IP3R Protein Research Articles

Functional Inositol 1,4,5-Trisphosphate Receptors Assembled from Concatenated Homo- and Heteromeric Subunits

Vertebrate genomes code for three subtypes of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R1, -2, and -3). Individual IP3R monomers are assembled to form homo- and heterotetrameric channels that mediate Ca(2+) release from intracellular stores. IP3R subtypes are regulated differentially by IP3, Ca(2+), ATP, and various other cellular factors and events. IP3R subtypes are seldom expressed in isolation in individual cell types, and cells often express different complements of IP3R subtypes. When multiple subtypes of IP3R are co-expressed, the subunit composition of channels cannot be specifically defined. Thus, how the subunit composition of heterotetrameric IP3R channels contributes to shaping the spatio-temporal properties of IP3-mediated Ca(2+) signals has been difficult to evaluate. To address this question, we created concatenated IP3R linked by short flexible linkers. Dimeric constructs were expressed in DT40-3KO cells, an IP3R null cell line. The dimeric proteins were localized to membranes, ran as intact dimeric proteins on SDS-PAGE, and migrated as an ∼1100-kDa band on blue native gels exactly as wild type IP3R. Importantly, IP3R channels formed from concatenated dimers were fully functional as indicated by agonist-induced Ca(2+) release. Using single channel "on-nucleus" patch clamp, the channels assembled from homodimers were essentially indistinguishable from those formed by the wild type receptor. However, the activity of channels formed from concatenated IP3R1 and IP3R2 heterodimers was dominated by IP3R2 in terms of the characteristics of regulation by ATP. These studies provide the first insight into the regulation of heterotetrameric IP3R of defined composition. Importantly, the results indicate that the properties of these channels are not simply a blend of those of the constituent IP3R monomers.

Diurnal and nutritional adjustments of intracellular Ca2+ release channels and Ca2+ ATPases associated with restricted feeding schedules in the rat liver

BackgroundIntracellular calcium is a biochemical messenger that regulates part of the metabolic adaptations in the daily fed-fast cycle. The aim of this study was to characterize the 24-h variations of the liver ryanodine and IP3 receptors (RyR and IP3R) as well as of the endoplasmic-reticulum and plasma-membrane Ca2+-ATPases (SERCA and PMCA) in daytime restricted feeding protocol.MethodsA biochemical and immunohistochemical approach was implemented in this study: specific ligand-binding for RyR and IP3R, enzymatic activity (SERCA and PMCA), and protein levels and zonational hepatic-distribution were determined by immunoblot and immunohistochemistry respectively under conditions of fasting, feeding, and temporal food-restriction.ResultsBinding assays and immunoblots for IP3R1 and 2 showed a peak at the light/dark transition in the ad-libitum (AL) group, whereas in the restricted-feeding (RF) group the peak shifted towards the food-access time. In the case of RyR binding experiments, both AL and RF groups showed a modest elevation during the dark period, with the RF rats exhibiting increased binding in response to feeding. The AL group showed 24-h rhythmicity in SERCA level; in contrast, RF group showed a pronounced amplitude elevation and a peak phase-shift during the light-period in SERCA level and activity. The activity of PMCA was constant along day in both groups; PMCA1 levels showed a 24-h rhythmicity in the RF rats (with a peak in the light period), meanwhile PMCA4 protein levels showed rhythmicity in both groups. The fasted condition promoted an increase in IP3R binding and protein level; re-feeding increased the amount of RyR; neither the activity nor expression of SERCA and PMCA protein was affected by fasting–re-feeding conditions. Histochemical experiments showed that the distribution of the Ca2+-handling proteins, between periportal and pericentral zones of the liver, varied with the time of day and the feeding protocol.ConclusionsOur findings show that RF influences mainly the phase and amplitude of hepatic IP3R and SERCA rhythms as well as discrete zonational distribution for RyR, IP3Rs, SERCA, and PMCA within the liver acinus, suggesting that intracellular calcium dynamics could be part of the rheostatic adaptation of the liver due to diurnal meal entrainment/food entrained oscillator expression.

Tespa1 protein is phosphorylated in response to store-operated calcium entry

We previously reported that Tespa1 (thymocyte-expressed, positive selection-associated gene 1) protein expressed in lymphocytes physically interacts with IP3R (Inositol 1,4,5-trisphosphate receptor), a Ca2+ channel protein spanning endoplasmic reticulum (ER) membrane. However, the biochemical characterization of Tespa1 protein remains unknown. In this study, we have found that Tespa1 protein was posttranslationally modified upon intracellular Ca2+ increase in thymocytes. Through the analyses using various inhibitors, store-operated Ca2+ entry (SOCE) was found to be an essential factor for the Tespa1 protein modification induced by T cell receptor (TCR)-stimulation. Remarkably, the Ca2+-dependent Tespa1 protein modification was restored by in vitro protein phosphatase treatment, indicating that this modification was due to phosphorylation. Moreover, we examined whether Ca2+-dependent phosphorylation of Tespa1 protein would affect the physical association between Tespa1 and IP3R proteins, revealing that physical association of these proteins is maintained regardless of the presence or absence of phosphorylation of Tespa1. In addition, KRAP protein which represents substantial amino acid sequence homology to Tespa1 was also posttranslationally phosphorylated by intracellular Ca2+ increase in HCT116 human colon cancer cells and HEK293 human embryonic kidney cells, suggesting that common signaling mechanism(s) may contribute to the molecular modification of Tespa1 and KRAP in different cellular processes. All these results suggested a novel molecular modification of Tespa1 and the existence of the regulatory pathway that SOCE affects the Tespa1–IP3R molecular complex.

Imaging the Motility of Inositol Trisphosphate Receptors in Intact Mammalian Cells using Single Particle Tracking Photoactivated Localization Microscopy (Sptpalm)

Inositol trisphosphate receptors (IP3Rs) are calcium-permeable channels in the membrane of the endoplasmic reticulum (ER) that liberate calcium to generate cytosolic calcium signals that control diverse cellular functions including gene expression, secretion and synaptic plasticity. The spatial distribution of these channels is crucial in determining the patterning of intracellular calcium signals. The mechanisms underlying the aggregation and maintenance of IP3Rs into clusters are controversial. Local calcium puffs reflecting concerted openings of clustered IP3Rs arise at just a few, fixed locations within a cell, suggesting clusters are stable entities; and calcium blips generated by ‘lone’ IP3Rs are also immotile. In contrast, GFP-tagged or immunostained IP3Rs show a dense distribution throughout a cell. Moreover, the majority IP3Rs can diffuse freely within the ER membrane, and aggregate into clusters following activation of IP3 signaling and/or cytosolic calcium elevation. These apparently different behaviors may be explained because calcium imaging detects only functional IP3Rs, whereas immunostained or GFP-tagged IP3Rs report the entire population of IP3R proteins. We therefore hypothesize that most IP3Rs are motile, but functionally unresponsive. Local calcium signals arise, instead, from a small subset of IP3Rs that are anchored, individually or in clusters, by association with static cytoskeletal structures and possibly as a consequence of this anchoring, display high sensitivity to IP3 to produce calcium signals. To test this hypothesis we expressed type 1 IP3R tagged with a photoactivatable genetically encoded protein to track the motility of thousands of individual IP3R molecules with nanoscale spatial and millisecond temporal resolution. We find that IP3Rs can be distinguished into two groups with relatively high or low motility, and that the apparently immotile IP3Rs are preferentially grouped within tight clusters. Support: NIH GM40871 (IP) and GM100201 (IFS).

Structural Insights into the Assembly and Function of the IP3 Receptor Ligand-Binding Module

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are calcium-releasing channels that are involved in numerous physiological functions such as learning and memory, muscle contraction and cell proliferation. Dysfunction of IP3Rs and IP3R-interacting proteins has been implicated in several diseases including neurodegenerative diseases and arrhythmia. The activation of IP3Rs requires the binding of both IP3 and Ca2+. The IP3-binding module is composed of two sub-regions: the suppressor domain and the IP3-binding core. Previous studies showed that the suppressor domain reduces the affinity of the IP3-binding core for IP3 and that the isoform-specific IP3-binding affinity of IP3R is due to the different interactions between these two domains.We crystallized and solved the structure of the whole N-terminal ligand-binding module of IP3R isoform 3 by X-ray crystallography to 3.4 A. This structure, which represents the closed state of the channel, shows a clear conformational change in the IP3-binding core compared to previous open state structures. The suppressor domain interacts with IP3-binding core mainly through a hydrophilic interface. Comparing this interface with the one from IP3R isoform 1, we identified the structural element that is crucial for the isoform-specific IP3-binding affinity. Docking into the IP3R electron microscopy map places the module on the cytoplasmic part of the channel around the four-fold symmetry axis. Four ligand-binding modules form a cluster at the apex of the mushroom-shaped IP3R protein. The crystal structure and the localization of IP3R ligand-binding module resemble another type of intracellular Ca2+-release channel, ryanodine receptors. We propose a model showing that these two channels share a common allosteric gating mechanism.

Abstract P4-01-07: Endoplasmic Reticulum-Associated Calcium Regulates Resistance of the Anti-Apoptotic Protein Bcl-2 to Taxol-Induced Apoptosis in Breast Cancer Cells

Abstract Background: The unique anti-apoptotic function of Bcl-2 results in cellular resistance to many chemotherapeutic agents used in solid tumor treatments. However, there is a controversy regarding the relationship between Bcl-2 resistance and the mechanism of action for Taxol, an anticancer agent commonly used in breast cancer treatment. Although previous studies showed both Bcl-2 and Taxol may have effects on calcium homeostasis independently, no studies have addressed whether calcium is the keystone in the relationship between Bcl-2 resistance and Taxol action. Objective: Since most internal calcium is stored in the endoplasmic reticulum (ER), this study focused on the role of ER calcium changes in Bcl-2-activated resistance to Taxol-induced apoptosis. The ER calcium channel IP3R and its relationship with Bcl-2 and Taxol were investigated in order to clarify the mechanism underlying calcium regulation. Methods and Materials: For this study, MDA-MB-468, a Bcl-2 negative breast carcinoma cell line, its Bcl-2 stable transfectant and MCF-7, a Bcl-2 positive breast carcinoma cell line were used. The dynamic calcium changes induced by Taxol in these cells were determined by measuring free cytosolic calcium fluctuations using Fluo4-AM and ER calcium changes by the calcium cameleon, D1ER. Taxol-induced apoptosis was measured using the Annexin V-FITC assay. The calcium interfering agents BAPTA-AM and 2-APB were applied to evaluate whether Taxol-induced calcium changes were related to this apoptotic event. Western blot was used to detect changes induced by Taxol treatment in Bcl-2 and IP3R protein levels. Results: Taxol induced a rapid ER calcium release followed by a gradual ER calcium depletion in both Bcl-2 positive and negative cell lines, indicating that ER calcium store provides a direct target for Taxol, independently of Bcl-2 expression. Interfering with these calcium changes inhibited the Taxol-induced apoptosis, suggesting ER calcium release promotes Taxol-induced apoptosis. Our results also showed that Bcl-2 inhibited the basal ER calcium release. Taxol stimulated ER calcium release and thus attenuated Bcl-2-initiated resistance to apoptosis. Relevance: This research addressed the question of whether ER calcium changes play a critical or marginal role in mediating resistance of Bcl-2 to Taxol-induced apoptosis. Elucidating the role of calcium in this process will not only help to clarify the mechanism of drug resistance associated with Bcl-2, but also aid in more effective application of Taxol in breast cancer treatment. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-01-07.

Recording single-channel activity of inositol trisphosphate receptors in intact cells with a microscope, not a patch clamp.

Optical single-channel recording is a novel tool for the study of individual Ca2+-permeable channels within intact cells under minimally perturbed physiological conditions. As applied to the functioning and spatial organization of IP3Rs, this approach complements our existing knowledge, which derives largely from reduced systems - such as reconstitution into lipid bilayers and patch clamping of IP3Rs on the membrane of excised nuclei - where the spatial arrangement and interactions among IP3Rs via CICR are disrupted. The ability to image the activity of single IP3R channels with millisecond resolution together with localization of their positions with a precision of a few tens of nanometers both raises several intriguing questions and holds promise of answers. In particular, what mechanism underlies the anchoring of puffs and blips to static locations; why do these Ca2+ release events appear to involve only a very small fraction of the IP3Rs within a cell; and how can we reconcile the relative immotility of functional IP3Rs with numerous studies reporting free diffusion of IP3R protein in the ER membrane?

Regulation of Inositol 1,4,5 Trisphosphate Receptors by InsP3 Receptor-Associated cGMP Kinase Substrate (IRAG)

Various factors interact with IP3R, regulating Ca2+ release and thus serve to define the spatial and temporal characteristics of the cytosolic Ca2+ signal. IP3R-associated cGMP kinase substrate (IRAG) has been reported to bind IP3R type-1 and inhibit Ca2+ mobilization in smooth muscle cells. No information is however available as to whether IRAG interacts or has functional effects on other IP3R family members. In this study, we examined whether IRAG binds to and regulates Ca2+ release via IP3R type-2 or type-3. cDNA encoding IP3R type-1, IRAG-GFP, and protein kinase G1β (PKG1β) were transiently transfected into COS cells. Following immunoprecipitation from cell lysates with an anti-GFP antibody, IP3R type-1 was detected by immunoblotting. In contrast, an IRAG-GFP construct (IRAGΔE12-GFP) in which 40 amino acids required for binding with IP3R was deleted, failed to interact with IP3R type-1, but was still capable of binding to PKG1β, an additional cognate binding partner of IRAG. Similarly, IP3R type-2 or IP3R type-3 could be shown to interact with IRAG-GFP but not IRAGΔE12-GFP in COS cells. Next, we investigated if IRAG regulates IP3-induced Ca-release using DT40-3KO cell lines stably expressing mammalian IP3R type-2 or type-3 in isolation. In DT40-3KO cells stably expressing IP3R type-2, and transiently expressing Muscarinic M3 receptor, IRAG-GFP and PKG1β, cell permeable PKG activators reduced the muscarinic agonist carbachol (CCh)-induced Ca2+-release. Ca2+ oscillations induced by low concentrations of CCh or by stimulating the endogenous B cell receptor were similarly attenuated. No inhibitory effect was evident in cells expressing IRAGΔE12-GFP or in the absence of IRAG-GFP. Similar results were obtained with DT40-3KO cells stably expressing IP3R type-3. These results indicate that Ca2+ release through all Inositol 1,4,5 trisphosphate receptors are inhibited by an interaction with IRAG and PKG1β.

Altered Calcium Handling Between Healthy And Atherosclerotic Vascular Smooth Muscle

In order for smooth muscle (SM) contraction and relaxation to proceed efficiently, Ca2+ handling is under tight regulation. The cyclic strain associated with hypertension is thought to play an initiating role in atherosclerosis, suggesting dysregulation of SM Ca2+ handling may be a contributing factor. Peroxynitrite (ONOO−), the reaction product of superoxide and nitric oxide, forms in diseased vessels and has been demonstrated to induce SM cell relaxation. In this study, we assessed function and expression levels of sarcoplasmic reticulum Ca2+ handling proteins; the inositol 1,4,5-trisphosphate receptor (IP3R) and the Ca2+ATPase (SERCA) in both healthy and atherosclerotic aorta. ONOO− dose-dependently relaxed U46619 pre-contracted aorta from both control and atherosclerotic ApoE−/− mice (2 months high fat diet) [51.2 ± 4.7% and 78.5 ± 4.3% maximal relaxation, respectively (3 × 10−5 ONOO−)]. This relaxation was antagonised in both C57 and ApoE−/− by the addition of either 3μM thapsigargin (TG), a SERCA inhibitor, or 60μM 2-aminoethoxydiphenyl borate (2-APB), an IP3R blocker. In control aorta, relaxation was 4.3 ± 5% (TG), p<0.001; n=7 and 14.6 ± 6.2% (2-APB) p=0.001; n=9. In ApoE−/− aorta, % relaxation was 22.09 ± 3.1 (TG) p<0.001; n=8 and 7 ± 5.9% (2-APB) p<0.001; n=7. There was no significant difference between endothelial denuded or intact vessels. These data indicate an alteration in the effect of Ca2+ handling protein inhibitors between control and ApoE−/− mice. This has been further correlated to expression of both SERCA and IP3R proteins. Studies with the potassium channel blocker tetraethylammonium (TEA) indicate plasma membrane hyperpolarisation is an effector of ONOO− induced relaxation [31.2% relaxation reduction with TEA in C57 aorta, p=0.002 vs 35.5% reduction in ApoE−/−, p=0.022]. We provide additional evidence, through myography and biochemical analysis, of a time-dependent correlation between atherosclerotic development and SM Ca2+ handling machinery modulation.

Comparison of IP3R and RyR Expression and Ca2+ Release Characteristics in Isolated Cardiac Nuclei

In cardiac muscle, the role of the inositol trisphosphate receptor (IP3R) and its regulation is not fully understood. A contribution to nuclear Ca2+ signalling has been proposed. This study compares expression and Ca2+ release characteristics of the IP3R and the ryanodine receptor (RyR) in purified functional cardiac nuclei. It also examines whether the IP3R may exist as a multi-protein complex in these preparations. Quantitative immunoblotting of IP3R and RyR protein levels in isolated nuclei demonstrated greater expression of the IP3R; nucleolin was used as an internal control for quantification. Ca2+ release in response to IP3 and caffeine from single isolated nuclei was used to compare IP3R and RyR activity. Changes in nuclear [Ca2+] were measured as fluorescence signals from nuclei loaded with 10μM Fluo 5N-AM. IP3 or caffeine was applied by hydrostatic pressure ejection and signals expressed as ratios (F/F0) of fluorescence counts relative to baseline. Ca2+ release in response to IP3 (10μM) was significantly greater than that released in response to caffeine (10mM) (0.12 ± 0.02 v's 0.017 ± 0.002 [Ca2+]Nuc (F/F0) for IP3 and caffeine respectively, n=6). When tetracaine (100μM) was applied to the nuclei, IP3-mediated Ca2+ release was unaffected but the response to caffeine was abolished, suggesting RyR activation does not contribute to IP3-mediated nuclear Ca2+ release. The potential for other nuclear proteins interacting with the nuclear IP3R was also investigated. Immunoblot analysis demonstrated expression of both FKBP12 and calcineurin in cardiac nuclei. These proteins are known to interact with the IP3R in other tissue types. Co-immunoprecipitation experiments using an anti-IP3R (type II) antibody suggest IP3R/calcineurin/FKBP12 interaction specifically at the nucleus. These results highlight the existence of a nuclear multi-protein IP3R complex, providing further scope for regulation of cardiac nuclear Ca2+ release.

Tumor Necrosis Factor-α Potentiates Intraneuronal Ca2+ Signaling via Regulation of the Inositol 1,4,5-Trisphosphate Receptor

Inflammatory events have long been implicated in initiating and/or propagating the pathophysiology associated with a number of neurological diseases. In addition, defects in Ca2+-handling processes, which shape membrane potential, influence gene transcription, and affect neuronal spiking patterns, have also been implicated in disease progression and cognitive decline. The mechanisms underlying the purported interplay that exists between neuroinflammation and Ca2+ homeostasis have yet to be defined. Herein, we describe a novel neuron-intrinsic pathway in which the expression of the type-1 inositol 1,4,5-trisphosphate receptor is regulated by the potent pro-inflammatory cytokine tumor necrosis factor-alpha. Exposure of primary murine neurons to tumor necrosis factor-alpha resulted in significant enhancement of Ca2+ signals downstream of muscarinic and purinergic stimulation. An increase in type-1 inositol 1,4,5-trisphosphate receptor mRNA and protein steady-state levels following cytokine exposure positively correlated with this alteration in Ca2+ homeostasis. Modulation of Ca2+ responses arising from this receptor subtype and its downstream effectors may exact significant consequences on neuronal function and could underlie the compromise in neuronal activity observed in the setting of chronic neuroinflammation, such as that associated with Parkinson disease and Alzheimer disease.

Knockdown of the type 2 and 3 inositol 1,4,5-trisphosphate receptors suppresses muscarinic antinociception in mice

The involvement of central endoplasmic inositol 1,4,5-trisphosphate receptors (IP 3R) in muscarinic antinociception was investigated in the mouse hot plate test. Selective knockdown of type 1, 2 and 3 IP 3R was obtained by means of an antisense oligonucleotide (aODN) strategy. A selective IP 3R protein level reduction of approximately 30–50% produced by aODN administration for each receptor subtype investigated was demonstrated by Western blotting experiments. I.c.v. pretreatment with an aODN complementary to the sequence of the type 2 IP 3R (0.1–3 nmol per mouse i.c.v.) prevented the antinociception induced by physostigmine (0.15 mg kg −1 s.c.) and oxotremorine (60 μg kg −1 s.c.). Similarly, an aODN against type 3 IP 3R (0.1–3 nmol per mouse i.c.v.) antagonized cholinergic antinociception. A shift to the right of the physostigmine dose–response curve was obtained after anti-type 2 IP 3R2 and anti-type 3 IP 3R treatments. Conversely, pretreatment with an aODN complementary to the sequence of type 1 IP 3R (0.1–5 nmol per mouse i.c.v.) did not modify the antinociception induced by physostigmine and oxotremorine. Mice undergoing treatment with aODNs did not show any impairment of the locomotor activity, spontaneous motility and exploratory activity as revealed by the rota-rod and hole board tests. These results indicate a selective involvement of type 2 and 3 IP 3R in central muscarinic antinociception in mice.

The Calponin Homology Domain of Vav1 Associates with Calmodulin and Is Prerequisite to T Cell Antigen Receptor-induced Calcium Release in Jurkat T Lymphocytes

Vav1 is a guanine nucleotide exchange factor that is expressed specifically in hematopoietic cells and plays important roles in T cell development and activation. Vav1 consists of multiple structural domains so as to facilitate both its guanine nucleotide exchange activity and scaffold function following T cell antigen receptor (TCR) engagement. Previous studies demonstrated that the calponin homology (CH) domain of Vav1 is required for TCR-stimulated calcium mobilization and thus downstream activation of nuclear factor of activated T cells. However, it remained obscure how Vav1 functions in regulating calcium flux. In an effort to explore molecules interacting with Vav1, we found that calmodulin bound to Vav1 in a calcium-dependent and TCR activation-independent manner. The binding site was mapped to the CH domain of Vav1. Reconstitution of vav1-null Jurkat T cells (J.Vav1) with CH-deleted Vav1 exhibited a severe deficiency in calcium release to the same extent as that of Jurkat cells treated with the calmodulin inhibitor or J.Vav1 cells. The defect persisted even when phospholipase-Cgamma1 was fully activated, indicating a prerequisite role of Vav1 CH domain in calcium signaling. The results suggest that Vav1 and calmodulin function cooperatively to potentiate TCR-induced calcium release. This study unveiled a mechanism by which the Vav1 CH domain is involved in calcium signaling and provides insight into our understanding of the role of Vav1 in T cell activation.

DANGER, New Regulator of IP 3 R Calcium-Release Channel

The inositol 1,4,5-trisphosphate receptor (IP 3 R) functions as a sensor for the second messenger IP3 and as a channel protein that allows release of Ca 2+ from intracellular stores in response to activated receptors that are linked to production of IP 3 . However, there are some 2000 extra amino acids in the large IP 3 R protein in between the region that confers IP 3 binding and the Ca 2+ channel itself, and a number of proteins have been identified that interact with the IP 3 R in this region to regulate its function. Van Rossum et al . used a yeast 2-hybrid screen to search for other IP 3 R-interacting proteins in mammalian cDNA libraries and identified a new one they call DANGER. The authors confirmed interaction of the endogenous proteins by immunoprecipitation from cultured PC12 and showed colocalization with IP 3 Rs near the plasma membrane by immunofluorescence microscopy. Addition of glutathione S -transferase­-tagged DANGER to microsomal fractions from COS7 cells expressing transfected IP 3 R enhanced the sensitivity of the channel to inhibitory feedback by Ca 2+ . Normally, supraphysiological concentrations of Ca 2+ are needed to decrease IP 3 -dependent release of Ca 2+ through the receptor, but when DANGER was present, moderate concentrations of Ca 2+ (0.8 to 3.8 μM) almost abolished Ca 2+ release. Single-channel recordings of IP 3 R activity showed that DANGER reduced the channel open probability by decreasing the opening rate of the channel. The authors propose that DANGER appears to cause allosteric regulation of the IP 3 R that enhances the Ca 2+ sensitivity of the receptor such that Ca 2+ becomes a major regulator of the IP 3 R in cells expressing DANGER. D. B. van Rossum, R. L. Patterson, K.-H. Cheung, R. K. Barrow, V. Syrovatkina, G. S. Gessell, S. G. Burkholder, D. N. Watkins, J. K. Foskett, S. H. Snyder, DANGER, a novel regulatory protein of inositol 1,4,5-trisphosphate-receptor activity. J. Biol. Chem. 281 , 37111-37116 (2006). [Abstract] [Full Text]

Changes in IP3 Receptor Are Associated With Altered Calcium Response to Cholecystokinin in Diabetic Rat Pancreatic Acini

Pancreatic acini of diabetic rats release amylase less than normal acini on cholecystokinin (CCK) stimulation. Pancreatic enzyme secretion by CCK is closely related to the second messenger inositol 1,4,5-trisphosphate (IP3), which mobilizes intracellular calcium stores via the endoplasmic reticulum-located receptor IP3 (IP3R). Recently, we observed altered intracellular calcium response on CCK-8 stimulation in streptozotocin (STZ)- treated diabetic rat acini. To determine whether IP3R is involved in altered calcium response, we measured inositol phosphate (IP) formation and the expression and phosphorylation of type III IP3R protein in diabetic acini. Also, CCK receptor mRNA expression was examined to determine whether the changes in IP formation and IP3R protein phosphorylation in diabetic acini might result from the defect at the postreceptor level. CCK-8-induced IP formation at all concentrations used was significantly reduced in diabetic acini, though IP formation was increased in a concentration-dependent manner. The expression of type III IP3R protein was significantly reduced in diabetic acini. Additionally, CCK-8-stimulated phosphorylation of type III IP3R protein was not observed in diabetic acini. However, the reduction of CCK receptor mRNA expression was not detected in diabetic acini. Our results indicate that altered calcium response to CCK-8 in diabetic acini might be associated with a post-CCK receptor defect including the changes in IP formation, type III IP3R protein expression, and phosphorylation of type III IP3R protein.

Bcl-X(L) affects Ca(2+) homeostasis by altering expression of inositol 1,4,5-trisphosphate receptors.

An oligonucleotide-based microarray analysis of 9,500 genes and expressed sequence tags (ESTs) demonstrated that the type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R) was significantly down-regulated in Bcl-X(L)-expressing as compared with control cells. This result was confirmed at the mRNA and protein levels by Northern and Western blot analyses of two independent hematopoietic cell lines and murine primary T cells. Bcl-X(L) expression resulted in a dose-dependent decrease in IP(3)R protein. IP(3)R expression is regulated as part of a mitochondrion-to-nucleus stress-responsive pathway. The uncoupling of mitochondrial oxidative phosphorylation resulted in induction of binding of the transcription factor NFATc2 to the IP(3)R promoter and transcriptional activation of IP(3)R. Expression of Bcl-X(L) led to a decreased induction of both NFATc2 DNA binding to the IP(3)R promoter and IP(3)R expression in response to the inhibition of mitochondrial oxidative phosphorylation. The Bcl-X(L)-dependent decrease in IP(3)R expression also correlated with a reduced T cell antigen receptor ligation-induced Ca(2+) flux in Bcl-X(L) transgenic murine T cells, and microsomal vesicles prepared from Bcl-X(L)-overexpressing cells exhibited lower IP(3)-mediated Ca(2+) release capacity. Furthermore, reintroducing IP(3)R into Bcl-X(L)-transfected cells partially reversed Bcl-X(L)-dependent anti-apoptotic activity. These results suggest that even under non-apoptotic conditions, expression of Bcl-2-family proteins influences a signaling network that links changes in mitochondrial metabolism to alterations in nuclear gene expression.

Proteasomal activation mediates down-regulation of inositol 1,4,5-trisphosphate receptor and calcium mobilization in rat pancreatic islets.

Inositol 1,4,5-trisphosphate receptor (IP3R) protein levels in isolated rat pancreatic islets were investigated in response to carbachol (CCh) and sulfated cholecystokinin 26-33 amide stimulation. Within 2 h, CCh reduced IP3R-I protein levels by 22% and IP3R-II and -III levels to 65% or more below basal. Sulfated cholecystokinin 26-33 amide decreased the levels of IP3R-I, -II, and -III by 34%, 60%, and 66% below basal, respectively. The effect of CCh was concentration- and time-dependent, with a persistent decline in IP3R levels for up to 6 h after the onset of stimulation. CCh-pretreated islets also showed an inhibition of glucose-stimulated insulin secretion. Proteasome inhibition completely blocked the down-regulatory effects of CCh on IP3Rs and significantly increased the insulin secretory response to glucose stimulation in the presence of CCH: Islet stimulation by glucose, alpha-ketoisocaproic acid, and tolbutamide completely protected IP3Rs against the down-regulatory effects of CCH: 2-deoxyglucose and 3-O-methyl glucose failed to affect CCh-induced IP3R down-regulation. The protective effects of glucose on IP3R down-regulation were completely inhibited by the Ca(2+) channel-blocking agent nimodipine. Intracellular Ca(2+) ([Ca(2+)](i)) levels in Fura-2 (fluorescent Ca(2+) indicator)-loaded islets, in the absence of extracellular Ca(2+), increased in response to glucose stimulation; but in islets pretreated with CCh, glucose did not increase [Ca(2+)](i) above basal levels. However, in islets pretreated with CCh and the proteasomal inhibitor MG-132 (carbobenzoxyl-leucinyl-leucinyl-leucinyl-H), the glucose-stimulated increase in [Ca(2+)](i) was significantly higher than the change observed for glucose-stimulated [Ca(2+)](i) in the absence of MG-132. The results suggest that muscarinic receptor stimulation modulates IP3R protein levels in islets through a proteasomal activation pathway, and that down-regulation of IP3Rs has a profound effect on Ca(2+) mobilization in islets that may relate to insulin secretory responsiveness.

Inositol 1,4,5-trisphosphate receptor isoform expression in mouse pancreatic islets: effects of carbachol

1 The inositol 1,4,5-trisphosphate receptors (IP3Rs) are ligand-gated Ca 2+ channels that regulate intracellular Ca 2+ mobilization. Among the IP3R mRNA isoforms I, II, and III, IP3R-I mRNA was expressed in mouse islets and the β-cell line βTC3, and was quantitatively the most abundant isoform as determined by reverse transcriptase–polymerase chain reaction. IP3R-II and -III mRNAs were expressed at similar levels in mouse islets, but neither isoform was detected in βTC3 cells. Culture of mouse islets for 30 min and 2 hr at 20 mM glucose, or for 7 days at 11 mM glucose did not affect IP3R-I mRNA expression compared with islets cultured in 5.5 mM glucose. Culture of islets or βTC3 cells with carbachol (0.5 mM) reduced IP3R-I mRNA expression levels below control. Mouse islet α- and β-cells expressed IP3R-I and -III proteins, but IP3R-II protein was not detected by immunoblot or double-label immunohistochemistry. Culture of islets for up to 6 hr with carbachol reduced IP3R-I and -III protein expression in a time-dependent manner with a half-maximal effect on type I at 1 hr. Glucose (20 mM) stimulation for 2 hr did not affect IP3R-1 levels. The carbachol-induced decrease in IP3R-I and -III protein expression was reversed by carbobenzoxyl-leucinyl-leucinyl-leucinyl-H (MG-132), a proteasome inhibitor. Thus, glucose failed to regulate mouse islet IP3R mRNA expression, whereas carbachol stimulation down-regulated IP3R mRNA and protein. A proteasomal protein degradative pathway appeared to mediate the muscarinic receptor-induced effects on IP3R-I and -III.

Cardioprotection induced by AT1R blockade after reperfused myocardial infarction: association with regional increase in AT2R, IP3R and PKCepsilon proteins and cGMP.

We hypothesized that the cardioprotective effect of angiotensin II (AngII) type 1 receptor (AT(1)R) blockade during in vivo ischemia-reperfusion (IR) might be associated with an increase in AngII type 2 receptor (AT(2)R) protein, as well as 1,4,5-inositol trisphosphate type 2 receptor (IP(3)R) and protein kinase C(epsilon) (PKC(epsilon)) proteins and cyclic guanosine 3',5' monophosphate (cGMP). We studied the effects of the AT(1)R blocker, candesartan, on in vivo left ventricular (LV) systolic and diastolic function and remodeling (echocardiogram/Doppler) and hemodynamics during canine reperfused anterior infarction (90-minute ischemia, 120-minute reperfusion), and ex vivo infarct size and AT(1)R/AT(2)R, IP(3)R, and PKC(epsilon) proteins (immunoblots), and cGMP (enzyme immunoassay). Compared with controls, candesartan (1 mg/kg intravenously over 30-minute preischemia) inhibited the AngII pressor response, decreased preload and afterload, improved LV systolic and diastolic function, limited LV remodeling, decreased infarct size (55% vs 27% risk; P <.000003), markedly increased AT(2)R, IP(3)R, and PKC(epsilon) proteins in the infarct zone, but not the AT(1)R protein, and increased infarct more than noninfarct cGMP. The overall results suggest that cardioprotective effects of AT(1)R blockade on acute IR injury might involve AT(2)R activation and downstream signaling via IP(3)R, PKC(epsilon), and cGMP.

Enhanced expression of angiotensin II type 2 receptor, inositol 1,4, 5-trisphosphate receptor, and protein kinase cepsilon during cardioprotection induced by angiotensin II type 2 receptor blockade.

We hypothesized that the cardioprotective effect of angiotensin II type 2 receptor (AT(2)R) blockade with PD 123,319 (PD) on the recovery of left ventricular (LV) mechanical function after ischemia/reperfusion (IR) in the isolated working rat heart is associated with the enhanced expression of AT(2)R protein and mRNA as well as an increase in inositol 1,4,5-trisphosphate type 2 receptor (IP(3)R) and protein kinase Cepsilon (PKCepsilon) proteins. We assessed AT(2)R, angiotensin II type 1 receptor (AT(1)R), IP(3)R, and PKCepsilon protein expression (Western blots) and AT(2)R mRNA levels (Northern blots) in myocardium from isolated working rat hearts that were subjected to global ischemia (30 minutes) followed by reperfusion (30 minutes). Groups of adult rat hearts (n=6) were exposed to no IR, no IR+PD (0.3 micromol/L), IR, and IR+PD. Compared with no IR and no IR+PD, IR decreased (P<0.05) functional recovery and AT(2)R mRNA and protein, as well as AT(1)R mRNA (not protein) and IP(3)R and PKCepsilon proteins. Compared with IR, PD+IR improved LV functional recovery (P<0.05) and markedly increased AT(2)R mRNA and protein (P<0.001). However, PD did not change AT(1)R mRNA or protein. More importantly, PD+IR markedly increased IP(3)R and PKCepsilon proteins. The downregulation of AT(2)R mRNA and protein with IR and their upregulation with PD indicate that the effects of PD are AT(2)R specific. The overall results suggest that the cardioprotective effect of acute PD treatment on LV functional recovery after IR in the isolated working rat heart is specifically due to AT(2)R blockade and is associated with enhanced downstream IP(3)R and PKCepsilon signaling.