellular level of PtdIns(4,5)P2, which was both time- and concentration-dependent. In contrast, the tyrosine kinase inhibitors, genistein and tyrphostin 23, caused a rapid and specific fall in the cellular PtdIns(4,5)P2 level and prevented the stimulatory effect of pervanadate on PtdIns(4,5)P2 formation. Inactivation of Rho proteins by Clostridium difficile toxin B caused a similar fall in the HEK-293 cell PtdIns(4,5)P2 level, which was not altered by additional genistein treatment. Furthermore, toxin B treatment abolished the pervanadate-induced increase in PtdIns(4,5)P2 levels. As PtdIns(4,5)P2 is an essential stimulatory cofactor for phospholipase D (PLD) enzymes, we finally examined the effects of the agents regulating PtdIns(4,5)P2 levels on PLD activity in HEK-293 cells. Inhibition of tyrosine phosphatases by pervanadate caused an increase in PLD activity, which was susceptible to genistein and tyrphostin 23, and which was abolished by prior treatment with toxin B. In conclusion, the data presented indicate that the cellular level of the multifunctional phospholipid, PtdIns(4,5)P2, in HEK-293 cells is controlled by a tyrosine-kinase-dependent mechanism and that this process apparently involves Rho proteins, as found similarly for tyrosine-phosphorylation-induced PLD activation.

COOH-terminal association of human smooth muscle calcium channel Cav1.2b with Src kinase protein binding domains: effect of nitrotyrosylation

2007 ◽  
Vol 293 (6) ◽  
pp. C1983-C1990 ◽  
Author(s):  
Minho Kang ◽  
Gracious R. Ross ◽  
Hamid I. Akbarali
Keyword(s):  
Smooth Muscle ◽  
Calcium Channel ◽  
Tyrosine Phosphorylation ◽  
Src Kinase ◽  
Sh2 Domain ◽  
Calcium Currents ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Binding Domains ◽  
293 Cells

The carboxyl terminus of the calcium channel plays an important role in the regulation of calcium entry, signal transduction, and gene expression. Potential protein-protein interaction sites within the COOH terminus of the L-type calcium channel include those for the SH3 and SH2 binding domains of c-Src kinase that regulates calcium currents in smooth muscle. In this study, we examined the binding sites involved in Src kinase-mediated phosphorylation of the human voltage-gated calcium channel (Cav) 1.2b (hCav1.2b) and the effect of nitrotyrosylation. Cotransfection of human embryonic kidney (HEK)-293 cells with hCav1.2b and c-Src resulted in tyrosine phosphorylation of the calcium channel, which was prevented by nitration of tyrosine residues by peroxynitrite. Whole cell calcium currents were reduced by 58 + 5% by the Src kinase inhibitor PP2 and 64 + 6% by peroxynitrite. Nitrotyrosylation prevented Src-mediated regulation of the currents. Glutathione S-transferase fusion protein of the distal COOH terminus of hCav1.2b (1809-2138) bound to SH2 domain of Src following tyrosine phosphorylation, while binding to SH3 required the presence of the proline-rich motif. Site-directed mutation of Y2134 prevented SH2 binding and resulted in reduced phosphorylation of hCav1.2b. Within the distal COOH terminus, single, double, or triple mutations of Y1837, Y1861, and Y2134 were constructed and expressed in HEK-293 cells. The inhibitory effects of PP2 and peroxynitrite on calcium currents were significantly reduced in the double mutant Y1837-2134F. These data demonstrate that the COOH terminus of hCav1.2b contains sites for the SH2 and SH3 binding of Src kinase. Nitrotyrosylation of these sites prevents Src kinase regulation and may be importantly involved in calcium influx regulation during inflammation.

scholarly journals Ca2+ entry through NaV channels generates submillisecond axonal Ca2+ signaling

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Naomi AK Hanemaaijer ◽  
Marko A Popovic ◽  
Xante Wilders ◽  
Sara Grasman ◽  
Oriol Pavón Arocas ◽  
...  
Keyword(s):  
Calcium Channels ◽  
High Speed ◽  
Initial Segment ◽  
Hek 293 ◽  
Voltage Gated ◽  
Voltage Gated Calcium Channels ◽  
293 Cells ◽  
Axonal Initial Segment ◽  
Nav Channels ◽  
Activity Dependent

Calcium ions (Ca2+) are essential for many cellular signaling mechanisms and enter the cytosol mostly through voltage-gated calcium channels. Here, using high-speed Ca2+ imaging up to 20 kHz in the rat layer five pyramidal neuron axon we found that activity-dependent intracellular calcium concentration ([Ca2+]i) in the axonal initial segment was only partially dependent on voltage-gated calcium channels. Instead, [Ca2+]i changes were sensitive to the specific voltage-gated sodium (NaV) channel blocker tetrodotoxin. Consistent with the conjecture that Ca2+ enters through the NaV channel pore, the optically resolved ICa in the axon initial segment overlapped with the activation kinetics of NaV channels and heterologous expression of NaV1.2 in HEK-293 cells revealed a tetrodotoxin-sensitive [Ca2+]i rise. Finally, computational simulations predicted that axonal [Ca2+]i transients reflect a 0.4% Ca2+ conductivity of NaV channels. The findings indicate that Ca2+ permeation through NaV channels provides a submillisecond rapid entry route in NaV-enriched domains of mammalian axons.

Fyn Tyrosine Kinase Reduces the Ethanol Inhibition of Recombinant NR1/NR2A but Not NR1/NR2B NMDA Receptors Expressed in HEK 293 Cells

2001 ◽  
Vol 72 (4) ◽  
pp. 1389-1393 ◽  
Author(s):  
Douglas L. Anders ◽  
Tana Blevins ◽  
Greg Sutton ◽  
Sheridan Swope ◽  
L. Judson Chandler ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Nmda Receptors ◽  
Ethanol Inhibition ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Fyn Tyrosine Kinase

Phosphoinositide 3-kinase-dependent phosphorylation of the dual adaptor for phosphotyrosine and 3-phosphoinositides by the Src family of tyrosine kinase

2000 ◽  
Vol 349 (2) ◽  
pp. 605-610 ◽  
Author(s):  
Simon DOWLER ◽  
Leire MONTALVO ◽  
Doreen CANTRELL ◽  
Nick MORRICE ◽  
Dario R. ALESSI
Keyword(s):  
Growth Factor ◽  
Tyrosine Kinase ◽  
Active Form ◽  
Adaptor Protein ◽  
Sh2 Domain ◽  
Ph Domain ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Phosphoinositide 3 Kinase

We recently identified a novel adaptor protein, termed dual adaptor for phosphotyrosine and 3-phosphoinositides (DAPP1), that possesses a Src homology (SH2) domain and a pleckstrin homology (PH) domain. DAPP1 exhibits a high-affinity interaction with PtdIns(3,4,5)P3 and PtdIns(3,4)P2, which bind to the PH domain. In the present study we show that when DAPP1 is expressed in HEK-293 cells, the agonists insulin, insulin-like growth factor-1 and epidermal growth factor induce the phosphorylation of DAPP1 at Tyr139. Treatment of cells with phosphoinositide 3-kinase (PI 3-kinase) inhibitors or expression of a dominant-negative PI 3-kinase prevent phosphorylation of DAPP1 at Tyr139, and a PH-domain mutant of DAPP1, which does not interact with PtdIns(3,4,5)P3 or PtdIns(3,4)P2, is not phosphorylated at Tyr139 following agonist stimulation of cells. Overexpression of a constitutively active form of PI 3-kinase induced the phosphorylation of DAPP1 in unstimulated cells. We demonstrated that Tyr139 of DAPP1 is likely to be phosphorylated in vivo by a Src-family tyrosine kinase, since the specific Src-family inhibitor, PP2, but not an inactive variant of this drug, PP3, prevented the agonist-induced tyrosine phosphorylation of DAPP1. Src, Lyn and Lck tyrosine kinases phosphorylate DAPP1 at Tyr139in vitro at similar rates in the presence or absence of PtdIns(3,4,5)P3, and overexpression of these kinases in HEK-293 cells induces the phosphorylation of Tyr139. These findings indicate that, following activation of PI 3-kinases, PtdIns(3,4,5)P3 or PtdIns(3,4)P2 bind to DAPP1, recruiting it to the plasma membrane where it becomes phosphorylated at Tyr139 by a Src-family tyrosine kinase.

scholarly journals Further evidence that the tyrosine phosphorylation of glycogen synthase kinase-3 (GSK3) in mammalian cells is an autophosphorylation event

2004 ◽  
Vol 377 (1) ◽  
pp. 249-255 ◽  
Author(s):  
Adam COLE ◽  
Sheelagh FRAME ◽  
Philip COHEN
Keyword(s):  
Tyrosine Phosphorylation ◽  
Mammalian Cells ◽  
Protein Tyrosine Kinase ◽  
Kinase Inhibitor ◽  
Glycogen Synthase ◽  
Glycogen Synthase Kinase ◽  
Hek 293 ◽  
293 Cells ◽  
General Protein ◽  
The Stability

Phosphorylation of the endogenous GSK3α (glycogen synthase kinase-3α) at Tyr279 and GSK3β at Tyr216 was suppressed in HEK-293 or SH-SY5Y cells by incubation with pharmacological inhibitors of GSK3, but not by an Src-family inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), or a general protein tyrosine kinase inhibitor (genistein). GSK3β transfected into HEK-293 cells or Escherichia coli became phosphorylated at Tyr216, but catalytically inactive mutants did not. GSK3β expressed in insect Sf 21 cells or E. coli was extensively phosphorylated at Tyr216, but the few molecules lacking phosphate at this position could autophosphorylate at Tyr216in vitro after incubation with MgATP. The rate of autophosphorylation was unaffected by dilution and was suppressed by the GSK3 inhibitor kenpaullone. Wild-type GSK3β was unable to catalyse the tyrosine phosphorylation of catalytically inactive GSK3β lacking phosphate at Tyr216. Our results indicate that the tyrosine phosphorylation of GSK3 is an intramolecular autophosphorylation event in the cells that we have studied and that this modification enhances the stability of the enzyme.

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