scholarly journals Subtype-selective regulation of IP3 receptors by thimerosal via cysteine residues within the IP3-binding core and suppressor domain

2013 ◽  
Vol 451 (2) ◽  
pp. 177-184 ◽  
Author(s):  
Samir A. Khan ◽  
Ana M. Rossi ◽  
Andrew M. Riley ◽  
Barry V. L. Potter ◽  
Colin W. Taylor
Keyword(s):  
Partial Agonist ◽  
Ryanodine Receptors ◽  
Cysteine Residues ◽  
Ip3 Receptors ◽  
Systematic Analysis ◽  
Low Concentrations ◽  
Thiol Reagent ◽  
Binding Core ◽  
Subtype Selective ◽  
Dt40 Cells

IP3R (IP3 [inositol 1,4,5-trisphosphate] receptors) and ryanodine receptors are the most widely expressed intracellular Ca2+ channels and both are regulated by thiol reagents. In DT40 cells stably expressing single subtypes of mammalian IP3R, low concentrations of thimerosal (also known as thiomersal), which oxidizes thiols to form a thiomercurylethyl complex, increased the sensitivity of IP3-evoked Ca2+ release via IP3R1 and IP3R2, but inhibited IP3R3. Activation of IP3R is initiated by IP3 binding to the IBC (IP3-binding core; residues 224–604) and proceeds via re-arrangement of an interface between the IBC and SD (suppressor domain; residues 1–223). Thimerosal (100 μM) stimulated IP3 binding to the isolated NT (N-terminal; residues 1–604) of IP3R1 and IP3R2, but not to that of IP3R3. Binding of a competitive antagonist (heparin) or partial agonist (dimeric-IP3) to NT1 was unaffected by thiomersal, suggesting that the effect of thimerosal is specifically related to IP3R activation. IP3 binding to NT1 in which all cysteine residues were replaced by alanine was insensitive to thimerosal, so too were NT1 in which cysteine residues were replaced in either the SD or IBC. This demonstrates that thimerosal interacts directly with cysteine in both the SD and IBC. Chimaeric proteins in which the SD of the IP3R was replaced by the structurally related A domain of a ryanodine receptor were functional, but thimerosal inhibited both IP3 binding to the chimaeric NT and IP3-evoked Ca2+ release from the chimaeric IP3R. This is the first systematic analysis of the effects of a thiol reagent on each IP3R subtype. We conclude that thimerosal selectively sensitizes IP3R1 and IP3R2 to IP3 by modifying cysteine residues within both the SD and IBC and thereby stabilizing an active conformation of the receptor.

Dynamic clustering of IP3 receptors by IP3

2012 ◽  
Vol 40 (2) ◽  
pp. 325-330 ◽  
Author(s):  
Taufiq Rahman
Keyword(s):  
Inhibitory Influence ◽  
Dynamic Clustering ◽  
Ip3 Receptors ◽  
Patch Clamp Recording ◽  
Open Probability ◽  
Low Concentrations ◽  
Inositol 1,4,5 Trisphosphate ◽  
Small Clusters ◽  
Dt40 Cells ◽  
Intracellular Messenger

The versatility of Ca2+ as an intracellular messenger stems largely from the impressive, but complex, spatiotemporal organization of the Ca2+ signals. For example, the latter when initiated by IP3 (inositol 1,4,5-trisphosphate) in many cells manifest hierarchical recruitment of elementary Ca2+ release events (‘blips’ and then ‘puffs’) en route to global regenerative Ca2+ waves as the cellular IP3 concentration rises. The spacing of IP3Rs (IP3 receptors) and their regulation by Ca2+ are key determinants of these spatially organized Ca2+ signals, but neither is adequately understood. IP3Rs have been proposed to be pre-assembled into clusters, but their composition, geometry and whether clustering affects IP3R behaviour are unknown. Using patch-clamp recording from the outer nuclear envelope of DT40 cells expressing rat IP3R1 or IP3R3, we have recently shown that low concentrations of IP3 cause IP3Rs to aggregate rapidly and reversibly into small clusters of approximately four IP3Rs. At resting cytosolic Ca2+ concentrations, clustered IP3Rs open independently, but with lower open probability, shorter open duration and lesser IP3-sensitivity than lone IP3Rs. This inhibitory influence of clustering on IP3R is reversed when the [Ca2+]i (cytosolic free Ca2+ concentration) increases. The gating of clustered IP3Rs exposed to increased [Ca2+]i is coupled: they are more likely to open and close together, and their simultaneous openings are prolonged. Dynamic clustering of IP3Rs by IP3 thus exposes them to local Ca2+ rises and increases their propensity for a CICR (Ca2+-induced Ca2+ rise), thereby facilitating hierarchical recruitment of the elementary events that underlie all IP3-evoked Ca2+ signals.

scholarly journals Deregulation of Ca2+-Signaling Systems in White Adipocytes, Manifested as the Loss of Rhythmic Activity, Underlies the Development of Multiple Hormonal Resistance at Obesity and Type 2 Diabetes

2021 ◽  
Vol 22 (10) ◽  
pp. 5109
Author(s):  
Egor A. Turovsky ◽  
Maria V. Turovskaya ◽  
Vladimir V. Dynnik
Keyword(s):  
Cell Size ◽  
Ryanodine Receptors ◽  
Fluorescent Microscopy ◽  
Rhythmic Activity ◽  
Ip3 Receptors ◽  
Signaling Systems ◽  
White Adipocytes ◽  
Inhibitory Analysis ◽  
Hormonal Resistance ◽  
The Impact

Various types of cells demonstrate ubiquitous rhythmicity registered as simple and complex Ca2+-oscillations, spikes, waves, and triggering phenomena mediated by G-protein and tyrosine kinase coupled receptors. Phospholipase C/IP3-receptors (PLC/IP3R) and endothelial NO-synthase/Ryanodine receptors (NOS/RyR)–dependent Ca2+ signaling systems, organized as multivariate positive feedback generators (PLC-G and NOS-G), underlie this rhythmicity. Loss of rhythmicity at obesity may indicate deregulation of these signaling systems. To issue the impact of cell size, receptors’ interplay, and obesity on the regulation of PLC-G and NOS-G, we applied fluorescent microscopy, immunochemical staining, and inhibitory analysis using cultured adipocytes of epididumal white adipose tissue of mice. Acetylcholine, norepinephrine, atrial natriuretic peptide, bradykinin, cholecystokinin, angiotensin II, and insulin evoked complex [Ca2+]i responses in adipocytes, implicating NOS-G or PLC-G. At low sub-threshold concentrations, acetylcholine and norepinephrine or acetylcholine and peptide hormones (in paired combinations) recruited NOS-G, based on G proteins subunits interplay and signaling amplification. Rhythmicity was cell size- dependent and disappeared in hypertrophied cells filled with lipids. Contrary to control cells, adipocytes of obese hyperglycemic and hypertensive mice, growing on glucose, did not accumulate lipids and demonstrated hormonal resistance being non responsive to any hormone applied. Preincubation of preadipocytes with palmitoyl-L-carnitine (100 nM) provided accumulation of lipids, increased expression and clustering of IP3R and RyR proteins, and partially restored hormonal sensitivity and rhythmicity (5–15% vs. 30–80% in control cells), while adipocytes of diabetic mice were not responsive at all. Here, we presented a detailed kinetic model of NOS-G and discussed its control. Collectively, we may suggest that universal mechanisms underlie loss of rhythmicity, Ca2+-signaling systems deregulation, and development of general hormonal resistance to obesity.

Ca2+ signals mediated by Ins(1,4,5)P3-gated channels in rat ureteric myocytes

2000 ◽  
Vol 349 (1) ◽  
pp. 323-332 ◽  
Author(s):  
François-Xavier BOITTIN ◽  
Frédéric COUSSIN ◽  
Jean-Luc MOREL ◽  
Guillaume HALET ◽  
Nathalie MACREZ ◽  
...  
Keyword(s):  
Ryanodine Receptor ◽  
Time Course ◽  
Binding Capacity ◽  
Ryanodine Receptors ◽  
Initiation Site ◽  
Variable Number ◽  
Spatial Spread ◽  
Receptor Antibody ◽  
Low Concentrations ◽  
High Level

Localized Ca2+-release signals (puffs) and propagated Ca2+ waves were characterized in rat ureteric myocytes by confocal microscopy. Ca2+ puffs were evoked by photorelease of low concentrations of Ins(1,4,5)P3 from a caged precursor and by low concentrations of acetylcholine; they were also observed spontaneously in Ca2+-overloaded myocytes. Ca2+ puffs showed some variability in amplitude, time course and spatial spread, suggesting that Ins(1,4,5)P3-gated channels exist in clusters containing variable numbers of channels and that within these clusters a variable number of channels can be recruited. Immunodetection of Ins(1,4,5)P3 receptors revealed the existence of several spots of fluorescence in the confocal cell sections, supporting the existence of clusters of Ins(1,4,5)P3 receptors. Strong Ins(1,4,5)P3 photorelease and high concentrations of acetylcholine induced Ca2+ waves that originated from an initiation site and propagated in the whole cell by spatial recruitment of neighbouring Ca2+-release sites. Both Ca2+ puffs and Ca2+ waves were blocked selectively by intracellular applications of heparin and an anti-Ins(1,4,5)P3-receptor antibody, but were unaffected by ryanodine and intracellular application of an anti-ryanodine receptor antibody. mRNAs encoding for the three subtypes of Ins(1,4,5)P3 receptor and subtype 3 of ryanodine receptor were detected in these myocytes, and the maximal binding capacity of [3H]Ins(1,4,5)P3 was 10- to 12-fold higher than that of [3H]ryanodine. These results suggest that Ins(1,4,5)P3-gated channels mediate a continuum of Ca2+ signalling in smooth-muscle cells expressing a high level of Ins(1,4,5)P3 receptors and no subtypes 1 and 2 of ryanodine receptors.

In Vivo Characterization and Dynamic Receptor Occupancy Imaging of TPA023B, an α2/α3/α5 Subtype Selective γ-Aminobutyric Acid–A Partial Agonist

2008 ◽  
Vol 64 (2) ◽  
pp. 153-161 ◽  
Author(s):  
Koen Van Laere ◽  
Guy Bormans ◽  
Sandra M. Sanabria-Bohórquez ◽  
Tjibbe de Groot ◽  
Patrick Dupont ◽  
...  
Keyword(s):  
Partial Agonist ◽  
Receptor Occupancy ◽  
Aminobutyric Acid ◽  
In Vivo Characterization ◽  
Subtype Selective

Regulation of Ca2+-release-activated Ca2+ current (Icrac) by ryanodine receptors in inositol 1,4,5-trisphosphate-receptor-deficient DT40 cells

2001 ◽  
Vol 360 (1) ◽  
pp. 17-22 ◽  
Author(s):  
Kirill KISELYOV ◽  
Dong Min SHIN ◽  
Nikolay SHCHEYNIKOV ◽  
Tomohiro KUROSAKI ◽  
Shmuel MUALLEM
Keyword(s):  
Time Course ◽  
Ryanodine Receptors ◽  
Cell Types ◽  
Ruthenium Red ◽  
General Mechanism ◽  
Inward Rectification ◽  
Wild Type ◽  
Ip3 Receptor ◽  
Inositol 1,4,5 Trisphosphate ◽  
Dt40 Cells

Persistence of capacitative Ca2+ influx in inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)-deficient DT40 cells (DT40IP3R-/−) raises the question of whether gating of Ca2+-release activated Ca2+ current (Icrac) by conformational coupling to Ca2+-release channels is a general mechanism of gating of these channels. In the present work we examined the properties and mechanism of activation of Icrac Ca2+ current in wild-type and DT40IP3R-/− cells. In both cell types passive depletion of internal Ca2+ stores by infusion of EGTA activated a Ca2+ current with similar characteristics and time course. The current was highly Ca2+-selective and showed strong inward rectification, all typical of Icrac. The activator of ryanodine receptor (RyR), cADP-ribose (cADPR), facilitated activation of Icrac, and the inhibitors of the RyRs, 8-N-cADPR, ryanodine and Ruthenium Red, all inhibited Icrac activation in DT40IP3R-/− cells, even after complete depletion of intracellular Ca2+ stores by ionomycin. Wild-type and DT40IP3R-/− cells express RyR isoforms 1 and 3. RyR levels were adapted in DT40IP3R-/− cells to a lower RyR3/RyR1 ratio than in wild-type cells. These results suggest that IP3Rs and RyRs can efficiently gate Icrac in DT40 cells and explain the persistence of Icrac gating by internal stores in the absence of IP3Rs.

scholarly journals Clusters of calcium release channels harness the Ising phase transition to confine their elementary intracellular signals

2017 ◽  
Vol 114 (29) ◽  
pp. 7525-7530 ◽  
Author(s):  
Anna V. Maltsev ◽  
Victor A. Maltsev ◽  
Michael D. Stern
Keyword(s):  
Phase Transition ◽  
Statistical Physics ◽  
Calcium Release ◽  
Cell Function ◽  
Particle System ◽  
Signal To Noise Ratio ◽  
Ryanodine Receptors ◽  
High Amplitude ◽  
Ip3 Receptors ◽  
Cooperative Action

Intracellular Ca signals represent a universal mechanism of cell function. Messages carried by Ca are local, rapid, and powerful enough to be delivered over the thermal noise. A higher signal-to-noise ratio is achieved by a cooperative action of Ca release channels such as IP3 receptors or ryanodine receptors arranged in clusters (release units) containing a few to several hundred release channels. The channels synchronize their openings via Ca-induced Ca release, generating high-amplitude local Ca signals known as puffs in neurons and sparks in muscle cells. Despite the positive feedback nature of the activation, Ca signals are strictly confined in time and space by an unexplained termination mechanism. Here we show that the collective transition of release channels from an open to a closed state is identical to the phase transition associated with the reversal of magnetic field in an Ising ferromagnet. Our simple quantitative criterion closely predicts the Ca store depletion level required for spark termination for each cluster size. We further formulate exact requirements that a cluster of release channels should satisfy in any cell type for our mapping to the Ising model and the associated formula to remain valid. Thus, we describe deterministically the behavior of a system on a coarser scale (release unit) that is random on a finer scale (release channels), bridging the gap between scales. Our results provide exact mapping of a nanoscale biological signaling model to an interacting particle system in statistical physics, making the extensive mathematical apparatus available to quantitative biology.

scholarly journals Maintenance of homeostatic plasticity at the Drosophila neuromuscular synapse requires continuous IP3-directed signaling

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Thomas D James ◽  
Danielle J Zwiefelhofer ◽  
C Andrew Frank
Keyword(s):  
Ryanodine Receptors ◽  
Neuromuscular Synapse ◽  
Synaptic Function ◽  
Homeostatic Plasticity ◽  
Editorial Note ◽  
Ip3 Receptors ◽  
Two Phases ◽  
Larval Neuromuscular Junction ◽  
Retrograde Signal ◽  
Maintenance Process

Synapses and circuits rely on neuroplasticity to adjust output and meet physiological needs. Forms of homeostatic synaptic plasticity impart stability at synapses by countering destabilizing perturbations. The Drosophila melanogaster larval neuromuscular junction (NMJ) is a model synapse with robust expression of homeostatic plasticity. At the NMJ, a homeostatic system detects impaired postsynaptic sensitivity to neurotransmitter and activates a retrograde signal that restores synaptic function by adjusting neurotransmitter release. This process has been separated into temporally distinct phases, induction and maintenance. One prevailing hypothesis is that a shared mechanism governs both phases. Here, we show the two phases are separable. Combining genetics, pharmacology, and electrophysiology, we find that a signaling system consisting of PLCβ, inositol triphosphate (IP3), IP3 receptors, and Ryanodine receptors is required only for the maintenance of homeostatic plasticity. We also find that the NMJ is capable of inducing homeostatic signaling even when its sustained maintenance process is absent.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (<xref ref-type="decision-letter" rid="SA1">see decision letter</xref>).

scholarly journals Mini-dystrophin Expression Down-regulates IP3-mediated Calcium Release Events in Resting Dystrophin-deficient Muscle Cells

2006 ◽  
Vol 128 (2) ◽  
pp. 219-230 ◽  
Author(s):  
Haouaria Balghi ◽  
Stéphane Sebille ◽  
Ludivine Mondin ◽  
Anne Cantereau ◽  
Bruno Constantin ◽  
...  
Keyword(s):  
Calcium Signaling ◽  
Signaling Pathway ◽  
Intracellular Signaling ◽  
Calcium Release ◽  
Ryanodine Receptors ◽  
Release Site ◽  
Cell Types ◽  
Ip3 Receptors ◽  
Calcium Signaling Pathway ◽  
Gi Protein

We present here evidence for the enhancement, at rest, of an inositol 1,4,5-trisphosphate (IP3)–mediated calcium signaling pathway in myotubes from dystrophin-deficient cell lines (SolC1(−)) as compared to a cell line from the same origin but transfected with mini-dystrophin (SolD(+)). With confocal microscopy, the number of sites discharging calcium (release site density [RSD]) was quantified and found more elevated in SolC1(−) than in SolD(+) myotubes. Variations of membrane potential had no significant effect on this difference, and higher resting [Ca2+]i in SolC1(−) (Marchand, E., B. Constantin, H. Balghi, M.C. Claudepierre, A. Cantereau, C. Magaud, A. Mouzou, G. Raymond, S. Braun, and C. Cognard. 2004. Exp. Cell Res. 297:363–379) cannot explain alone higher RSD. The exposure with SR Ca2+ channel inhibitors (ryanodine and 2-APB) and phospholipase C inhibitor (U73122) significantly reduced RSD in both cell types but with a stronger effect in dystrophin-deficient SolC1(−) myotubes. Immunocytochemistry allowed us to localize ryanodine receptors (RyRs) as well as IP3 receptors (IP3Rs), IP3R-1 and IP3R-2 isoforms, indicating the presence of both RyRs-dependent and IP3-dependent release systems in both cells. We previously reported evidence for the enhancement, through a Gi protein, of the IP3-mediated calcium signaling pathway in SolC1(−) as compared to SolD(+) myotubes during a high K+ stimulation (Balghi, H., S. Sebille, B. Constantin, S. Patri, V. Thoreau, L. Mondin, E. Mok, A. Kitzis, G. Raymond, and C. Cognard. 2006. J. Gen. Physiol. 127:171–182). Here we show that, at rest, these regulation mechanisms are also involved in the modulation of calcium release activities. The enhancement of resting release activity may participate in the calcium overload observed in dystrophin-deficient myotubes, and our findings support the hypothesis of the regulatory role of mini-dystrophin on intracellular signaling.

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