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 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.

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

2006 ◽  
Vol 290 (5) ◽  
pp. H2155-H2162 ◽  
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.

Role of RYR3 splice variants in calcium signaling in mouse nonpregnant and pregnant myometrium

2007 ◽  
Vol 293 (3) ◽  
pp. C848-C854 ◽  
Author(s):  
Fabrice Dabertrand ◽  
Nicolas Fritz ◽  
Jean Mironneau ◽  
Nathalie Macrez ◽  
Jean-Luc Morel
Keyword(s):  
Alternative Splicing ◽  
Antisense Oligonucleotides ◽  
Splice Variants ◽  
Pregnant Mouse ◽  
Receptor Subtype ◽  
Hek 293 ◽  
Physiological Regulation ◽  
293 Cells ◽  
Subtype 3

Alternative splicing of ryanodine receptor subtype 3 (RYR3) may generate a short isoform (RYR3S) without channel function and a functional full-length isoform (RYR3L). The RYR3S isoform has been shown to negatively regulate the native RYR2 subtype in smooth muscle cells as well as the RYR3L isoform when both isoforms were coexpressed in HEK-293 cells. Mouse myometrium expresses only the RYR3 subtype, but the role of RYR3 isoforms obtained by alternative splicing and their activation by cADP-ribose during pregnancy have never been investigated. Here, we show that both RYR3S and RYR3L isoforms are differentially expressed in nonpregnant and pregnant mouse myometrium. The use of antisense oligonucleotides directed against each isoform indicated that only RYR3L was activated by caffeine and cADP-ribose in nonpregnant myometrium. These RYR3L-mediated Ca2+ releases were negatively regulated by RYR3S expression. At the end of pregnancy, the relative expression of RYR3L versus RYR3S and its ability to respond to cADP-ribose were increased. Therefore, our results suggest that physiological regulation of RYR3 alternative splicing may play an essential role at the end of pregnancy.

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

2012 ◽  
Vol 303 (12) ◽  
pp. C1260-C1268 ◽  
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.

scholarly journals Intracellular Ca2+ oscillations generated via the Ca2+-sensing receptor are mediated by negative feedback by PKCα at Thr888

2014 ◽  
Vol 306 (3) ◽  
pp. C298-C306 ◽  
Author(s):  
Steven H. Young ◽  
Osvaldo Rey ◽  
James Sinnett-Smith ◽  
Enrique Rozengurt
Keyword(s):  
Negative Feedback ◽  
Small Interfering Rna ◽  
Phosphorylation Site ◽  
Hek 293 ◽  
Pkc Inhibitor ◽  
Identical Response ◽  
293 Cells ◽  
Intracellular Ca ◽  
Interfering Rna

To clarify the mechanism(s) underlying intracellular Ca2+ concentration ([Ca2+]i) oscillations induced by an elevation in extracellular Ca2+ concentration ([Ca2+]e) via the extracellular Ca2+-sensing receptor (CaR), we analyzed the pattern of [Ca2+]i response in multiple (2,303) individual HEK-293 cells transfected with the human CaR. An increase in the [Ca2+]e from 1.5 to 3 mM produced oscillatory fluctuations in [Ca2+]i in 70% of the cell population. To determine the role of PKC in the generation of [Ca2+]i oscillations, cells were exposed to increasing concentrations (0.5–5 μM) of the preferential PKC inhibitor Ro-31-8220 before stimulation by extracellular Ca2+. Ro-31-8220 at 3–5 μM completely eliminated the [Ca2+]e-evoked [Ca2+]i oscillations and transformed the pattern to a peak and sustained plateau response. Treatment with other broad PKC inhibitors, including GFI or Gö6983, produced an identical response. Similarly, treatment with Ro-31-8220 or GFI eliminated [Ca2+]e-evoked [Ca2+]i oscillations in colon-derived SW-480 cells expressing the CaR. Treatment with inhibitors targeting classic PKCs, including Gö6976 and Ro-32-0432 as well as small interfering RNA-mediated knockdown of PKCα, strikingly reduced the proportion of cell displaying [Ca2+]e-evoked [Ca2+]i oscillations. Furthermore, none of the cells analyzed expressing a CaR mutant in which the major PKC phosphorylation site Thr888 was converted to alanine (CaRT888A) showed [Ca2+]i oscillations after CaR activation. Our results show that [Ca2+]i oscillations induced by activation of the CaR in response to an increase in extracellular Ca2+ or exposure to the calcimimetic R-568 result from negative feedback involving PKCα-mediated phosphorylation of the CaR at Thr888.

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 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 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.

Molecular identification of a TTX-sensitive Ca2+current

2001 ◽  
Vol 280 (5) ◽  
pp. C1327-C1339 ◽  
Author(s):  
Silvia Guatimosim ◽  
Eric A. Sobie ◽  
Jader dos Santos Cruz ◽  
Laura A. Martin ◽  
W. J. Lederer
Keyword(s):  
Cardiac Myocytes ◽  
Ion Selectivity ◽  
Inactivation Kinetics ◽  
Experimental Conditions ◽  
Guinea Pig Heart ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Intracellular Ca ◽  
Rat Myocytes

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.”

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.

Sign in / Sign up

Export Citation Format

Share Document