scholarly journals Phosphoregulation of the intracellular termini of K+-Cl− cotransporter 2 (KCC2) enables flexible control of its activity

2018 ◽  
Vol 293 (44) ◽  
pp. 16984-16993 ◽  
Author(s):  
Antje Cordshagen ◽  
Wiebke Busch ◽  
Michael Winklhofer ◽  
Hans Gerd Nothwang ◽  
Anna-Maria Hartmann
Keyword(s):  
Intrinsic Activity ◽  
Hek 293 ◽  
Flexible Control ◽  
Mediated Effects ◽  
293 Cells ◽  
Bona Fide ◽  
Graded Activity ◽  
First Time ◽  
Increased Activity

The pivotal role of K+-Cl− cotransporter 2 (KCC2) in inhibitory neurotransmission and severe human diseases fosters interest in understanding posttranslational regulatory mechanisms such as (de)phosphorylation. Here, the regulatory role of the five bona fide phosphosites Ser31, Thr34, Ser932, Thr999, and Thr1008 was investigated by the use of alanine and aspartate mutants. Tl+-based flux analyses in HEK-293 cells demonstrated increased transport activity for S932D (mimicking phosphorylation) and T1008A (mimicking dephosphorylation), albeit to a different extent. Increased activity was due to changes in intrinsic activity, as it was not caused by increased cell-surface abundance. Substitutions of Ser31, Thr34, or Thr999 had no effect. Additionally, we show that the indirect actions of the known KCC2 activators staurosporine and N-ethylmaleimide (NEM) involved multiple phosphosites. S31D, T34A, S932A/D, T999A, or T1008A/D abrogated staurosporine mediated stimulation, and S31A, T34D, or S932D abolished NEM-mediated stimulation. This demonstrates for the first time differential effects of staurosporine and NEM on KCC2. In addition, the staurosporine-mediated effects involved both KCC2 phosphorylation and dephosphorylation with Ser932 and Thr1008 being bona fide target sites. In summary, our data reveal a complex phosphoregulation of KCC2 that provides the transporter with a toolbox for graded activity and integration of different signaling pathways.

scholarly journals Species-Specific Regulation of TRPM2 by PI(4,5)P2 via the Membrane Interfacial Cavity

2021 ◽  
Vol 22 (9) ◽  
pp. 4637
Author(s):  
Daniel Barth ◽  
Andreas Lückhoff ◽  
Frank J. P. Kühn
Keyword(s):  
Amino Acid Residues ◽  
Hek 293 ◽  
Complete Inactivation ◽  
293 Cells ◽  
Activation Mechanisms ◽  
Specific Regulation ◽  
Species Specific ◽  
Inside Out ◽  
Positively Charged

The human apoptosis channel TRPM2 is stimulated by intracellular ADR-ribose and calcium. Recent studies show pronounced species-specific activation mechanisms. Our aim was to analyse the functional effect of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), commonly referred to as PIP2, on different TRPM2 orthologues. Moreover, we wished to identify the interaction site between TRPM2 and PIP2. We demonstrate a crucial role of PIP2, in the activation of TRPM2 orthologues of man, zebrafish, and sea anemone. Utilizing inside-out patch clamp recordings of HEK-293 cells transfected with TRPM2, differential effects of PIP2 that were dependent on the species variant became apparent. While depletion of PIP2 via polylysine uniformly caused complete inactivation of TRPM2, restoration of channel activity by artificial PIP2 differed widely. Human TRPM2 was the least sensitive species variant, making it the most susceptible one for regulation by changes in intramembranous PIP2 content. Furthermore, mutations of highly conserved positively charged amino acid residues in the membrane interfacial cavity reduced the PIP2 sensitivity in all three TRPM2 orthologues to varying degrees. We conclude that the membrane interfacial cavity acts as a uniform PIP2 binding site of TRPM2, facilitating channel activation in the presence of ADPR and Ca2+ in a species-specific manner.

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.

scholarly journals Anoctamin 5/TMEM16E facilitates muscle precursor cell fusion

2018 ◽  
Vol 150 (11) ◽  
pp. 1498-1509 ◽  
Author(s):  
Jarred M. Whitlock ◽  
Kuai Yu ◽  
Yuan Yuan Cui ◽  
H. Criss Hartzell
Keyword(s):  
Cell Fusion ◽  
Ionic Current ◽  
Ionic Currents ◽  
Muscle Physiology ◽  
Muscle Repair ◽  
Hek 293 ◽  
293 Cells ◽  
Phospholipid Scrambling ◽  
Muscle Precursor Cell

Limb-girdle muscular dystrophy type 2L (LGMD2L) is a myopathy arising from mutations in ANO5; however, information about the contribution of ANO5 to muscle physiology is lacking. To explain the role of ANO5 in LGMD2L, we previously hypothesized that ANO5-mediated phospholipid scrambling facilitates cell–cell fusion of mononucleated muscle progenitor cells (MPCs), which is required for muscle repair. Here, we show that heterologous overexpression of ANO5 confers Ca2+-dependent phospholipid scrambling to HEK-293 cells and that scrambling is associated with the simultaneous development of a nonselective ionic current. MPCs isolated from adult Ano5−/− mice exhibit defective cell fusion in culture and produce muscle fibers with significantly fewer nuclei compared with controls. This defective fusion is associated with a decrease of Ca2+-dependent phosphatidylserine exposure on the surface of Ano5−/− MPCs and a decrease in the amplitude of Ca2+-dependent outwardly rectifying ionic currents. Viral introduction of ANO5 in Ano5−/− MPCs restores MPC fusion competence, ANO5-dependent phospholipid scrambling, and Ca2+-dependent outwardly rectifying ionic currents. ANO5-rescued MPCs produce myotubes having numbers of nuclei similar to wild-type controls. These data suggest that ANO5-mediated phospholipid scrambling or ionic currents play an important role in muscle repair.

Extracellular signal-regulated kinase activation by parathyroid hormone in distal tubule cells

2007 ◽  
Vol 292 (3) ◽  
pp. F1028-F1034 ◽  
Author(s):  
W. Bruce Sneddon ◽  
Yanmei Yang ◽  
Jianming Ba ◽  
Lisa M. Harinstein ◽  
Peter A. Friedman
Keyword(s):  
Conditioned Media ◽  
Kidney Cells ◽  
Wild Type ◽  
Egfr Transactivation ◽  
Hek 293 ◽  
Erk Activation ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Extracellular Signal

The PTH receptor (PTH1R) activates multiple signaling pathways, including extracellular signal-regulated kinases 1 and 2 (ERK1/2). The role of epidermal growth factor receptor (EGFR) transactivation in ERK1/2 activation by PTH in distal kidney cells, a primary site of PTH action, was characterized. ERK1/2 phosphorylation was stimulated by PTH and blocked by the EGFR inhibitor, AG1478. Upon PTH stimulation, metalloprotease cleavage of membrane-bound heparin-binding fragment (HB-EGF) induced EGFR transactivation of ERK. Conditioned media from PTH-treated distal kidney cells activated ERK in HEK-293 cells. AG1478 added to HEK-293 cells ablated transactivation by conditioned media. HB-EGF directly activated ERK1/2 in HEK-293 cells. Pretreatment of distal kidney cells with the metalloprotease inhibitor GM-6001 abolished transactivation of ERK1/2 by PTH. The role of the PTH1R COOH terminus in PTX-sensitive ERK1/2 activation was characterized in HEK-293 cells transfected with wild-type PTH1R, with a PTH1R mutated at its COOH terminus, or with PTH1R truncated at position 480. PTH stimulated ERK by wild-type, mutated and truncated PTH1Rs 21-, 27- and 57-fold, respectively. Thus, the PTH1R COOH terminus exerts an inhibitory effect on ERK activation. EBP50, a scaffolding protein that binds to the PDZ recognition domain of the PTH1R, impaired PTH but not isoproterenol or calcitonin-induced ERK activation. Pertussis toxin inhibited PTH-stimulated ERK1/2 by mutated and truncated PTH1Rs and abolished ERK1/2 activation by wild-type PTH1R. We conclude that ERK phosphorylation in distal kidney cells by PTH requires PTH1R activation of Gi, which leads to stimulation of metalloprotease-mediated cleavage of HB-EGF and transactivation of the EGFR and is regulated by EBP50.

scholarly journals The Role of Endogenous Human Trp4 in Regulating Carbachol-induced Calcium Oscillations in HEK-293 Cells

2002 ◽  
Vol 277 (16) ◽  
pp. 13597-13608 ◽  
Author(s):  
Xiaoyan Wu ◽  
György Babnigg ◽  
Tatiana Zagranichnaya ◽  
Mitchel L. Villereal
Keyword(s):  
Calcium Oscillations ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

scholarly journals Role of integrin αVβ3 in the production of recombinant adenoviruses in HEK-293 cells

Gene Therapy ◽  
2002 ◽  
Vol 9 (14) ◽  
pp. 907-914 ◽  
Author(s):  
WLW Ling ◽  
RL Longley ◽  
DL Brassard ◽  
L Armstrong ◽  
EJ Schaefer
Keyword(s):  
Integrin Αvβ3 ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

scholarly journals Influence of intracellular Ca2+ and alternative splicing on the pharmacological profile of ANO1 channels

2016 ◽  
Vol 311 (3) ◽  
pp. C437-C451 ◽  
Author(s):  
Tae Sik Sung ◽  
Kate O'Driscoll ◽  
Haifeng Zheng ◽  
Nicholas J. Yapp ◽  
Normand Leblanc ◽  
...  
Keyword(s):  
Splice Variants ◽  
Channel Blockers ◽  
Anoctamin 1 ◽  
Patch Clamp Technique ◽  
Hek 293 ◽  
Naturally Occurring ◽  
293 Cells ◽  
Intracellular Ca ◽  
Inside Out

Anoctamin-1 (ANO1) is a Ca2+-activated Cl− channel expressed in many types of cells. Splice variants of ANO1 have been shown to influence the biophysical properties of conductance. It has been suggested that several new antagonists of ANO1 with relatively high affinity and selectivity might be useful for experimental and, potentially, therapeutic purposes. We investigated the effects of intracellular Ca2+ concentration ([Ca2+]i) at 100-1,000 nM, a concentration range that might be achieved in cells during physiological activation of ANO1 channels, on blockade of ANO1 channels expressed in HEK-293 cells. Whole cell and excised patch configurations of the patch-clamp technique were used to perform tests on a variety of naturally occurring splice variants of ANO1. Blockade of ANO1 currents with aminophenylthiazole (T16Ainh-A01) was highly dependent on [Ca2+]i. Increasing [Ca2+]i reduced the potency of this blocker. Similar Ca2+-dependent effects were also observed with benzbromarone. Experiments on excised, inside-out patches showed that the diminished potency of the blockers caused by intracellular Ca2+ might involve a competitive interaction for a common binding site or repulsion of the blocking drugs by electrostatic forces at the cytoplasmic surface of the channels. The degree of interaction between the channel blockers and [Ca2+]i depends on the splice variant expressed. These experiments demonstrate that the efficacy of ANO1 antagonists depends on [Ca2+]i, suggesting a need for caution when ANO1 blockers are used to determine the role of ANO1 in physiological functions and in their use as therapeutic agents.

scholarly journals The cytoplasmic domain is essential for transport function of the integral membrane transport protein SLC4A11

2016 ◽  
Vol 310 (2) ◽  
pp. C161-C174 ◽  
Author(s):  
Sampath K. Loganathan ◽  
Chris M. Lukowski ◽  
Joseph R. Casey
Keyword(s):  
Membrane Transport ◽  
Fluorescent Protein ◽  
Strong Association ◽  
Transport Protein ◽  
Transport Function ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Membrane Transport Protein ◽  
293 Cells

Large cytoplasmic domains (CD) are a common feature among integral membrane proteins. In virtually all cases, these CD have a function (e.g., binding cytoskeleton or regulatory factors) separate from that of the membrane domain (MD). Strong associations between CD and MD are rare. Here we studied SLC4A11, a membrane transport protein of corneal endothelial cells, the mutations of which cause genetic corneal blindness. SLC4A11 has a 41-kDa CD and a 57-kDa integral MD. One disease-causing mutation in the CD, R125H, manifests a catalytic defect, suggesting a role of the CD in transport function. Expressed in HEK-293 cells without the CD, MD-SLC4A11 is retained in the endoplasmic reticulum, indicating a folding defect. Replacement of CD-SLC4A11 with green fluorescent protein did not rescue MD-SLC4A11, suggesting some specific role of CD-SLC4A11. Homology modeling revealed that the structure of CD-SLC4A11 is similar to that of the Cl−/HCO3−exchange protein AE1 (SLC4A1) CD. Fusion to CD-AE1 partially rescued MD-SLC4A11 to the cell surface, suggesting that the structure of CD-AE1 is similar to that of CD-SLC4A11. The CD-AE1-MD-SLC4a11 chimera, however, had no functional activity. We conclude that CD-SLC4A11 has an indispensable role in the transport function of SLC4A11. CD-SLC4A11 forms insoluble precipitates when expressed in bacteria, suggesting that the domain cannot fold properly when expressed alone. Consistent with a strong association between CD-SLC4A11 and MD-SLC4A11, these domains specifically associate when coexpressed in HEK-293 cells. We conclude that SLC4A11 is a rare integral membrane protein in which the CD has strong associations with the integral MD, which contributes to membrane transport function.

scholarly journals Processing of Pro–B-Type Natriuretic Peptide: Furin and Corin as Candidate Convertases2

2010 ◽  
Vol 56 (7) ◽  
pp. 1166-1176 ◽  
Author(s):  
Alexander G Semenov ◽  
Natalia N Tamm ◽  
Karina R Seferian ◽  
Alexander B Postnikov ◽  
Natalia S Karpova ◽  
...  
Keyword(s):  
Heart Failure ◽  
Natriuretic Peptide ◽  
Spectrometric Analysis ◽  
Precursor Molecule ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Lovo Cells ◽  
Interfering Rna

Abstract Background: B-type natriuretic peptide (BNP) and its N-terminal fragment (NT-proBNP) are the products of the enzyme-mediated cleavage of their precursor molecule, proBNP. The clinical significance of proBNP-derived peptides as biomarkers of heart failure has been explored thoroughly, whereas little is known about the mechanisms of proBNP processing. We investigated the role of 2 candidate convertases, furin and corin, in human proBNP processing. Methods: We measured proBNP expression in HEK 293 and furin-deficient LoVo cells. We used a furin inhibitor and a furin-specific small interfering RNA (siRNA) to explore the implication of furin in proBNP processing. Recombinant proBNPs were incubated with HEK 293 cells transfected with the corin-expressing plasmid. We applied mass spectrometry to analyze the products of furin- and corin-mediated cleavage. Results: Reduction of furin activity significantly impaired proBNP processing in HEK 293 cells. Furin-deficient LoVo cells were unable to process proBNP, whereas coexpression with furin resulted in effective proBNP processing. Mass spectrometric analysis revealed that the furin-mediated cleavage of proBNP resulted in BNP 1–32, whereas corin-mediated cleavage led to the production of BNP 4–32. Some portion of proBNP in the plasma of heart failure patients was not glycosylated in the cleavage site region and was susceptible to furin-mediated cleavage. Conclusions: Both furin and corin are involved in the proBNP processing pathway, giving rise to distinct BNP forms. The significance of the presence of unprocessed proBNP in circulation that could be cleaved by the endogenous convertases should be further investigated for better understanding BNP physiology.

Essential role for Ca2+in regulation of IL-1β secretion by P2X7nucleotide receptor in monocytes, macrophages, and HEK-293 cells

2003 ◽  
Vol 285 (2) ◽  
pp. C286-C299 ◽  
Author(s):  
Lalitha Gudipaty ◽  
Jonathan Munetz ◽  
Philip A. Verhoef ◽  
George R. Dubyak
Keyword(s):  
Essential Role ◽  
Signal Sequence ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Nucleotide Receptors ◽  
Hek 293 ◽  
External Stimulation ◽  
Hek 293 Cells ◽  
293 Cells

Interleukin (IL)-1β is a proinflammatory cytokine that elicits the majority of its biological activity extracellularly, but the lack of a secretory signal sequence prevents its export via classic secretory pathways. Efficient externalization of IL-1β in macrophages and monocytes can occur via stimulation of P2X7nucleotide receptors with extracellular ATP. However, the exact mechanisms by which the activation of these nonselective cation channels facilitates secretion of IL-1β remain unclear. Here we demonstrate a pivotal role for a sustained increase in cytosolic Ca2+to potentiate secretion of IL-1β via the P2X7receptors. Using HEK-293 cells engineered to coexpress P2X7receptors with mature IL-1β (mIL-1β), we show that activation of P2X7receptors results in a rapid secretion of mIL-1β by a process(es) that is dependent on influx of extracellular Ca2+and a sustained rise in cytosolic Ca2+. Moreover, reduction in extracellular Ca2+attenuates ∼90% of P2X7receptor-mediated IL-1β secretion but has no effect on enzymatic processing of precursor IL-1β (proIL-1β) to mIL-1β by caspase-1. Similar experiments with THP-1 human monocytes and Bac1.2F5 murine macrophages confirm the unique role of Ca2+in P2X7receptor-mediated secretion of IL-1β. In addition, we report that cell surface expression of P2X7receptors in the absence of external stimulation also results in enhanced release of IL-1β and that this can be repressed by inhibitors of P2X7receptors. We clarify an essential role for Ca2+in ATP-induced IL-1β secretion and indicate an additional role of P2X7receptors as enhancers of the secretory apparatus by which IL-1β is released.

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