Presence Of IP3 Research Articles

Store-Operated Ca2+ Entry in Oocytes Modulate the Dynamics of IP3 -Dependent Ca2+ Release From Oscillatory to Tonic.

Ca2+ signaling is ubiquitous and mediates various cellular functions encoded in its spatial, temporal, and amplitude features. Here, we investigate the role of store-operated Ca2+ entry (SOCE) in regulating the temporal dynamics of Ca2+ signals in Xenopus oocytes, which can be either oscillatory or tonic. Oscillatory Ca2+ release from intracellular stores is typically observed at physiological agonist concentration. When Ca2+ release leads to Ca2+ store depletion, this triggers the activation of SOCE that translates into a low-amplitude tonic Ca2+ signal. SOCE has also been implicated in fueling Ca2+ oscillations when activated at low levels. Here, we show that sustained SOCE activation in the presence of IP3 to gate IP3 receptors (IP3 R) results in a pump-leak steady state across the endoplasmic reticulum (ER) membrane that inhibits Ca2+ oscillations and produces a tonic Ca2+ signal. Tonic signaling downstream of SOCE activation relies on focal Ca2+ entry through SOCE ER-plasma membrane (PM) junctions, Ca2+ uptake into the ER, followed by release through open IP3 Rs at distant sites, a process we refer to as "Ca2+ teleporting." Therefore, sustained SOCE activation in the presence of an IP3 -dependent "leak" pathway at the ER membrane results in a switch from oscillatory to tonic Ca2+ signaling. J. Cell. Physiol. 232: 1095-1103, 2017. © 2016 Wiley Periodicals, Inc.

Structural Studies of IP3R by Cryoem

Inositol 1,4,5-trisphosphate receptors (IP3Rs) play important roles in a battery of cellular activities. Structural study of the receptors is therefore very important for understanding how they are gated by their natural ligands and are modulated by their intracellular partners. In the past several years, multiple groups have generated very disparate reconstructions of the type 1 IP3R. Striking structural variations have been reported for the receptors in different detergents and for the receptors prepared from native and sf9 cells by the same research groups, suggesting that biochemical preparations of the receptors might have had significant variations and that the heterogeneity in the samples could be a limiting factor in reaching accordant results. To resolve such discrepancies, we developed a three- layered strategy to enhance biochemical homogeneity and structural agreements. We collected two cryoEM datasets of the receptors in the absence and presence of IP3 and Ca2+ and calculated two separate reconstructions. The two structures not only agree with each other in many aspects, but also reveal a conformational change at the top of the cytosolic domain that may lead to some reorganization of the channel pore. In order to verify the structural details and solidify the conformational changes in the pore domain, we need higher resolution structures. We are using the high-end facilities with direct electron detectors to collect near-atomic resolution data in order to further improve the resolutions of our 3D reconstructions.

Spatio-Temporal Properties of IP3 Receptor-Mediated Ca Release in Cardiac Myocytes

In cardiac myocytes cytosolic Ca increase and subsequent cell contraction are mainly determined by ryanodine receptor (RyR) mediated Ca release. However atrial cells are also equipped with IP3 receptors (IP3Rs), a second type of Ca release channels. We investigated IP3R-mediated Ca release events and their subcellular spatio-temporal properties in single membrane-permeabilized rabbit atrial myocytes. Local Ca release events were detected by confocal microscopy (fluo-4, longitudinal line scan mode). Local IP3R-mediated Ca release events were evoked by exposure to inositol-1,4,5-triphosphate (IP3) in the presence of tetracaine to inhibit RyR-mediated Ca spark activity, and could be inhibited by the IP3R blocker 2-APB. We classified IP3R-mediated Ca release events as subsarcolemmal, perinuclear (events 5μm) or nuclear. Nuclear events were considered membrane associated when occurring at a distance <1μm from the nuclear envelope or membranes of the nucleoplasmic reticulum. With tetracaine the Ca spark frequency decreased to 0.7±0.3 (from 8.8±1.2 in control), whereas the frequency of events detected after subsequent addition of IP3 increased to 2.9±1.3 (events/100μm/s). Subsequent exposure to 2-APB reduced the frequency to 1.8±0.8. Compared to Ca sparks, local Ca release events in the presence of IP3 were lower in amplitude, prolonged in duration and had a slower rise time. The increase in frequency of local Ca release events was particularly pronounced in the perinucler regions compared to the cytosol or the subsarcolemmal space. Furthermore, IP3-mediated Ca release events could also be detected within the nucleus. These nuclear events occurred at a higher incidence at locations that were closely associated with membrane structures of the nuclear envelope or the nucleoplasmic reticulum. In conclusion, in atrial myocytes IP3R-mediated Ca release is spatially inhomogeneous and preferentially occurs in the nuclear and perinuclear regions.

PKA Enhances the Sensitivity of IP3 Receptors in Swine Airway Submucosal Gland Cells.

Increased sensitivity of airway submucosal gland cells (SMGC) to ACh by PKA activation results in increased transepithelial ion movement. The mechanism of the increased sensitivity induced by PKA is unclear. Since ACh‐induced transepithelial ion movement is dependent on the IP3/Ca2+ signaling pathway, we hypothesized that PKA enhances sensitivity of IP3 receptors in airway SMGC, resulting in increased [Ca2+]i. Patch clamp of swine airway SMGC was performed in these studies. We showed that forskolin decreased the EC50 for IP3 about 4‐fold measured as the peak KCa current induced upon whole cell formation with IP3 in the pipette. VIP (10 nM), a co‐neurotransmitter present in cholinergic airway nerve fibers, enhanced the response to 0.3 µM IP3. Neither VIP nor IP3 (0.3 µM) induced KCa current when applied alone. VIP induced a significant KCa current in SMGC only when the internal solution contained IP3. Removing external Ca2+ did not abolish the VIP‐induced sensitization of the peak IP3 response although the plateau response was lost. This suggests that Ca2+ release is sensitized as well as Ca2+ influx. Elevating external Ca2+ to 5 mM revealed a KCa current induced only in the presence of IP3 (0.3 µM). Thus Ca2+ entry may also be increased by IP3 (0.3 µM). These results suggest that PKA increases the sensitivity of IP3 receptors by phosphorylation, resulting in the increased intracellular Ca2+ release and Ca2+ entry.

IRBIT Suppresses IP3 Receptor Activity by Competing with IP3 for the Common Binding Site on the IP3 Receptor

The inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are IP3-gated intracellular Ca2+ channels. We previously identified an IP3R binding protein, IRBIT, which binds to the IP3 binding domain of IP3R and is dissociated from IP3R in the presence of IP3. In the present study, we showed that IRBIT suppresses the activation of IP3R by competing with IP3 by [3H]IP3 binding assays, in vitro Ca2+ release assays, and Ca2+ imaging of intact cells. Multiserine phosphorylation of IRBIT was essential for the binding, and 10 of the 12 key amino acids in IP3R for IP3 recognition participated in binding to IRBIT. We propose a unique mode of IP3R regulation in which IP3 sensitivity is regulated by IRBIT acting as an endogenous "pseudoligand" whose inhibitory activity can be modulated by its phosphorylation status.

Single-channel recording of inositol trisphosphate receptor in the isolated nucleus of a muscle cell line

Nuclear calcium appears to have an important role in the regulation of gene expression in many cells, but the mechanisms involved in controlling nuclear Ca2+ signaling are controversial and still poorly understood. We have described the presence of inositol 1,4,5 trisphosphate (IP3) receptors in the nuclei of skeletal muscle cells. Now, we have characterized the properties of the IP3 receptors channels present in the nuclei of the 1B5 cell line, which do not express any isoforms of the ryanodine receptor. Immunocytochemistry of isolated nuclei confirmed the presence of IP3R in the nuclear envelope and fluorescence measurements in nuclei suspensions allowed us to document ATP-dependent calcium loading by the nucleus and its release upon IP3 addition. Patch clamp of nuclear membranes was performed, and single-channel activity recorded was dependent on the presence of IP3 in the pipette; single-channel conductance was in the range reported in the literature for these channels, and the open probability was shown to be dependent on IP3 concentration. The presence of functional IP3 receptors in the nuclear envelope membrane is likely to have an important function in the regulation of nucleoplasmic calcium concentration and consequently in the regulation of transcription in muscle cells.

The Role of Mitochondria for Ca2+ Refilling of the Endoplasmic Reticulum

Endoplasmic reticulum (ER) Ca2+ refilling is an active process to ensure an appropriate ER Ca2+ content under basal conditions and to maintain or restore ER Ca2+ concentration during/after cell stimulation. The mechanisms to achieve successful ER Ca2+ refilling are multiple and built on a concerted action of processes that provide a suitable reservoir for Ca2+ sequestration into the ER. Despite mitochondria having been found to play an essential role in the maintenance of capacitative Ca2+ entry by buffering subplasmalemmal Ca2+, their contribution to ER Ca2+ refilling was not subjected to detailed analysis so far. Thus, this study was designed to elucidate the involvement of mitochondria in Ca2+ store refilling during and after cell stimulation. ER Ca2+ refilling was found to be accomplished even during continuous inositol 1,4,5-trisphosphate (IP3)-triggered ER Ca2+ release by an agonist. Basically, ER Ca2+ refilling depended on the presence of extracellular Ca2+ as the source and sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) activity. Interestingly, in the presence of an IP3-generating agonist, ER Ca2+ refilling was prevented by the inhibition of trans-mitochondrial Ca2+ flux by CGP 37157 (7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one) that precludes the mitochondrial Na+/Ca2+ exchanger as well as by mitochondrial depolarization using a mixture of oligomycin and antimycin A. In contrast, after the removal of the agonist, ER refilling was found to be largely independent of trans-mitochondrial Ca2+ flux. Under these conditions, ER Ca2+ refilling took place even without an associated Ca2+ elevation in the deeper cytosol, thus, indicating that superficial ER domains mimic mitochondrial Ca2+ buffering and efficiently sequester subplasmalemmal Ca2+ and consequently facilitate capacitative Ca2+ entry. Hence, these data point to different contribution of mitochondria in the process of ER Ca2+ refilling based on the presence or absence of IP3, which represents the turning point for the dependence or autonomy of ER Ca2+ refilling from trans-mitochondrial Ca2+ flux.

Bcl-2 in Partnership with IP 3 R

Inositol 1,4,5-trisphosphate receptor (IP 3 R)-mediated release of calcium from endoplasmic reticulum (ER) stores has been implicated in various apoptotic processes. Although the antiapoptotic protein Bcl-2 (found in both mitochondrial and ER membranes) can quell oscillations in intracellular calcium, the mechanisms through which Bcl-2 affects calcium signaling--and the precise role of ER Bcl-2--have remained unclear. Chen et al. found that WEHI7.2 T cells transfected with either human Bcl-2 or a Bcl-2 mutant targeted to the ER showed an attenuated increase in cytosolic calcium in response to stimulation with an antibody to CD3. Bcl-2 also inhibited the increase in cytosolic calcium produced by a membrane-permeant IP 3 ester but had no effect on calcium release after SERCA (sarcoendoplasmic reticulum Ca 2+ -ATPase) inhibition with thapsigargin nor did it affect calcium concentration in the ER lumen (measured with Fura-2FF). The affinity of IP 3 for the IP 3 R in microsomes isolated from cells expressing Bcl-2 was lower than that in microsomes from cells that did not express Bcl-2; however, Bcl-2 inhibited calcium release from the ER even in permeabilized cells exposed to saturating concentrations of IP 3 . Adding purified Bcl-2 to IP 3 Rs incorporated into planar lipid bilayers decreased their open probability in the presence of IP 3 . Western analysis combined with either blue native gel electrophoresis of ER membranes from WEHI7.2 cells expressing Bcl-2 or with coimmunoprecipitation using transfected WEHI7.2 cells or a T cell line that expressed endogenous Bcl-2 indicated that Bcl-2 and IP 3 R formed a complex in vivo. Thus, ER Bcl-2 appears to play a role in regulating IP 3 R-mediated calcium release from the ER. R. Chen, I. Valencia, F. Zhong, K. S. McColl, H. L. Roderick, M. D. Bootman, M. J. Berridge, S. J. Conway, A. B. Holmes, G. A. Mignery, P. Velez, C. W. Distelhorst, Bcl-2 functionally interacts with inositol 1,4,5-trisphosphate receptors to regulate calcium release from the ER in response to inositol 1,4,5-trisphosphate. J. Cell Biol. 166 , 193-203 (2004). [Abstract] [Full Text]

IP3 receptors

What are they? Members of a family of intracellular ligand-gated ion channels that allow Ca2+ release from intracellular stores. The endoplasmic reticulum (ER), or specialised regions of it, contains ATP-driven Ca2+ pumps which generate a high Ca2+ concentration in the ER lumen. When inositol 1,4,5-trisphosphate (IP3) binds to IP3 receptors, the channel region of the receptor opens, allowing Ca2+ to flood out into the cytosol. How and when did they become famous? In 1983, Hans Streb, Robin Irvine, Mike Berridge and Irene Schulz showed that IP3 released Ca2+ from stores in permeabilised cells. A variety of cell types were found to have high-affinity IP3 binding sites on their intracellular membranes. The high-affinity IP3 binding protein was found to be identical with a high-abundance protein, P400, previously found in the cerebellum and to be a cation channel which was opened in the presence of IP3IP3. What do they look like? The receptor has four subunits, each of around 2700 amino acid residues. The carboxy-terminal region of each subunit probably has six transmembrane segments, and these interact to form the channel region. Each subunit has a large cytoplasmic domain containing a variety of regulatory sites. The IP3 binding site is close to the amino terminus, and there is a long regulatory region between there and the channel. Very little of the protein projects into the lumen of the ER. In mammals and birds, there are three IP3 receptor subunit isoforms, types 1, 2 and 3, plus some splice variants. Type 1 is present at a high level in neuronal tissue, particularly Purkinje cells. Receptors can occur as either homotetramers or heterotetramers. Any living relatives? Its overall structure and some of its properties are similar to those of the ryanodine receptor, another ligand-gated Ca2+-release channel. Ryanodine receptors are gated by Ca2+ and some isoforms are sensitised by cyclic ADP ribose; they are the main routes for Ca2+ release in muscle cells, but are also present in other cell types. Various other intracellular Ca2+-release channels have been identified, for example those gated by NAADP. In many cases, IP3 receptors and ryanodine receptors act together to generate Ca2+ signals. So IP3 is the signal for Ca2+ release… Not quite as simple as that! IP3 receptors are also potently activated by Ca2+ on the cytosolic side, so like ryanodine receptors they also show Ca2+-stimulated Ca2+ release – but only in the presence of IP3. IP3 and Ca2+ act as co-agonists to cause Ca2+ release. There is an essential glutamate residue, close to the channel region, which is required for Ca2+ stimulation. Ca2+ at higher concentrations inhibits IP3 receptors, so they show a bell-shaped dependence on cytosolic [Ca2+] (Figure 1). The complex interplay between positive and negative feedback from Ca2+ that has just been released from the channel is responsible for the generation of local Ca2+ signals and for conversion of local signals into intracellular and intercellular Ca2+ waves. ATP greatly increases the open-probability of IP3 receptors in the presence of IP3 and Ca2+. Known associates… IP3 receptors have a very well-documented – but poorly understood – interaction with calmodulin. In general, Ca2+–calmodulin inhibits Ca2+ release, but this is not necessarily the same Ca2+ inhibition as the one we have just been talking about. A lot of other proteins have also been found lurking suspiciously in the neighbourhood: FKBP12 and calcineurin (phosphoprotein phosphatase 2B) are two suspects. The neuronal Ca2+-binding protein CaBP1 has been shown to activate IP3 receptor channels, even in the absence of IP3. In secretory granules of neuroendocrine cells, luminal chromogranin A increases channel activation by IP3, and in the nematode Caenorhabditis elegans, an interaction has been demonstrated between myosin II and IP3 receptors. Particularly significant is a possible association with members of the Trp family of plasma membrane Ca2+ channels that might control Ca2+ entry into cells. Other endearing properties… Very complex kinetic behaviour, which causes successive packets of Ca2+ to be released in response to small increments of IP3 concentration – so-called quantal Ca2+ release – has provided years of fun for a brave band of Ca2+ kineticists.

A model of cytosolic calcium regulation and autacoids production in vascular endothelial cell.

A model of vascular endothelial cell is proposed to describe the mechanisms by which cytosolic calcium (Cai) is modulated and endothelium-derived relaxing factor (EDRF) and prostacyclin (PGI2) are released when the cell is stimulated by agonist. The intracellular Ca2+ store of the model cell is comprised of a superficial (sc) and a deep (dc) compartment. The dc Ca2+ content is refilled by the sc whose [Ca2+] is the same as extracellular Ca2+. Inositol (1,4,5)-trisphosphate (IP3) produced by agonist modifies the dc permeability which discharges its Ca2+ to the cytosol. The increase of Cai induces Ca2+ released from the sc. Ca(2+)-activated K+ current hyperpolarises the cell. The raised Cai releases PGI2 in the presence of IP3 while EDRF is released by Cai. The model explains satisfactorily the Ca2+ transient and autacoids production of the aortic endothelial cell without the need of calcium influx from extracellular space. The cytoplasmic Ca2+ oscillations observed in human endothelial cell from umbilical veins were reproduced by the model. Production of EDRF by the artery due to increase in pressure was also simulated.

Influence of inositol 1,4,5-trisphosphate and guanine nucleotides on intracellular calcium release within the N1E-115 neuronal cell line.

The Ca2+ accumulating properties of a nonmitochondrial intracellular organelle within cultured N1E-115 neuroblastoma cells containing an (ATP + Mg2+)-dependent Ca2+ pump were recently described in detail (Gill, D. L., and Chueh, S. H. (1985) J. Biol. Chem. 260, 9289-9297). Using both saponin-permeabilized N1E-115 cells and microsomal membranes from cells, this report describes the effectiveness of both inositol 1,4,5-trisphosphate (IP3) and guanine nucleotides in mediating Ca2+ release from this internal organelle, believed to be endoplasmic reticulum. Using permeabilized N1E-115 cells, 2 microM IP3 effects rapid release (t1/2 less than 20 s) of approximately 40% of accumulated Ca2+ releasable with 5 microM A23187. Half-maximal Ca2+ release occurs with 0.5 microM IP3, and maximal release with 3 microM IP3. Using a frozen microsomal membrane fraction isolated from lysed cells, 2 microM IP3 rapidly releases (t1/2 less than 30 s) 10-20% of A23187-releasable Ca2+ accumulated within nonmitochondrial Ca2+-pumping vesicles, although only in the presence of 3% polyethylene glycol (PEG). 10 microM GTP, but not guanosine 5'-(beta, gamma-imido)triphosphate (GMPPNP), increases the extent of release in the presence of IP3. Importantly, however, GTP alone induces a substantial release of Ca2+ (up to 40% of releasable Ca2+) with a t1/2 value (60-90 s) slightly longer than that for IP3. The effects of IP3 and GTP are approximately additive, and both effects require 3% PEG. Half-maximal Ca2+ release occurs with 1 microM GTP, with maximal release at 3-5 microM GTP; 20 microM GMPPNP has no effect on release and only slightly inhibits 5 microM GTP; 20 microM GDP promotes full release, but only after a 90-s lag, and initially inhibits the action of 5 microM GTP. Using permeabilized N1E-115 cells, 5 microM GTP with 3% PEG releases greater than 50% of releasable Ca2+; without PEG, GTP still mediates approximately 30% release of Ca2+ from cells. Neither IP3, GTP, or both together (with or without PEG) effects release of Ca2+ accumulated within synaptic plasma membrane vesicles. The profound effectiveness of GTP on Ca2+ release has important implications for intracellular Ca2+ regulation and is probably related to Ca2+ release mediated by IP3.