Inositol 1,4,5-trisphosphate receptor 1, a widespread Ca2+ channel, is a novel substrate of polo-like kinase 1 in eggs

To initiate embryo development, the sperm induces in the egg release of intracellular calcium ([Ca²⁺]_i). During oocyte maturation, the inositol 1,4,5-trisphosphate receptor (IP₃R1), the channel implicated, undergoes modifications that enhance its function. We found that IP₃R1 becomes phosphorylated during maturation at an MPM-2 epitope and that this persists until the fertilization-associated [Ca²⁺]_i responses cease. We also reported that maturation without ERK activity diminishes IP₃R1 MPM-2 reactivity and [Ca²⁺]_i responses. Here, we show that IP₃R1 is a novel target for Polo-like kinase1 (Plk1), a conserved M-phase kinase, which phosphorylates it at an MPM-2 epitope. Plk1 and IP₃R1 interact in an M-phase preferential manner, and they exhibit close co-localization in the spindle/spindle poles area. This co-localization is reduced in the absence of ERK activity, as the ERK pathway regulates spindle organization and IP₃R1 cortical re-distribution. We propose that IP₃R1 phosphorylation by Plk1, and possibly by other M-phase kinases, underlies the delivery of spatially and temporally regulated [Ca²⁺]_i signals during meiosis/mitosis and cytokinesis.     

Inositol(1,4,5)trisphosphate signal triggers a receptor-mediated ATP release

Intracellular signal transduction pathways involved in ATP release evoked by angiotensin II (Ang II) were investigated in cultured guinea pig Taenia coli smooth muscle cells. Ang II (0.3-1 microM) elicited substantial release of ATP from the cells, but not from a human fibroblast cell line. However, Ang II even at 10 microM failed to cause a leakage of lactate dehydrogenase (LDH) from the smooth muscle cells. The release of ATP by Ang II was suppressed by 10 microM SC52458, an AT1 receptor antagonist, not by 10 microM PD123319, an AT2 receptor antagonist. The evoked release of ATP was almost completely inhibited in the presence of 10 microM U73122, a phospholipase C inhibitor, and 0.5 microM thapsigargin, a Ca2+-ATPase inhibitor. Furthermore, the release was hampered by 50 microM BAPTA/AM, an intracellular Ca2+ chelator, but not by 0.1 microM nifedipine, a voltage gated Ca2+ channel inhibitor. The basal release of ATP was increased by BAPTA/AM, but was reduced by U-73122. Ang II enhanced instantaneously inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) accumulation in the cells. The enhancing effect was perfectly antagonized by SC52458. These findings suggest that intracellular Ca2+ signals activated via stimulation of Ins(1,4,5)P3 receptor are involved in the release of ATP evoked by Ang II.     

The type III inositol 1,4,5-trisphosphate receptor is associated with aggressiveness of colorectal carcinoma

The inositol 1,4,5-trisphosphate receptor (InsP3R) mediates Ca(2+) signaling in epithelia and regulates cellular functions such as secretion, apoptosis and cell proliferation. Loss of one or more InsP3R isoform has been implicated in disease processes such as cholestasis. Here we examined whether gain of expression of InsP3R isoforms also may be associated with development of disease. Expression of all three InsP3R isoforms was evaluated in tissue from colorectal carcinomas surgically resected from 116 patients. Type I and II InsP3Rs were seen in both normal colorectal mucosa and colorectal cancer, while type III InsP3R was observed only in colorectal cancer. Type III InsP3R expression in the advancing margins of tumors correlated with depth of invasion, lymph node metastasis, liver metastasis, and TNM stage. Heavier expression of type III InsP3R also was associated with decreased 5-year survival. shRNA knockdown of type III InsP3R in CACO-2 colon cancer cells enhanced apoptosis, while over-expression of the receptor decreased apoptosis. Thus, type III InsP3R becomes expressed in colon cancer, and its expression level is directly related to aggressiveness of the tumor, which may reflect inhibition of apoptosis by the receptor. These findings suggest a previously unrecognized role for Ca(2+) signaling via this InsP3R isoform in colon cancer.     

Functional characterization of the P1059L mutation in the inositol 1,4,5-trisphosphate receptor type 1 identified in a Japanese SCA15 family

Spinocerebellar ataxia type 15 (SCA15) is a group of human neurodegenerative disorders characterized by a slowly progressing pure cerebellar ataxia. The inositol 1,4,5-trisphosphate (IP₃) receptor type 1 (IP₃R1) is an intracellular IP₃-induced Ca²⁺ release channel that was recently identified as a causative gene for SCA15. In most case studies, a heterozygous deletion of the IP₃R1 gene was identified. However, one Japanese SCA15 family was found to have a Pro to Leu (P1059L) substitution in IP₃R1. To investigate the effect of the P1059L mutation, we analyzed the channel properties of the mutant human IP₃R1 by expressing it in an IP₃R-deficient B lymphocyte cell line. The P1059L mutant was a functional Ca²⁺ release channel with a twofold higher IP₃ binding affinity compared to wild-type IP₃R1. The cooperative dependence of the Ca²⁺ release activity of the mutant on IP₃ concentration was reduced, but both wild-type and mutant receptors produced similar B cell receptor-induced Ca²⁺ signals. These results demonstrate that the Ca²⁺ release properties of IP₃R1 are largely unaffected by the P1059L mutation.     

An inside job: Annexin 1A-Inositol 1,4,5-trisphosphate receptor interaction conveys endoplasmic reticulum luminal Ca2+ sensitivity

Cytoplasmic Ca²⁺ is a pivotal regulator of IP₃R activity. It is however controversial whether the [Ca²⁺] in the Endoplasmic Reticulum lumen also directly regulates channel function. We highlight a recent paper that demonstrates that luminal [Ca²⁺] potently inhibits IP₃R activity. This regulation occurs indirectly by an interaction mediated through a binding partner, likely Annexin 1A.     

KRAS-induced actin-interacting protein regulates inositol 1,4,5-trisphosphate-receptor-mediated calcium release

KRAS-induced actin-interacting protein (KRAP) was originally characterized as a filamentous- actin-interacting protein. We have recently found that KRAP is an associated molecule with inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R) and is responsible for the proper subcellular localization of IP₃R. Since it remains unknown whether KRAP regulates the IP₃R-mediated Ca²⁺ signaling, we herein examined the effects of KRAP on the IP₃R-mediated Ca²⁺ release by Ca²⁺ imagings in the cultured HEK293 or MCF7 cells. Reduction of KRAP protein by KRAP-specific siRNA diminishes ATP-induced Ca²⁺ release and the ATP-induced Ca²⁺ release is completely quenched by the pretreatment with the IP₃R inhibitor but not with the ryanodine receptor inhibitor, indicating that KRAP regulates IP₃R-mediated Ca²⁺ release. To further reveal mechanistic insights into the regulation of IP₃R-mediated Ca²⁺ release by KRAP, we examined the effects of the KRAP-knockdown on the releasable Ca²⁺ content of intracellular Ca²⁺ stores. Consequently, reduction of KRAP does not affect the amount of ionophore- or Ca²⁺-ATPase inhibitor-induced Ca²⁺ release in the HEK293 cells, indicating that releasable Ca²⁺ content of intracellular Ca²⁺ stores is not altered by KRAP. Thus, KRAP is involved in the proper regulation of IP₃R-mediated Ca²⁺ release.     

Inositol 1,4,5-trisphosphate-induced calcium release from canine aortic sarcoplasmic reticulum vesicles

Inositol 1,4,5-trisphosphate-induced calcium release from canine aortic smooth muscle sarcoplasmic reticulum vesicles was examined using the calcium indicator antipyrylazo III. Calcium release was initiated by addition of inositol 1,4,5-trisphosphate (IP₃) to aortic vesicles 7 min after initiation of ATP-supported calcium uptake. Half-maximal calcium release occurred at 1 μM IP₃, with maximal calcium release amounting to 25±2% of the intravesicular calcium (n=12, 9 preparations). Ruthenium red (10–20 μM), which has been reported to block IP₃-induced calcium release from skeletal muscle sarcoplasmic reticulum, did not inhibit aortic IP₃-induced calcium release. Elevation of Mg²⁺ concentration from 0.06 to 7.8 mM inhibited aortic IP₃-induced calcium release 75%, which contrasts with the Mg²⁺-insensitive IP₃-induced calcium release from platelet reticular membranes. The IP₃-dependence of aortic calcium release suggested that Mg²⁺ acted as a noncompetitive inhibitor. Thus, aortic sarcoplasmic reticulum vesicles contain an IP₃-sensitive calcium pathway which is inhibited by millimolar concentrations of Mg²⁺, but which is not inhibited by Ruthenium red and so differs from the previously described IP₃-sensitive calcium pathways in skeletal muscle and platelet reticular membranes.     

Developmental and Regional Studies of the Metabolism of Inositol 1,4,5-Trisphosphate in Rat Brain

Coupling of CNS receptors to phosphoinositide turnover has previously been found to vary with both age and brain region. To determine whether the metabolism of the second messenger inositol 1,4,5-trisphosphate also displays such variations, activities of inositol 1,4,5-trisphosphate 5'-phosphatase and 3'-kinase were measured in developing rat cerebral cortex and adult rat brain regions. The 5'-phosphatase activity was relatively high at birth (approximately 50% of adult values) and increased to adult levels by 2 weeks postnatal. In contrast, the 3'-kinase activity was low at birth and reached approximately 50% of adult levels by 2 weeks postnatal. In the adult rat, activities of the 3'-kinase were comparable in the cerebral cortex, hippocampus, and cerebellum, whereas much lower activities were found in hypothalamus and pons/medulla. The 5'-phosphatase activities were similar in cerebral cortex, hippocampus, hypothalamus, and pons/medulla, whereas 5- to 10-fold higher activity was present in the cerebellum. The cerebellum is estimated to contain 50-60% of the total inositol 1,4,5-trisphosphate 5'-phosphatase activity present in whole adult rat brain. The localization of the enriched 5'-phosphatase activity within the cerebellum was examined. Application of a histochemical lead-trapping technique for phosphatase indicated a concentration of inositol 1,4,5-trisphosphate 5'-phosphatase activity in the cerebellar molecular layer. Further support for this conclusion was obtained from studies of Purkinje cell-deficient mutant mice, in which a marked decrement of cerebellar 5'-phosphatase was observed. These results suggest that the metabolic fate of inositol 1,4,5-trisphosphate depends on both brain region and stage of development.     

Alzheimer's presenilin-1 mutation potentiates inositol 1,4,5-trisphosphate-mediated calcium signaling in Xenopus oocytes

Perturbations in intracellular Ca2+ signaling may represent one mechanism underlying Alzheimer's disease (AD). The presenilin-1 gene (PS1), associated with the majority of early onset familial AD cases, has been implicated in this signaling pathway. Here we used the Xenopus oocyte expression system to investigate in greater detail the role of PS1 in intracellular Ca2+ signaling pathways. Treatment of cells expressing wild-type PS1 with a cell surface receptor agonist to stimulate the phosphoinositide second messenger pathway evoked Ca2+-activated Cl- currents that were significantly potentiated relative to controls. To determine which elements of the signal transduction pathway are responsible for the potentiation, we used photolysis of caged inositol 1,4,5-trisphosphate (IP3) and fluorescent Ca2+ imaging to demonstrate that PS1 potentiates IP3-mediated release of Ca2+ from internal stores. We show that an AD-linked mutation produces a potentiation in Ca2+ signaling that is significantly greater than that observed for wild-type PS1 and that cannot be attributed to differences in protein expression levels. Our findings support a role for PS1 in modulating IP3-mediated Ca2+ liberation and suggest that one pathophysiological mechanism by which PS1 mutations contribute to AD neurodegeneration may involve perturbations of this function.     

Mammalian target of rapamycin (mTOR) phosphorylates inositol 1,4,5-trisphosphate receptor type 2 and increases its Ca release activity

There is substantial evidence that crosstalk between the proliferation and Ca²⁺-signaling pathways plays a critical role in the regulation of normal physiological functions as well as in the pathogenesis of a variety of abnormal processes. In non-excitable cells, intracellular Ca²⁺ is mobilized through inositol 1,4,5-trisphosphate sensitive Ca²⁺ channels (IP₃R) expressed on the endoplasmic reticulum. Here we report that mTOR, a point of convergence for signals from mitogenic growth factors, nutrients and cellular energy levels, phosphorylates the IP₃R-2, the predominant isoform of IP₃R in AR4-2J cells. Pretreatment with the mTOR inhibitor rapamycin, decreased carbachol-induced Ca²⁺ release in AR4-2J cells. Rapamycin also decreased IP₃-induced Ca²⁺ release in permeabilized AR4-2J cells. We also showed that IGF-1 potentiates carbachol-induced Ca²⁺ release in AR4-2J cells, an effect that was prevented by rapamycin. Rapamycin also decreased carbachol-induced Ca²⁺ release in HEK 293A cells in which IP₃R-1 and IP₃R-3 had been knocked down. These results suggest that mTOR potentiates the activity of IP₃R-2 by a phosphorylation mechanism. This conclusion supports the concept of crosstalk between Ca²⁺ signaling and proliferation pathways and thus provides another way by which intracellular Ca²⁺ signals are finely encoded.     

The activation state of the inositol 1,4,5-trisphosphate receptor regulates the velocity of intracellular Ca2+ waves in bovine aortic endothelial cells

Ca(2+) is a highly versatile second messenger that plays a key role in the regulation of many cell processes. This versatility resides in the fact that different signals can be encoded spatio-temporally by varying the frequency and amplitude of the Ca(2+) response. A typical example of an organized Ca(2+) signal is a Ca(2+) wave initiated in a given area of a cell that propagates throughout the entire cell or within a specific subcellular region. In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP(3) R) is responsible for the release of Ca(2+) from the endoplasmic reticulum. IP(3) R activity can be directly modulated in many ways, including by interacting molecules, proteins, and kinases such as PKA, PKC, and mTOR. In the present study, we used a videomicroscopic approach to measure the velocity of Ca(2+) waves in bovine aortic endothelial cells under various conditions that affect IP(3) R function. The velocity of the Ca(2+) waves increased with the intensity of the stimulus while extracellular Ca(2+) had no significant impact on wave velocity. Forskolin increased the velocity of IP(3) R-dependent Ca(2+) waves whereas PMA and rapamycin decreased the velocity. We used scatter plots and Pearson's correlation test to visualize and quantify the relationship between the Ca(2+) peak amplitude and the velocity of Ca(2+) waves. The velocity of IP(3) R-dependent Ca(2+) waves poorly correlated with the amplitude of the Ca(2+) response elicited by agonists in all the conditions evaluated, indicating that the velocity depended on the activation state of IP(3) R, which can be modulated in many ways.     

Solubilization of rat liver inositol 1,4,5-trisphosphate receptor

High affinity Ins(1,4,5)P₃-binding sites of permeabilized hepatocytes are probably the ligand recognition sites of the receptors that mediate the effects of Ins91,4,5)P₃ on intracellular Ca²⁺ mobilization. We have now solubilized these sites from rat liver membranes in the zwitterionic detergent, CHAPS, and shown that the solubilized bind Ins(1,4,5)P₃ with an affinity (K_d = 7.26 ± 0.52 nM, Hill coefficient H = 1.05 ± 0.06) similar to that of the sites in native membranes (K_d = 6.02 ± 0.02). ATP and a range of inositol phosphates (Ins(2,4,5)P₃ Ins(4,5)P₂, and inositol 1,4,5-trisphosphorothioate) also bound with similar affinities to the native and solubilized sites. Solubilization of the liver InsP₃ receptor will allow its further characterization, purification, and comparison of its properties with those of InsP₃ receptors already purified from cerebellum and smooth muscle.     

The NK-1 Receptor and a Calcium-Phospholipid Pathway: Inositol Trisphosphate Production and Calcium Movements Induced by Selective Agonists of Neurokinin Receptors in Rat Parotid Glands

In the rat parotid gland, substance P has been shown to induce a phosphatidylinositol bisphosphate breakdown resulting in an inositol trisphosphate production. These data suggested that substance P activated a phospholipase C and thus mediated its effects through the calcium-phospholipid pathway. To determine which neurokinin (NK) receptor was involved in the substance P response, we have used selective agonists of the different NK receptors and examined their effects on both inositol trisphosphate production and calcium movements. A selective NK-1 receptor agonist, [Sar9Met(O2)11]-substance P, evoked an [3H]inositol trisphosphate production and a rapid and transient 45Ca2+ efflux. On the other hand, selective NK-2 and NK-3 receptor agonists, [beta-Ala8]-NKA(4-10) and [MePhe7]-NKB, respectively, were without effect. We conclude that, in the rat parotid glands, only the NK-1 receptors are coupled to the calcium-phospholipid pathway. The C-terminal part of substance P appeared to be sufficient to stimulate this route because the C-terminal octapeptide, substance P(4-11), mimicked substance P effects on both inositol trisphosphate production and calcium movements. The NK-2 and NK-3 receptors, if present in the rat parotid glands, are not associated with the calcium-phospholipid pathway.     

Models of the inositol trisphosphate receptor

The inositol (1,4,5)-trisphosphate receptor (IPR) plays a crucial role in calcium dynamics in a wide range of cell types, and is often a central feature in quantitative models of calcium oscillations and waves. We review deterministic and stochastic mathematical models of the IPR, from the earliest ones of the 1970s and 1980s, to the most recent. The effects of IPR stochasticity on Ca2+ dynamics are briefly discussed.     

Pivotal role of type-1 inositol 1,4,5-trisphosphate receptor for glucagon-induced gluconeogenesis

We highlight a recent paper which documents the important role that Ca²⁺ release through type-1 Inositol 1,4,5-trisphosphate receptor (IP₃R1) plays in the acute regulation by glucagon of gluconeogenesis in hepatocytes. The specificity is likely the result of discrete localization close to mitochondria and PKA-dependent phosphorylation of IP₃R1 which enhances Ca²⁺ release.     

Reduced Striatal [3H]Inositol 1,4,5-Trisphosphate Binding in Huntington''s Disease

Specific [3H]inositol 1,4,5-trisphosphate [( 3H]InsP3) binding was studied in regions of postmortem brain from 15 patients with Huntington's disease (HD) and 13 nonneurological controls. Single-point binding analyses, using 5.0 nM InsP3, showed statistically significant reductions in specific [3H]InsP3 binding in the caudate (-71%) and putamen (-75%) of HD patients compared with controls. Frontal and occipital cortical [3H]InsP3 binding was not significantly different between HD and controls, a finding suggesting that the reduced [3H]InsP3 binding parallels the brain regional specificity of the neuropathological changes in HD. Scatchard analyses of data from [3H]InsP3 competition binding assays performed on caudate nucleus revealed that the reductions found using single-point binding assays were due to a decrease in both binding density (-57%) and affinity (-50%) in HD brain compared with controls. The concomitant changes in InsP3 receptor density and affinity in HD brain suggest that these alterations may be produced by processes in addition to cell loss. These results suggest the possibility that disturbances in InsP3 receptor function, possibly resulting in altered intracellular calcium flux and homeostasis, occur in HD and may participate in the pathogenesis of this neurodegenerative disorder.     

μ-Opioid Receptor Stimulation of Inositol (1,4,5)Trisphosphate Formation via a Pertussis Toxin-Sensitive G Protein

Abstract: The cellular mechanisms underlying opioid action remain to be fully determined, although there is now growing indirect evidence that some opioid receptors may be coupled to phospholipase C. Using SH-SY5Y human neuroblastoma cells (expressing both μ-and δ-opioid receptors), we demonstrated that fentanyl, a μ-preferring opioid, caused a dose-dependent (EC₅₀= 16 n M ) monophasic increase in inositol (1,4,5)trisphosphate mass formation that peaked at 15 s and returned to basal within 1–2 min. This response was of similar magnitude (25.4 ± 0.8 pmol/mg of protein for 0.1 μ M fentanyl) to that found in the plateau phase (5 min) following stimulation with 1 m M carbachol (18.3 ± 1.4 pmol/mg of protein), and was naloxone-, but not naltrindole-(a δ antagonist), reversible. Further studies using [ d -Ala², MePhe⁴, Gly(ol)⁵]enkephalin and [ d -Pen^2,5]enkephalin confirmed that the response was specific for the μ receptor. Incubation with Ni²⁺ (2.5 m M ) or in Ca²⁺-free buffer abolished the response, as did pretreatment (100 ng/ml for 24 h) with pertussis toxin (control plus 0.1 μ M fentanyl, 26.9 ± 1.5 pmol/mg of protein; pertussis-treated plus 0.1 μ M fentanyl, 5.1 ± 1.3 pmol/mg of protein). In summary, we have demonstrated a μ-opioid receptor-mediated activation of phospholipase C, via a pertussis toxin-sensitive G protein, that is Ca²⁺-dependent. This stimulatory effect of opioids on phospholipase C, and the potential inositol (1,4,5)trisphosphate-mediated rises in intracellular Ca²⁺, could play a part in the cellular mechanisms of opioid action.     

Characterisation of Distinct Inositol 1,4,5-Trisphosphate-Sensitive and Caffeine-Sensitive Calcium Stores in Digitonin-Permeabilised Adrenal Chromaffin Cells

The effect of inositol 1,4,5-trisphosphate [Ins-(1,4,5)P3] and caffeine on Ca2+ release from digitonin-permeabilised bovine adrenal chromaffin cells was examined by using the Ca2+ indicator fura-2 to monitor [Ca2+]. Permeabilised cells accumulated Ca2+ in the presence of ATP and addition of either Ins(1,4,5)P3 or caffeine released 17% or 40-50%, respectively, of the accumulated Ca2+, indicated by sustained rises in [Ca2+] in the cell suspension. Prior addition of Ins(1,4,5)P3 had no effect on the magnitude of the response to a subsequent addition of caffeine. The response to Ins(1,4,5)P3 was prevented by prior addition of caffeine or CaCl2, indicating that the Ins(1,4,5)P3 response was blocked by elevated [Ca2+]. The responses were essentially identical in the presence of the proton ionophore carbonyl cyanide m-chlorophenylhydrazone, indicating that the Ca2+ release was not from mitochondria or secretory granules and that a proton gradient was not required for Ca2+ accumulation into the Ins(1,4,5)P3- or caffeine-sensitive stores. Ca2+ release from the caffeine-sensitive store was selectively blocked by ryanodine. The Ins(1,4,5)P3-sensitive store was emptied by thapsigargin, which had no effect on caffeine responses. These data suggest that permeabilised chromaffin cells possess two distinct nonoverlapping Ca2+ stores sensitive to either Ins(1,4,5)P3 or caffeine and support previous conclusions that these stores possess different Ca2(+)-ATPases.     

Muscarinic Cholinoceptor-Stimulated Synthesis and Degradation of Inositol 1,4,5-Trisphosphate in the Rat Cerebellar Granule Cell

Abstract: A detailed analysis of the generation and subsequent metabolism of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] following muscarinic cholinoceptor stimulation in primary cultures of rat cerebellar granule cells has been undertaken. Following incubation of cerebellar granule cell cultures with [³H]inositol for 48 h, labelling of the inositol phospholipid pool approached equilibrium. Significant basal labelling of inositol pentakisphosphate (InsP₅) and inositol hexakisphosphate (InsP₆), as well as inositol mono- to tetrakisphosphate, fractions was observed. Addition of carbachol (1 m M ) caused an immediate increase in level of Ins(1,4,5)P₃ (peak increase two-fold over basal by 60 s), which was well-maintained over the initial 300 s following agonist addition. In contrast, only a modest, more slowly developing, increase in inositol tetrakisphosphate accumulation was observed, whereas labelling of InsP₅ and InsP₆ was entirely unaffected by carbachol stimulation. Analysis of the products of Ins(1,4,5)P₃ and inositol 1,3,4,5-tetrakisphosphate metabolism in broken cell preparations strongly suggested that Ins(1,4,5)P₃ metabolism occurs predominantly via the inositol polyphosphate 5-phosphatase route, with metabolism via the Ins(1,4,5)P₃ 3-kinase being a relatively minor pathway. In view of the pattern of inositol (poly)phosphate metabolites observed on stimulation of the muscarinic receptor, it seems likely that, over the time course studied, the inositol polyphosphates are derived principally from phosphoinositide-specific phospholipase C hydrolysis of phosphatidylinositol 4,5-bisphosphate, although some hydrolysis of phosphatidylinositol 4-phosphate cannot be excluded.