InsP3R Activity Research Articles

The Amplification Of InsP3R Activity By NCS-1 Is Attenuated By Medications Used In The Treatment Of Bipolar Disorder

Neuronal Calcium Sensor-1 (NCS-1) is a high-affinity, low-capacity calcium-binding protein abundantly expressed in neuronal and neuroendocrine cells. We previously showed that NCS-1 interacts with the inositol 1,4,5-trisphosphate receptor (InsP3R) and modulates calcium signaling by enhancing InsP3-dependent InsP3R channel activity and intracellular calcium transients. Furthermore, it is known that NCS-1 is overexpressed in the prefrontal cortex of bipolar disorders and schizophrenic patients. Because we had reported that addition of lithium, a compound used for treatment of bipolar disorders, attenuates the NCS-1/InsP3R association, we hypothesized that other medications used for these disorders also might target the interaction between NCS-1 and the InsP3R.

Compensation of Inositol 1,4,5-Trisphosphate Receptor Function by Altering Sarco-Endoplasmic Reticulum Calcium ATPase Activity in theDrosophilaFlight Circuit

Ionic Ca2+ functions as a second messenger to control several intracellular processes. It also influences intercellular communication. The release of Ca2+ from intracellular stores through the inositol 1,4,5-trisphosphate receptor (InsP3R) occurs in both excitable and nonexcitable cells. In Drosophila, InsP3R activity is required in aminergic interneurons during pupal development for normal flight behavior. By altering intracellular Ca2+ and InsP3 levels through genetic means, we now show that signaling through the InsP3R is required at multiple steps for generating the neural circuit required in air puff-stimulated Drosophila flight. Decreased Ca2+ release in aminergic neurons during development of the flight circuit can be compensated by reducing Ca2+ uptake from the cytosol to intracellular stores. However, this mode of increasing intracellular Ca2+ is insufficient for maintenance of flight patterns over time periods necessary for normal flight. Our study suggests that processes such as maintenance of wing posture and formation of the flight circuit require InsP3 receptor function at a slow timescale and can thus be modulated by altering levels of cytosolic Ca2+ and InsP3. In contrast, maintenance of flight patterns probably requires fast modulation of Ca2+ levels, in which the intrinsic properties of the InsP3R play a pivotal role.

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.

Inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3Rs) are channels responsible for calcium release from the endoplasmic reticulum (ER). We show that the anti-apoptotic protein Bcl-2 (either wild type or selectively localized to the ER) significantly inhibited InsP3-mediated calcium release and elevation of cytosolic calcium in WEHI7.2 T cells. This inhibition was due to an effect of Bcl-2 at the level of InsP3Rs because responses to both anti-CD3 antibody and a cell-permeant InsP3 ester were decreased. Bcl-2 inhibited the extent of calcium release from the ER of permeabilized WEHI7.2 cells, even at saturating concentrations of InsP3, without decreasing luminal calcium concentration. Furthermore, Bcl-2 reduced the open probability of purified InsP3Rs reconstituted into lipid bilayers. Bcl-2 and InsP3Rs were detected together in macromolecular complexes by coimmunoprecipitation and blue native gel electrophoresis. We suggest that this functional interaction of Bcl-2 with InsP3Rs inhibits InsP3R activation and thereby regulates InsP3-induced calcium release from the ER.

Modification of phospholipase C-gamma-induced Ca2+ signal generation by 2-aminoethoxydiphenyl borate.

The mechanisms by which Ca(2+)-store-release channels and Ca(2+)-entry channels are coupled to receptor activation are poorly understood. Modification of Ca(2+) signals by 2-aminoethoxydiphenyl borate (2-APB), suggests the agent may target entry channels or the machinery controlling their activation. In DT40 B-cells and Jurkat T-cells, complete Ca(2+) store release was induced by 2-APB (EC(50) 10-20 microM). At 75 microM, 2-APB emptied stores completely in both lymphocyte lines, but had no such effect on other cells. In DT40 cells, 2-APB mimicked B-cell receptor (BCR) cross-linking, but no effect was observed in mutant DT40 lines devoid of inositol 1,4,5-trisphosphate (InsP(3)) receptors (InsP(3)Rs) or phospholipase C-gamma2 (PLC-gamma2). Like the BCR, 2-APB activated transfected TRPC3 (canonical transient receptor potential) channels, which acted as sensors for PLC-gamma2-generated diacylglycerol in DT40 cells. The action of 2-APB on InsP(3)Rs and TRPC3 channels was prevented by PLC-inhibition, and required PLC-gamma2 catalytic activity. However, unlike BCR activation, no increased InsP(3) level could be measured in response to 2-APB. Also, calyculin A-induced cytoskeletal reorganization prevented 2-APB-induced InsP(3)R and TRPC3-channel activation, but not that induced by the BCR. 2-APB still activated TRPC3 channels in DT40 cells with fully depleted Ca(2+) stores, indicating its action was not via Ca(2+) release. Significantly, 2-APB-induced InsP(3)R and TRPC3 activation was prevented in DT40 knockout cells devoid of the BCR- and PLC-gamma2-coupled adaptor/kinases, Syk, Lyn, Btk or BLNK. The results suggest that 2-APB activates Ca(2+) signals in lymphocytes by initiating and enhancing coupling between components of the BCR-PLC-gamma2 complex and both Ca(2+)-entry and Ca(2+)-release channels.

Characterization of neuropeptide Y-mediated corticotropin-releasing factor synthesis and release from human placental trophoblasts.

Neuropeptide Y (NPY) is a CRF secretagogue for human placental cells in culture. We have studied the involvement of intracellular calcium and calcium-dependent signaling in the NPY-induced CRF release in trophoblastic cells. The incubation of trophoblasts with NPY for 3 and 8 h led to a dose-dependent increase in CRF secretion. Also, NPY stimulated synthesis of this peptide hormone upon an 8-h incubation period. BIBP3226, a selective Y1 receptor antagonist, and pertussis toxin (PTX) eliminated these effects. NPY-stimulated CRF secretion was mostly prevented by loading cells with BAPTA-AM, suggesting that elevation of intracellular calcium is responsible for the increase of CRF secretion. However, this calcium chelator had no effect on CRF synthesis. Furthermore, U-73122, a phospholipase C-betas (PLC) inhibitor or xestospongin C, an inositol triphosphate receptor (InsP3-R) blocker, have partially prevented the effect of NPY on CRF synthesis and secretion. Therefore, the increase in CRF synthesis and secretion rely in part on the release of calcium from intracellular store. Interestingly, SKF 96365, an inhibitor of store operated calcium (SOC) influx, also partially blocked the NPY stimulatory effect on CRF release but not its synthesis, suggesting that calcium influx is also involved in this stimulation. In the syncytiotrophoblast, known to possess a NPY-activated protein kinase C (PKCs) activity, NPY also stimulated calcium calmodulin kinase II (CaMKII) and extracellular regulated kinase (ERK1/2) activities. In the present study, we observed that bisindolylmaleimide (BIM), a nonspecific PKCs inhibitor partially prevented the NPY-induced CRF release. On the other hand, autocamtide-2 related inhibitory peptide (AIP), a CaMKII inhibitor, prevented most of the stimulatory effect of NPY on both CRF synthesis and release. Go6976, an inhibitor of the conventional and mu PKCs and PD 098059, an inhibitor of the ERK cascade, had no effect on neither CRF synthesis nor release. Altogether, these results support a Y1 receptor-mediated PTX-sensitive induction on CRF synthesis and release by NPY from human placental trophoblasts. The stimulation of CRF synthesis by NPY seems to depend mainly on a PLC-beta to InsP3-R axis and on CaMKII activity. Also, the release of CRF depends on the PLC-beta to InsP3-R axis and CaMKII activity but also entails the participation of a calcium-independent PKCs.

Inositol 1,4,5-trisphosphate-induced Ca2+ release is inhibited by mitochondrial depolarization

We investigated the consequences of depolarizing the mitochondrial membrane potential (∆ψmit) on Ca2+ signals arising via inositol 1,4,5-trisphosphate receptors (InsP3R) in hormone-stimulated HeLa cells. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or a mixture of antimycin A+oligomycin were found to rapidly depolarize ∆ψmit. Mitochondrial depolarization enhanced the number of cells responding to a brief application of a Ca2+-mobilizing hormone and prolonged the recovery of cytosolic Ca2+ after washout of the hormone; effects consistent with the removal of a passive Ca2+ buffer. However, with repeated application of the same hormone concentration both the number of responsive cells and peak Ca2+ changes were observed to progressively decline. The inhibition of Ca2+ signalling was observed using different Ca2+-mobilizing hormones and also with a membrane-permeant Ins(1,4,5)P3 ester. Upon washout of FCCP, the Ca2+ signals recovered with a time course similar to the re-establishment of ∆ψmit. Global measurements indicated that none of the obvious factors such as changes in pH, ATP concentration, cellular redox state, permeability transition pore activation or reduction in Ca2+-store loading appeared to underlie the inhibition of Ca2+ signalling. We therefore suggest that local changes in one or more of these factors, as a consequence of depolarizing ∆ψmit, prevents InsP3R activation.

Protein–protein interactions in intracellular Ca2+-release channel function

Release of Ca2+ ions from intracellular stores can occur via two classes of Ca2+-release channel (CRC) protein, the inositol 1,4, 5-trisphosphate receptors (InsP3Rs) and the ryanodine receptors (RyRs). Multiple isoforms and subtypes of each CRC class display distinct but overlapping distributions within mammalian tissues. InsP3Rs and RyRs interact with a plethora of accessory proteins which modulate the activity of their intrinsic channels. Although many aspects of CRC structure and function have been reviewed in recent years, the properties of proteins with which they interact has not been comprehensively surveyed, despite extensive current research on the roles of these modulators. The aim of this article is to review the regulation of CRC activity by accessory proteins and, wherever possible, to outline the structural details of such interactions. The CRCs are large transmembrane proteins, with the bulk of their structure located cytoplasmically. Intra- and inter-complex protein-protein interactions between these cytoplasmic domains also regulate CRC function. Some accessory proteins modulate channel activity of all CRC subtypes characterized, whereas other have class- or even isoform-specific effects. Certain accessory proteins exert both direct and indirect forms of regulation on CRCs, occasionally with opposing effects. Others are themselves modulated by changes in Ca2+ concentration, thereby participating in feedback mechanisms acting on InsP3R and RyR activity. CRCs are therefore capable of integrating numerous signalling events within a cell by virtue of such protein-protein interactions. Consequently, the functional properties of InsP3Rs and RyRs within particular cells and subcellular domains are 'customized' by the accessory proteins present.

Functional Coupling of Phosphatidylinositol 4,5-Bisphosphate to Inositol 1,4,5-Trisphosphate Receptor

The inositol 1,4,5-trisphosphate receptor (InsP3R) plays a key role in intracellular Ca2+ signaling. InsP3R is activated by InsP3 produced from phosphatidylinositol 4,5-bisphosphate (PIP2) by phospholipase C cleavage. Using planar lipid bilayer reconstitution technique, we demonstrate here that rat cerebellar InsP3R forms a stable inhibitory complex with endogenous PIP2. Disruption of InsP3R-PIP2 interaction by specific anti-PIP2 monoclonal antibody resulted in 3-4-fold increase in InsP3R activity and 10-fold shift in apparent affinity for InsP3. Exogenously added PIP2 blocks InsP3 binding to InsP3R and inhibits InsP3R activity. Similar results were obtained with a newly synthesized water soluble analog of PIP2, dioctanoyl-(4,5)PIP2, indicating that insertion of PIP2 into membrane is not required to exert its inhibitory effects on the InsP3R. We hypothesize that the functional link between InsP3R and PIP2 described in the present report provides a basis for a local, rapid, and efficient coupling between phospholipase C activation, PIP2 hydrolysis, and intracellular Ca2+ wave initiation in neuronal and non-neuronal cells.

Single-Channel Properties of Inositol (1,4,5)-Trisphosphate Receptor Heterologously Expressed in HEK-293 Cells

The inositol (1,4,5)-trisphosphate receptor (InsP3R) mediates Ca2+ release from intracellular stores in response to generation of second messenger InsP3. InsP3R was biochemically purified and cloned, and functional properties of native InsP3-gated Ca2+ channels were extensively studied. However, further studies of InsP3R are obstructed by the lack of a convenient functional assay of expressed InsP3R activity. To establish a functional assay of recombinant InsP3R activity, transient heterologous expression of neuronal rat InsP3R cDNA (InsP3R-I, SI- SII+ splice variant) in HEK-293 cells was combined with the planar lipid bilayer reconstitution experiments. Recombinant InsP3R retained specific InsP3 binding properties (Kd = 60 nM InsP3) and were specifically recognized by anti-InsP3R-I rabbit polyclonal antibody. Density of expressed InsP3R-I was at least 20-fold above endogenous InsP3R background and only 2-3-fold lower than InsP3R density in rat cerebellar microsomes. When incorporated into planar lipid bilayers, the recombinant InsP3R formed a functional InsP3-gated Ca2+ channel with 80 pS conductance using 50 mM Ba2+ as a current carrier. Mean open time of recombinant InsP3-gated channels was 3.0 ms; closed dwell time distribution was double exponential and characterized by short (18 ms) and long (130 ms) time constants. Overall, gating and conductance properties of recombinant neuronal rat InsP3R-I were very similar to properties of native rat cerebellar InsP3R recorded in identical experimental conditions. Recombinant InsP3R also retained bell-shaped dependence on cytosolic Ca2+ concentration and allosteric modulation by ATP, similar to native cerebellar InsP3R. The following conclusions are drawn from these results. (a) Rat neuronal InsP3R-I cDNA encodes a protein that is either sufficient to produce InsP3-gated channel with functional properties identical to the properties of native rat cerebellar InsP3R, or it is able to form a functional InsP3-gated channel by forming a complex with proteins endogenously expressed in HEK-293 cells. (b) Successful functional expression of InsP3R in a heterologous expression system provides an opportunity for future detailed structure-function characterization of this vital protein.

The inositol 1,4,5-trisphosphate receptor of cerebellum. Mn2+ permeability and regulation by cytosolic Mn2+.

The inositol 1,4,5-trisphosphate receptor (InsP3R), an intracellular calcium release channel, is found in virtually all cells and is abundant in the cerebellum. We used Mn2+ as a tool to study two aspects of the cerebellar InsP3R. First, to investigate the structure of the ion pore, Mn2+ permeation through the channel was determined. We found that Mn2+ can pass through the InsP3R; the selectivity sequence for divalent cations is Ba2+ > Sr2+ > Ca2+ > Mg2+ > Mn2+. Second, to begin characterization of the cytosolic regulatory sites responsible for the Ca(2+)-dependent modulation of InsP3R function, the ability of Mn2+ to replace Ca2+ was investigated. We show that Mn2+, as Ca2+, modulates InsP3R activity with a bell-shaped dependence where the affinity of the activation site of the InsP3R is similar for both ions, but higher concentrations of Mn2+ were necessary to inhibit the channel. These results suggest that the two regulatory sites are structurally distinct. Our findings are also important for the understanding of cellular responses when Mn2+ is used to quench the intracellular fluorescence of Ca2+ indicator dyes.

Inositol 1,4,5-trisphosphate and ryanodine receptor distributions and patterns of acetylcholine- and caffeine-induced calcium release in cultured mouse hippocampal neurons

The distributions of inositol 1,4,5-trisphosphate and ryanodine receptors (InsP3R and RyR) and the patterns of increase in intracellular calcium ion concentration ([Ca2+]i) elicited by their activation were compared in cultured hippocampal neurons. InsP3R and RyR were labeled using specific antibodies and formed small aggregations in the somata and dendrites of pyramidally shaped neurons. Both receptors were densest in somata. In dendrites the InsP3R and RyR were not distributed homogeneously; InsP3R was found in all regions, while RyR was least dense in fine processes. Increases in [Ca2+]i elicited by acetylcholine (to activate InsP3 receptors via muscarinic receptors) and caffeine (to stimulate ryanodine receptors) were measured in dendrites using Ca(2+)-sensitive fluorescent dyes and confocal microscopy. Ca2+ responses to acetylcholine were transient and observed in proximal and distal dendritic regions. In contrast, caffeine-induced responses were sustained and restricted to proximal dendrites. Thus the patterns of calcium release in fine dendrites mirrored the distributions of InsP3R and RyR. Calcium responses to both acetylcholine and caffeine were observed in the absence of external calcium and thus were dependent on Ca2+ release. Ca2+ responses showed localized fluctuations and variations in response delay times. Sequential activation of InsP3R and RyR in somata resulted in mutual occlusion of Ca2+ release. The existence of InsP3-gated and Ca(2+)-induced Ca2+ release as spatially distinct, but mutually interacting, mechanisms may be important in the generation of oscillations and propagating Ca2+ waves in somata and dendrites of hippocampal neurons.