Protein kinase C decreases the apparent affinity of the inositol 1,4,5-trisphosphate receptor type 3 in RINm5F cells

In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular Ca2+ channel which plays a major role in Ca2+ signalling. Three isoforms of IP3R have been identified (IP3R-1, IP3R-2 and IP3R-3) and most cell types express different proportions of each isoform. The differences between the pharmacological and functional properties of the various isoforms of IP3R are poorly known. RINm5F cells who express almost exclusively (approximately 90%) the IP3R-3, represent an interesting model to study this particular isoform. Here, we investigated a regulatory mechanism by which protein kinase C (PKC) may influence IP3R-3-mediated Ca2+ release. With an immunoprecipitation approach we confirmed that RINm5F cells express almost exclusively the IP3R-3 isoform. With an in vitro phosphorylation approach, we showed that the immunopurified IP3R-3 was efficiently phosphorylated by exogenous PKC. With a direct in cellulo approach and an indirect in cellulo back-phosphorylation approach we showed that phorbol-12-myristate-13-acetate (PMA) causes the phosphorylation of IP3R-3 in intact RINm5F cells. In saponin-permeabilized RINm5F cells, 3-induced Ca2+ release was reduced after a pre-treatment with PMA. PMA also reduced the Ca2+ response of intact RINm5F cells stimulated with carbachol and EGF, two agonists that use different receptor types to activate phospholipase C. These results suggest the existence of a negative feedback mechanism involving two components of the Ca2+ signalling cascade, whereby activated PKC dampens IP3R-3 activity.     

The inositol 1,4,5-trisphosphate receptor (Itpr) gene family in Xenopus: identification of type 2 and type 3 inositol 1,4,5-trisphosphate receptor subtypes

Studies in the Xenopus model system have provided considerable insight into the developmental role of intracellular Ca2+ signals produced by activation of IP3Rs (inositol 1,4,5-trisphosphate receptors). However, unlike mammalian systems where three IP3R subtypes have been well characterized, our molecular understanding of the IP3Rs that underpin Ca2+ signalling during Xenopus embryogenesis relate solely to the original characterization of the 'Xenopus IP3R' cloned and purified from Xenopus laevis oocytes several years ago. In the present study, we have identified Xenopus type 2 and type 3 IP3Rs and report the full-length sequence, genomic architecture and developmental expression profile of these additional IP3R subtypes. In the light of the emerging genomic resources and opportunities for genetic manipulation in the diploid frog Xenopus tropicalis, these data will facilitate manipulations to resolve the contribution of IP3R diversity in Ca2+ signalling events observed during vertebrate development.     

Down-regulation of the inositol 1,4,5-trisphosphate receptor in mouse eggs following fertilization or parthenogenetic activation

Fertilization in mammalian eggs is characterized by the presence of intracellular calcium ([Ca(2+)]i) oscillations. In mouse eggs, these oscillations cease after a variable period of time and this is accompanied by a decrease in inositol 1,4,5-trisphosphate receptor (IP3R) responsiveness and down-regulation of the IP3R type 1 (IP3R-1). To investigate the signaling pathway responsible for inducing IP3R-1 down-regulation during fertilization, mouse eggs were exposed to or injected with several Ca(2+)-releasing agonists and the amounts of IP3R-1 immunoreactivity evaluated by Western blotting. Exposure to ethanol or ionomycin, which induce a single [Ca(2+)]i rise, failed to signal down-regulation of IP3R-1. However, [Ca(2+)]i oscillations induced by injection of boar sperm fractions (SF), which presumably stimulate production of IP3, or adenophostin A, an IP3R agonist, both induced down-regulation of IP3R-1 of a magnitude similar to or greater than that observed after fertilization. Exposure to thimerosal, an oxidizing agent that modifies the IP3R without stimulating production of IP3, also initiated down-regulation of IP3R-1, although oscillations initiated by SrCl(2) failed to evoke down-regulation of IP3R-1. The degradation of IP3R-1 in mouse eggs appears to be mediated by the proteasome pathway because it was inhibited by preincubation with lactacystin, a very specific proteasome inhibitor. We therefore suggest that persistent stimulation of the phosphoinositide pathway in mouse eggs by the sperm during fertilization or by injection of SF leads to down-regulation of the IP3R-1.     

Isoforms of the inositol 1,4,5-trisphosphate receptor are expressed in bovine oocytes and ovaries: the type-1 isoform is down-regulated by fertilization and by injection of adenophostin A.

Mammalian fertilization is characterized by the presence of long-lasting intracellular calcium ([Ca2+]i) oscillations that are required to induce oocyte activation. One of the Ca2+ channels that may mediate this Ca2+ release is the inositol 1,4, 5-trisphosphate receptor (IP(3)R). Three isoforms of the receptor have been described, but their expression in oocytes and possible roles in mammalian fertilization are not well known. Using isoform-specific antibodies against IP(3)R types 1, 2, and 3 and Western analysis, we determined the isoforms that are expressed in bovine metaphase II oocytes and ovaries. In oocytes, all isoforms are expressed, but type 1 is present in overwhelmingly larger amounts and is likely responsible for the majority of Ca2+ release at fertilization. In ovarian microsomes, all three isoforms appear well expressed, suggesting the participation of all IP(3)R isoforms in ovarian Ca2+ signaling. We then investigated whether the reported cessation/reduction in amplitude of fertilization-associated [Ca2+]i oscillations, which is observed as pronuclear formation approaches, corresponded with down-regulation of the IP(3)R-1 isoform. Fertilization resulted in approximately 40% reduction in the amount of receptor by 16 h postinsemination. In addition, injection of adenophostin A, a potent IP(3)R agonist that elicits high-frequency [Ca2+]i oscillations in mammalian oocytes, induced similar reduction in receptor numbers. Together, these data show that 1) the three IP(3)R isoforms are expressed in bovine oocytes; 2) IP(3)R-1 is likely to mediate most of the Ca2+ release during fertilization; 3) its down-regulation may explain the decline in amplitude of sperm-induced [Ca2+]i rises as fertilization progresses toward pronuclear formation; and 4) agonists of the IP(3)R induce down-regulation of the type-1 receptor in oocytes similar to that evoked by fertilization.     

Modeling the impact of store-operated Ca2+ entry on intracellular Ca2+ oscillations

Calcium (Ca2+) oscillations play fundamental roles in various cell signaling processes and have been the subject of numerous modeling studies. Here we have implemented a general mathematical model to simulate the impact of store-operated Ca2+ entry on intracellular Ca2+ oscillations. In addition, we have compared two different models of the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and their influences on intracellular Ca2+ oscillations. Store-operated Ca2+ entry following Ca2+ depletion of endoplasmic reticulum (ER) is an important component of Ca2+ signaling. We have developed a phenomenological model of store-operated Ca2+ entry via store-operated Ca2+ (SOC) channels, which are activated upon ER Ca2+ depletion. The depletion evokes a bi-phasic Ca2+ signal, which is also produced in our mathematical model. The IP3R is an important regulator of intracellular Ca2+ signals. This IP3 sensitive Ca2+ channel is also regulated by Ca2+. We apply two IP3R models, the Mak-McBride-Foskett model and the De Young and Keizer model, with significantly different channel characteristics. Our results show that the two separate IP3R models evoke intracellular Ca2+ oscillations with different frequencies and amplitudes. Store-operated Ca2+ entry affects the oscillatory behavior of these intracellular Ca2+ oscillations. The IP3 threshold is altered when store-operated Ca2+ entry is excluded from the model. Frequencies and amplitudes of intracellular Ca2+ oscillations are also altered without store-operated Ca2+ entry. Under certain conditions, when intracellular Ca2+ oscillations are absent, excluding store-operated Ca2+ entry induces an oscillatory response. These findings increase knowledge concerning store-operated Ca2+ entry and its impact on intracellular Ca2+ oscillations.     

Characterisation of serum-induced intracellular Ca2+ oscillations in primary bone marrow stromal cells

Intracellular Ca2+ signalling is pivotal to cell function and [Ca2+]i oscillations permit precise and prolonged modulation of an array of Ca2+-sensitive processes without the need for extended, global elevations in [Ca2+]i. We have studied [Ca2+]i signalling in primary rat marrow stromal cells exposed to foetal calf serum (FCS) constituents at concentrations up to those required to promote growth and differentiation in culture. Spontaneous [Ca2+]i signalling was not observed, but exposure to 1% FCS induced regular, sustained Ca2+ oscillations in 41 +/- 3% of cells. Incidence of FCS-induced oscillations was dose-dependent, saturating at 0.5%. These oscillations were arrested by disruption of Ca2+ stores with 100 nM-1 microM thapsigargin or discharge of mitochondrial membrane potential and were sensitive to blockade of IP3-receptors by 50 microM 2-amino-ethoxydiphenyl borate (2-APB) and inhibition of phospholipase C with 5 microM U73122. The oscillations decreased in frequency and amplitude following inhibition of Ca2+ influx with EGTA or La3+ but were poorly sensitive to nifedipine (1-10 microM) and Bay K 8644 (300 nM). The factor(s) responsible for inducing [Ca2+]i oscillations are heat stable, insensitive to disulphide bond reduction with 20 mM dithioerythritol and retained by a 30 kDa molecular weight filter. Serum is routinely present in culture medium at 10%-15% [v/v] and marrow stromal cells maintained under culture conditions exhibited sustained oscillations. This is the first demonstration of agonist-induced complex Ca2+ signals in marrow stromal cells. We conclude that Ca2+ oscillations occur constantly in these cells in culture and are potentially important regulators of cell proliferation and differentiation.     

Distinct roles of inositol 1,4,5-trisphosphate receptor types 1 and 3 in Ca2+ signaling

Three subtypes of inositol 1,4,5-trisphosphate receptor (IP(3)R1, IP(3)R2, and IP(3)R3) Ca(2+) release channel share basic properties but differ in terms of regulation. To what extent they contribute to complex Ca(2+) signaling, such as Ca(2+) oscillations, remains largely unknown. Here we show that HeLa cells express comparable amounts of IP(3)R1 and IP(3)R3, but knockdown by RNA interference of each subtype results in dramatically distinct Ca(2+) signaling patterns. Knockdown of IP(3)R1 significantly decreases total Ca(2+) signals and terminates Ca(2+) oscillations. Conversely, knockdown of IP(3)R3 leads to more robust and long lasting Ca(2+) oscillations than in controls. Effects of IP(3)R3 knockdown are surprisingly similar in COS-7 cells that predominantly (>90% of total IP(3)R) express IP(3)R3, suggesting that IP(3)R3 functions as an anti-Ca(2+)-oscillatory unit without contributing to peak amplitude of Ca(2+) signals, irrespective of its relative expression level. Therefore, differential expression of the IP(3)R subtype is critical for various forms of Ca(2+) signaling, and, particularly, IP(3)R1 and IP(3)R3 have opposite roles in generating Ca(2+) oscillations.     

An inositol 1,4,5-trisphosphate receptor-dependent cation entry pathway in DT40 B lymphocytes

We examined the roles of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) in calcium signaling using DT40 B lymphocytes, and a variant lacking the three IP3R isoforms (IP3R-KO). In wild-type cells, B cell receptor (BCR) stimulation activates a cation entry route that exhibits significantly greater permeability to Ba2+ than does capacitative calcium entry. This cation entry is absent in IP3R-KO cells. Expression of the type-3 IP3R (IP3R-3) in the IP3R-KO cells rescued not only agonist-dependent release of intracellular Ca2+, but also Ba2+ influx following receptor stimulation. Similar results were obtained with an IP3R-3 mutant carrying a conservative point mutation in the selectivity filter region of the channel (D2477E); however, an IP3R-3 mutant in which this same aspartate was replaced by alanine (D2477A) failed to restore either BCR-induced Ca2+ release or receptor-dependent Ba2+ entry. These results suggest that in DT40 B lymphocytes, BCR stimulation activates a novel cation entry across the plasma membrane that depends upon, or is mediated by, fully functional IP3R.     

IP3-independent signalling of OX1 orexin/hypocretin receptors to Ca2+ influx and ERK

OX1 orexin receptors (OX1R) have been shown to activate receptor-operated Ca2+ influx pathways as their primary signalling pathway; however, investigations are hampered by the fact that orexin receptors also couple to phospholipase C, and therewith inositol-1,4,5-trisphosphate (IP3)-dependent Ca2+ release. We have here devised a method to block the latter signalling in order to focus on the mechanism of Ca2+ influx activation by OX1R in recombinant systems. Transient expression of the IP3-metabolising enzymes IP3-3-kinase-A (inositol-1,4,5-trisphosphate-->inositol-1,3,4,5-tetrakisphosphate) and type I IP3-5-phosphatase (inositol-1,4,5-trisphosphate-->inositol-1,4-bisphosphate) almost completely attenuated the OX1R-stimulated IP3 elevation and Ca2+ release from intracellular stores. Upon attenuation of the IP3-dependent signalling, the receptor-operated Ca2+ influx pathway became the only source for Ca2+ elevation, enabling mechanistic studies on the receptor-channel coupling. Attenuation of the IP3 elevation did not affect the OX1R-mediated ERK (extracellular signal-regulated kinase) activation in CHO cells, which supports our previous finding of the major importance of receptor-operated Ca2+ influx for this response.     

Calcium sensing receptor regulates cardiomyocyte function through nuclear calcium

Nuclear Ca2+ plays a pivotal role in the regulation of gene expression. IP3 (inositol-1,4,5-trisphosphate) is an important regulator of nuclear Ca2+. We hypothesized that the CaR (calcium sensing receptor) stimulates nuclear Ca2+ release through IICR (IP3-induced calcium release) from perinuclear stores. Spontaneous Ca2+ oscillations and the spark frequency of nuclear Ca2+ were measured simultaneously in NRVMs (neonatal rat ventricular myocytes) using confocal imaging. CaR-induced nuclear Ca2+ release through IICR was abolished by inhibition of CaR and IP3Rs (IP3 receptors). However, no effect on the inhibition of RyRs (ryanodine receptors) was detected. The results suggest that CaR specifically modulates nuclear Ca2+ signalling through the IP3R pathway. Interestingly, nuclear Ca2+ was released from perinuclear stores by CaR activator-induced cardiomyocyte hypertrophy through the Ca2+-dependent phosphatase CaN (calcineurin)/NFAT (nuclear factor of activated T-cells) pathway. We have also demonstrated that the activation of the CaR increased the NRVM protein content, enlarged cell size and stimulated CaN expression and NFAT nuclear translocation in NRVMs. Thus, CaR enhances the nuclear Ca2+ transient in NRVMs by increasing fractional Ca2+ release from perinuclear stores, which is involved in cardiac hypertrophy through the CaN/NFAT pathway.     

Interplay between ryanodine and IP3 receptors in ATP-stimulated mouse luteinized-granulosa cells

In mouse luteinized-granulosa cells (MGLC), ATP induces an increase in intracellular Ca2+ concentration by stimulating phospholipase C (PLC) associated with purinergic receptors, leading to production of inositol 1,4,5-trisphosphate (IP3) and subsequent release of Ca2+ from intracellular stores. In this study, we examined the cross-talk between the ryanodine receptors (RyR) and IP3 receptors (IP3R) in response to ATP in MGLC. Specifically, the effect of RyR modulators on ATP response was examined. The results showed that ATP-induced intracellular calcium elevation was abolished by inhibitors of the RyR, such as dantrolene (25 microM) and ryanodine (80 microM). When the MGLC were stimulated with activators of RyR, 2 microM ryanodine and 10 mM caffeine, the ATP-elicited response was decreased. These actions were independent of IP3 production stimulated by ATP. Hence, ATP-induced intracellular Ca2+ mobilization involves the coordinated action of both types of calcium release channels (CRCs). Using fluorescent probes, it was shown that IP3R is uniformly distributed throughout the cell; in contrast, RyR is mainly found around the nuclei. It is concluded that the IP3R and the RyR are functionally associated, and both play a role in the pattern of Ca2+ increase observed during purinergic stimulation of MGLC. This coupling may provide a highly efficient amplification mechanism for ATP stimulation of Ca2+ mobilization.     

Adenine-nucleotide binding sites on the inositol 1,4,5-trisphosphate receptor bind caffeine, but not adenophostin A or cyclic ADP-ribose.

Binding of ATP to the inositol 1,4,5-trisphosphate receptor (IP3R) results in a more pronounced Ca2+ release in the presence of inositol 1,4,5-trisphosphate (IP3). We have expressed the cDNAs encoding two putative adenine-nucleotide binding sites of the neuronal form of IP3R-1 as glutathione S-transferase (GST)-fusion proteins in bacteria. Specific [alpha-32P]ATP binding was observed for the two GST-fusion proteins, representing aa 1710-1850 and aa 1944-2040 of IP3R-1. The ATP-binding sites in both fusion proteins had the same nucleotide specificity as found for the intact IP3R (ATP > ADP > AMP > GTP). Smaller GST-fusion proteins (aa 1745-1792 and aa 2005-2023) displayed a much weaker ATP-binding activity. CoA, which also potentiated IP3-induced Ca2+ release in A7r5 cells, interacted with the ATP-binding sites on the fusion proteins. Such interaction was not observed for 1,N6-etheno CoA and 3'-dephospho-CoA, which are much less effective in potentiating IP3-induced Ca2+ release. Since the adenine-containing compounds adenophostin A, caffeine and cyclic ADP-ribose modulate IP3-induced Ca2+ release, a possible effect of these compounds on the ATP-binding sites was examined. ATP stimulated adenophostin A- and IP3-induced Ca2+ release in A7r5 cells with an EC50 of respectively 21 and 20 microM. Also the threshold concentration of ATP for stimulating the release was similar for the two agonists. Adenophostin A (100 microM) and cyclic ADP-ribose (100 microM) were ineffective in displacing [alpha-32P]ATP from the binding sites of both GST-fusion proteins. Caffeine (50 mM), however, inhibited [alpha-32P]ATP binding to both fusion proteins by more than 50%. These data provide evidence for a direct interaction of caffeine but not of adenophostin A or cyclic ADP-ribose with the adenine-nucleotide binding sites of the IP3R.     

Differential modulation of inositol 1,4,5-trisphosphate receptor type 1 and type 3 by ATP

Binding of ATP to the inositol 1,4,5-trisphosphate receptor (IP(3)R) results in a more pronounced Ca(2+)release in the presence of inositol 1,4,5-trisphosphate (IP(3)). Two recently published studies demonstrated a different ATP sensitivity of IP(3)-induced Ca(2+)release in cell types expressing different IP(3)R isoforms. Cell types expressing mainly IP(3)R3 were less sensitive to ATP than cell types expressing mainly IP(3)R1 (Missiaen L, Parys JB, Sienaert I et al. Functional properties of the type 3 InsP(3)receptor in 16HBE14o- bronchial mucosal cells. J Biol Chem 1998;273: 8983-8986; Miyakawa T, Maeda A, Yamazawa T et al. Encoding of Ca(2+)signals by differential expression of IP(3)receptor subtypes. EMBO J 1999;18: 1303-1308). In order to investigate the difference in ATP sensitivity between IP(3)R isoforms at the molecular level, microsomes of Sf9 insect cells expressing full-size IP(3)R1 or IP(3)R3 were covalently labeled with ATP by using the photoaffinity label 8-azido[alpha-(32)P]ATP. ATP labeling of the IP(3)R was measured after immunoprecipitation of IP(3)Rs with isoform-specific antibodies, SDS-PAGE and Phosphorimaging. Unlabeled ATP inhibited covalent linking of 8-azido[alpha-(32)P]ATP to the recombinant IP(3)R1 and IP(3)R3 with an IC(50)of 1.6 microM and 177 microM, respectively. MgATP was as effective as ATP in displacing 8-azido[alpha-(32)P]ATP from the ATP-binding sites on IP(3)R1 and IP(3)R3, and in stimulating IP(3)-induced Ca(2+)release from permeabilized A7r5 and 16HBE14o- cells. The interaction of ATP with the ATP-binding sites on IP(3)R1 and IP(3)R3 was different from its interaction with the IP(3)-binding domains, since ATP inhibited IP(3)binding to the N-terminal 581 amino acids of IP(3)R1 and IP(3)R3 with an IC(50)of 353 microM and 4.0 mM, respectively. The ATP-binding sites of IP(3)R1 bound much better ATP than ADP, AMP and particularly GTP, while IP(3)R3 displayed a much broader nucleotide specificity. These results therefore provide molecular evidence for a differential regulation of IP(3)R1 and IP(3)R3 by ATP.     

ATP binding to a unique site in the type-1 S2- inositol 1,4,5-trisphosphate receptor defines susceptibility to phosphorylation by protein kinase A

The subtype- and splice variant-specific modulation of inositol 1,4,5-trisphosphate receptors (InsP3R) by interaction with cellular factors plays a fundamental role in defining the characteristics of Ca2+ release in individual cell types. In this study, we investigate the binding properties and functional consequences of the expression of a putative nucleotide binding fold (referred to as the ATPC site) unique to the S2- splice variant of the type-1 InsP3R (InsP3R-1), the predominant splice variant in peripheral tissue. A glutathione S-transferase fusion protein encompassing amino acids 1574-1765 of the S2- InsP3R-1 and including the glycine-rich motif Gly-Tyr-Gly-Glu-Lys-Gly bound ATP specifically as measured by fluorescent trinitrophenyl-ATP binding. This binding was completely abrogated by a point mutation (G1690A) in the nucleotide binding fold. The functional sensitivity of S2- InsP3R-1 constructs was evaluated in DT40-3KO-M3 cells, a null background for InsP3R, engineered to express muscarinic M3 receptors. The S2- InsP3R-1 containing the G1690A mutation was markedly less sensitive to agonist stimulation than wild type S2- InsP3R-1 or receptors containing a similar (Gly --> Ala) mutation in the established nucleotide binding sites in InsP3R-1 (the ATPA and ATPB sites). The ATP sensitivity of InsP3-induced Ca2+ release, however, was not altered by the G1690A mutation when measured in permeabilized DT40-3KO cells, suggesting a unique role for the ATPC site. Ca2+ release was dramatically potentiated following activation of cAMP-dependent protein kinase in DT40-3KO cells transiently expressing wild type S2- InsP3R or Gly --> Ala mutations in the ATPA and ATPB sites, but phosphorylation of the receptor and the potentiation of Ca2+ release were absent in cells expressing the G1690A mutation in S2- InsP3R. These data indicate that ATP binding specifically to the ATPC site in S2- InsP3R-1 controls the susceptibility of the receptor to protein kinase A-mediated phosphorylation, contributes to the functional sensitivity of the S2- InsP3R-1 and ultimately the sensitivity of cells to agonist stimulation.     

Minimal requirements for calcium oscillations driven by the IP3 receptor.

Hormones and neurotransmitters that act through inositol 1,4,5-trisphosphate (IP3) can induce oscillations of cytosolic Ca2+ ([Ca2+]c), which render dynamic regulation of intracellular targets. Imaging of fluorescent Ca2+ indicators located within intracellular Ca2+ stores was used to monitor IP3 receptor channel (IP3R) function and to demonstrate that IP3-dependent oscillations of Ca2+ release and re-uptake can be reproduced in single permeabilized hepatocytes. This system was used to define the minimum essential components of the oscillation mechanism. With IP3 clamped at a submaximal concentration, coordinated cycles of IP3R activation and subsequent inactivation were observed in each cell. Cycling between these states was dependent on feedback effects of released Ca2+ and the ensuing [Ca2+]c increase, but did not require Ca2+ re-accumulation. [Ca2+]c can act at distinct stimulatory and inhibitory sites on the IP3R, but whereas the Ca2+ release phase was driven by a Ca2+-induced increase in IP3 sensitivity, Ca2+ release could be terminated by intrinsic inactivation after IP3 bound to the Ca2+-sensitized IP3R without occupation of the inhibitory Ca2+-binding site. These findings were confirmed using Sr2+, which only interacts with the stimulatory site. Moreover, vasopressin induced Sr2+ oscillations in intact cells in which intracellular Ca2+ was completely replaced with Sr2+. Thus, [Ca2+]c oscillations can be driven by a coupled process of Ca2+-induced activation and obligatory intrinsic inactivation of the Ca2+-sensitized state of the IP3R, without a requirement for occupation of the inhibitory Ca2+-binding site.     

The Pro335 --> Leu polymorphism of type 3 inositol 1,4,5-trisphosphate receptor found in mouse inbred lines results in functional change

Inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular Ca2+ channel involved in various cellular signaling. Type 3 IP3R (IP3R3) retains ligand-gated Ca2+ channel properties differing from other subtypes in terms of IP3-binding affinity and regulation of its channel activity by effector molecules. In this study, we found the natural Pro335 --> Leu polymorphism of mouse IP3R3 between BALB/c and C57BL/6J. We investigated the functional differences between Pro335IP3R3 and Leu335IP3R3 with purified receptors reconstituted into proteoliposomes as well as with soluble ligand binding domains. Pro335IP3R3 exhibited significantly higher IP3-binding affinity and IP3-induced Ca2+ release than those of Leu335IP3R3 in both forms of the receptor. Moreover, the polymorphic change caused differences in the effect of external Ca2+ on IP3-induced Ca2+ release. The Pro335 --> Leu substitution alters the conformation of soluble ligand binding domain as revealed by intrinsic fluorescence and circular dichroism spectra with or without Ca2+. The results indicate that the polymorphism of IP3R3 causes changes in receptor function, presumably affecting intracellular Ca2+ signaling.     

ATP regulation of recombinant type 3 inositol 1,4,5-trisphosphate receptor gating

A family of inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) Ca2+ release channels plays a central role in Ca2+ signaling in most cells, but functional correlates of isoform diversity are unclear. Patch-clamp electrophysiology of endogenous type 1 (X-InsP3R-1) and recombinant rat type 3 InsP3R (r-InsP3R-3) channels in the outer membrane of isolated Xenopus oocyte nuclei indicated that enhanced affinity and reduced cooperativity of Ca2+ activation sites of the InsP3-liganded type 3 channel distinguished the two isoforms. Because Ca2+ activation of type 1 channel was the target of regulation by cytoplasmic ATP free acid concentration ([ATP](i)), here we studied the effects of [ATP]i on the dependence of r-InsP(3)R-3 gating on cytoplasmic free Ca2+ concentration ([Ca2+]i. As [ATP]i was increased from 0 to 0.5 mM, maximum r-InsP3R-3 channel open probability (Po) remained unchanged, whereas the half-maximal activating [Ca2+]i and activation Hill coefficient both decreased continuously, from 800 to 77 nM and from 1.6 to 1, respectively, and the half-maximal inhibitory [Ca2+]i was reduced from 115 to 39 microM. These effects were largely due to effects of ATP on the mean closed channel duration. Whereas the r-InsP3R-3 had a substantially higher Po than X-InsP3R-1 in activating [Ca2+]i (< 1 microM) and 0.5 mM ATP, the Ca2+ dependencies of channel gating of the two isoforms became remarkably similar in the absence of ATP. Our results suggest that ATP binding is responsible for conferring distinct gating properties on the two InsP3R channel isoforms. Possible molecular models to account for the distinct regulation by ATP of the Ca2+ activation properties of the two channel isoforms and the physiological implications of these results are discussed. Complex regulation by ATP of the types 1 and 3 InsP3R channel activities may enable cells to generate sophisticated patterns of Ca2+ signals with cytoplasmic ATP as one of the second messengers.     

Two neuropeptides recruit different messenger pathways to evoke Ca2+ signals in the same cell

Bombesin and cholecystokinin (CCK) peptides act as signalling molecules in both the central nervous system and gastrointestinal tract [1-4]. It was reported recently that nicotinic acid adenine dinucleotide phosphate (NAADP) releases Ca2+ from mammalian brain microsomes [5] and triggers Ca2+ signals in pancreatic acinar cells, where it is proposed to mediate CCK-evoked Ca2+ signals [6]. Here, for the first time, we have finely resolved bombesin-induced cytosolic Ca2+ oscillations in single pancreatic acinar cells by whole-cell patch-clamp monitoring of Ca2+-dependent ionic currents [6-8]. Picomolar concentrations of bombesin and CCK evoked similar patterns of cytosolic Ca2+ oscillations, but high, desensitising, NAADP concentrations selectively inhibited CCK, but not bombesin-evoked signals. Inhibiting inositol trisphosphate (IP3) receptors with a high concentration of caffeine blocked both types of oscillations. We further tested whether NAADP is involved in Ca2+ signals triggered by activation of the low-affinity CCK receptor sites. Nanomolar concentrations of CCK evoked non-oscillatory Ca2+ signals, which were not affected by desensitising NAADP receptors. Our results suggest that Ca2+-release channels gated by the novel Ca2+-mobilising molecule NAADP are only essential in specific Ca2+-mobilising pathways, whereas the IP3 receptors are generally required for Ca2+ signals. Thus, the same cell may use different combinations of intracellular Ca2+-releasing messengers to encode different external messages.     

Homer 2 tunes G protein-coupled receptors stimulus intensity by regulating RGS proteins and PLCbeta GAP activities

Homers are scaffolding proteins that bind G protein-coupled receptors (GPCRs), inositol 1,4,5-triphosphate (IP3) receptors (IP3Rs), ryanodine receptors, and TRP channels. However, their role in Ca2+ signaling in vivo is not known. Characterization of Ca2+ signaling in pancreatic acinar cells from Homer2-/- and Homer3-/- mice showed that Homer 3 has no discernible role in Ca2+ signaling in these cells. In contrast, we found that Homer 2 tunes intensity of Ca2+ signaling by GPCRs to regulate the frequency of [Ca2+]i oscillations. Thus, deletion of Homer 2 increased stimulus intensity by increasing the potency for agonists acting on various GPCRs to activate PLCbeta and evoke Ca2+ release and oscillations. This was not due to aberrant localization of IP3Rs in cellular microdomains or IP3R channel activity. Rather, deletion of Homer 2 reduced the effectiveness of exogenous regulators of G proteins signaling proteins (RGS) to inhibit Ca2+ signaling in vivo. Moreover, Homer 2 preferentially bound to PLCbeta in pancreatic acini and brain extracts and stimulated GAP activity of RGS4 and of PLCbeta in an in vitro reconstitution system, with minimal effect on PLCbeta-mediated PIP2 hydrolysis. These findings describe a novel, unexpected function of Homer proteins, demonstrate that RGS proteins and PLCbeta GAP activities are regulated functions, and provide a molecular mechanism for tuning signal intensity generated by GPCRs and, thus, the characteristics of [Ca2+]i oscillations.     

cAMP-dependent protein kinase enhances inositol 1,4,5-trisphosphate-induced Ca2+ release in AR4-2J cells

In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP(3)R), a ligand-gated Ca(2+) channel, plays an important role in the control of intracellular Ca(2+). There are three subtypes of IP(3)R that are differentially distributed among cell types. AR4-2J cells express almost exclusively the IP(3)R-2 subtype. The purpose of this study was to investigate the effect of cAMP-dependent protein kinase (PKA) on the activity of IP(3)R-2 in AR4-2J cells. We showed that immunoprecipitated IP(3)R-2 is a good substrate for PKA. Using a back-phosphorylation approach, we showed that endogenous PKA phosphorylates IP(3)R-2 in intact AR4-2J cells. Pretreatment with PKA enhanced IP(3)-induced Ca(2+) release in permeabilized AR4-2J cells. Pretreatment with the cAMP generating agent's forskolin and vasoactive intestinal peptide (VIP) enhanced carbachol (Cch)-induced and epidermal growth factor (EGF)-induced Ca(2+) responses in intact AR4-2J cells. Our results are consistent with an enhancing effect of PKA on IP(3)R-2 activity. This conclusion supports the emerging concept of crosstalk between Ca(2+) signaling and cAMP pathways and thus provides another way by which Ca(2+) signals are finely encoded within non-excitable cells.