IRBIT suppresses IP3 receptor activity by competing with IP3 for the common binding site on the IP3 receptor

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

Binding of IRBIT to the IP3 receptor: determinants and functional effects

IRBIT has previously been shown to interact with the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) in an IP3-sensitive way. So far it remained to be elucidated whether this interaction was direct or indirect, and whether it was functionally relevant. We now show that IRBIT can directly interact with the IP3R, and that both the suppressor domain and the IP3-binding core of the IP3R are essential for a strong interaction. Moreover, we identified a PEST motif and a PDZ-ligand on IRBIT which were critical for the interaction with the IP3R. Furthermore, we identified Asp-73 as a critical residue for this interaction. Finally, we demonstrated that this interaction functionally affects the IP3R: IRBIT inhibits both IP3 binding and IP3-induced Ca2+ release.     

Protein phosphatase-1 is a novel regulator of the interaction between IRBIT and the inositol 1,4,5-trisphosphate receptor

IRBIT is an IP3R [IP3 (inositol 1,4,5-trisphosphate) receptor]-binding protein that competes with IP3 for binding to the IP3R. Phosphorylation of IRBIT is essential for the interaction with the IP3R. The unique N-terminal region of IRBIT, residues 1-104 for mouse IRBIT, contains a PEST (Pro-Glu-Ser-Thr) domain with many putative phosphorylation sites. In the present study, we have identified a well-conserved PP1 (protein phosphatase-1)-binding site preceeding this PEST domain which enabled the binding of PP1 to IRBIT both in vitro and in vivo. IRBIT emerged as a mediator of its own dephosphorylation by associated PP1 and, hence, as a novel substrate specifier for PP1. Moreover, IRBIT-associated PP1 specifically dephosphorylated Ser68 of IRBIT. Phosphorylation of Ser68 was required for subsequent phosphorylation of Ser71 and Ser74, but the latter two sites were not targeted by PP1. We found that phosphorylation of Ser71 and Ser74 were sufficient to enable inhibition of IP3 binding to the IP3R by IRBIT. Finally, we have shown that mutational inactivation of the docking site for PP1 on IRBIT increased the affinity of IRBIT for the IP3R. This pinpoints PP1 as a key player in the regulation of IP3R-controlled Ca2+ signals.     

IP3 receptor/Ca2+ channel: from discovery to new signaling concepts

Inositol 1,4,5-trisphosphate (IP(3)) is a second messenger that induces the release of Ca(2+) from the endoplasmic reticulum (ER). The IP(3) receptor (IP(3)R) was discovered as a developmentally regulated glyco-phosphoprotein, P400, that was missing in strains of mutant mice. IP(3)R can allosterically and dynamically change its form in a reversible manner. The crystal structures of the IP(3)-binding core and N-terminal suppressor sequence of IP(3)R have been identified. An IP(3) indicator (known as IP(3)R-based IP(3) sensor) was developed from the IP(3)-binding core. The IP(3)-binding core's affinity to IP(3) is very similar among the three isoforms of IP(3)R; instead, the N-terminal IP(3) binding suppressor region is responsible for isoform-specific IP(3)-binding affinity tuning. Various pathways for the trafficking of IP(3)R have been identified; for example, the ER forms a meshwork upon which IP(3)R moves by lateral diffusion, and vesicular ER subcompartments containing IP(3)R move rapidly along microtubles using a kinesin motor. Furthermore, IP(3)R mRNA within mRNA granules also moves along microtubules. IP(3)Rs are involved in exocrine secretion. ERp44 works as a redox sensor in the ER and regulates IP(3)R1 activity. IP(3) has been found to release Ca(2+), but it also releases IRBIT (IP(3)R-binding protein released with IP(3)). IRBIT is a pseudo-ligand for IP(3) that regulates the frequency and amplitude of Ca(2+) oscillations through IP(3)R. IRBIT binds to pancreas-type Na, bicarbonate co-transporter 1, which is important for acid-base balance. The presence of many kinds of binding partners, like homer, protein 4.1N, huntingtin-associated protein-1A, protein phosphatases (PPI and PP2A), RACK1, ankyrin, chromogranin, carbonic anhydrase-related protein, IRBIT, Na,K-ATPase, and ERp44, suggest that IP(3)Rs form a macro signal complex and function as a center for signaling cascades. The structure of IP(3)R1, as revealed by cryoelectron microscopy, fits closely with these molecules.     

Inositol 1,4,5-trisphosphate receptor type 1 phosphorylation and regulation by extracellular signal-regulated kinase

Type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) is a widely expressed intracellular calcium-release channel found in many cell types. The operation of IP(3)R1 is regulated through phosphorylation by multiple protein kinases. Extracellular signal-regulated kinase (ERK) has been found involved in calcium signaling in distinct cell types, but the underlying mechanisms remain unclear. Here, we present evidence that ERK1/2 and IP(3)R1 bind together through an ERK binding motif in mouse cerebellum in vivo as well as in vitro. ERK-phosphorylating serines (Ser 436) was identified in mouse IP(3)R1 and Ser 436 phosphorylation had a suppressive effect on IP(3) binding to the recombinant N-terminal 604-amino acid residues (N604). Moreover, phosphorylation of Ser 436 in R(224-604) evidently enhance its interaction with the N-terminal "suppressor" region (N223). At last, our data showed that Ser 436 phosphorylation in IP(3)R1 decreased Ca(2+) releasing through IP(3)R1 channels.     

IP(3) receptors: the search for structure

Inositol (1,4,5)-trisphosphate receptors (IP(3)R) are intracellular Ca(2+) channels that are regulated by Ca(2+) and IP(3), and are modulated by many additional signals. They thereby allow both receptors that stimulate IP(3) formation and Ca(2+) to control release of Ca(2+) from intracellular stores. IP(3)Rs share many features with their close relatives, ryanodine receptors; each provides insight into the structure and function of the other. The structural basis of IP(3)R behaviour is beginning to emerge from intermediate-resolution structures of the complete IP(3)R, a 2.2-A structure of the IP(3)-binding core and comparisons with the pore structures of other tetrameric cation channels. The binding of IP(3) to a site towards the N-terminal of each IP(3)R subunit promotes binding of Ca(2+). This destabilizes an inhibitory interaction between N-terminal residues and a C-terminal 'gatekeeper' sequence, enabling the pore to open.     

Structural insights into the regulatory mechanism of IP3 receptor

Inositol 1,4,5-trisphosphate receptors (IP(3)R) are intracellular Ca(2+) release channels whose opening requires binding of two intracellular messengers IP(3) and Ca(2+). The regulation of IP(3)R function has also been shown to involve a variety of cellular proteins. Recent biochemical and structural analyses have deepened our understanding of how the IP(3)-operated Ca(2+) channel functions. Specifically, the atomic resolution structure of the IP(3)-binding region has provided a sound structural basis for the receptor interaction with the natural ligand. Electron microscopic studies have also shed light on the overall shape of the tetrameric receptor. This review aims to provide comprehensive overview of the current information available on the structure and function relationship of IP(3)R.     

80K-H interacts with inositol 1,4,5-trisphosphate (IP3) receptors and regulates IP3-induced calcium release activity

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular channel proteins that mediate calcium (Ca2+) release from the endoplasmic reticulum, and they are involved in many biological processes (e.g. fertilization, secretion, and synaptic plasticity). Recent reports show that IP3R activity is strictly regulated by several interacting molecules (e.g. IP3R binding protein released with inositol 1,4,5-trisphosphate, huntingtin, presenilin, DANGER, and cytochrome c), and perturbation of this regulation causes intracellular Ca2+ elevation leading to several diseases (e.g. Huntington disease and Alzheimer disease). In this study, we identified protein kinase C substrate 80K-H (80K-H) to be a novel molecule interacting with the COOH-terminal tail of IP3Rs by yeast two-hybrid screening. 80K-H directly interacted with IP3R type 1 (IP3R1) in vitro and co-immunoprecipitated with IP3R1 in cell lysates. Immunocytochemical and immunohistochemical staining revealed that 80K-H colocalized with IP3R1 in COS-7 cells and in hippocampal neurons. We also showed that the purified recombinant 80K-H protein directly enhanced IP3-induced Ca2+ release activity by a Ca2+ release assay using mouse cerebellar microsomes. Furthermore 80K-H was found to regulate ATP-induced Ca2+ release in living cells. Thus, our findings suggest that 80K-H is a novel regulator of IP3R activity, and it may contribute to neuronal functions.     

Mechanism of ER stress-induced brain damage by IP(3) receptor

Deranged Ca(2+) signaling and an accumulation of aberrant proteins cause endoplasmic reticulum (ER) stress, which is a hallmark of cell death implicated in many neurodegenerative diseases. However, the underlying mechanisms are elusive. Here, we report that dysfunction of an ER-resident Ca(2+) channel, inositol 1,4,5-trisphosphate receptor (IP(3)R), promotes cell death during ER stress. Heterozygous knockout of brain-dominant type1 IP(3)R (IP(3)R1) resulted in neuronal vulnerability to ER stress in?vivo, and IP(3)R1 knockdown enhanced ER stress-induced apoptosis via mitochondria in cultured cells. The IP(3)R1 tetrameric assembly was positively regulated by the ER chaperone GRP78 in an energy-dependent manner. ER stress induced IP(3)R1 dysfunction through an impaired IP(3)R1-GRP78 interaction, which has also been observed in the brain of Huntington's disease model mice. These results suggest that IP(3)R1 senses ER stress through GRP78 to alter the Ca(2+) signal to promote neuronal cell death implicated in neurodegenerative diseases.     

The IP3 receptor/Ca2+ channel and its cellular function

The IP3R [IP3 (inositol 1,4,5-trisphosphate) receptor] is responsible for Ca2+ release from the ER (endoplasmic reticulum). We have been working extensively on the P400 protein, which is deficient in Purkinje-neuron-degenerating mutant mice. We have discovered that P400 is an IP3R and we have determined the primary sequence. Purified IP3R, when incorporated into a lipid bilayer, works as a Ca2+ release channel and overexpression of IP3R shows enhanced IP3 binding and channel activity. Addition of an antibody blocks Ca2+ oscillations indicating that IP3R1 works as a Ca2+ oscillator. Studies on the role of IP3R during development show that IP3R is involved in fertilization and is essential for determination of dorso-ventral axis formation. We found that IP3R is involved in neuronal plasticity. A double homozygous mutant of IP3R2 (IP3R type 2) and IP3R3 (IP3R type 3) shows a deficit of saliva secretion and gastric juice secretion suggesting that IP3Rs are essential for exocrine secretion. IP3R has various unique properties: cryo-EM (electron microscopy) studies show that IP3R contains multiple cavities; IP3R allosterically and dynamically changes its form reversibly (square form-windmill form); IP3R is functional even though it is fragmented by proteases into several pieces; the ER forms a meshwork but also forms vesicular ER and moves along microtubules using a kinesin motor; X ray analysis of the crystal structure of the IP3 binding core consists of an N-terminal beta-trefoil domain and a C-terminal alpha-helical domain. We have discovered ERp44 as a redox sensor in the ER which binds to the luminal part of IP3R1 and regulates its activity. We have also found the role of IP3 is not only to release Ca2+ but also to release IRBIT which binds to the IP3 binding core of IP3R.     

A novel recombinant hyperaffinity inositol 1,4,5-trisphosphate (IP(3)) absorbent traps IP(3), resulting in specific inhibition of IP(3)-mediated calcium signaling.

We have developed a novel recombinant hyperaffinity inositol 1,4,5-trisphosphate (IP(3)) absorbent, called the "IP(3) sponge," which we constructed on the basis of the ligand-binding site of the mouse type 1 IP(3) receptor (IP(3)R1). The IP(3) sponge exhibited approximately 1000-fold higher affinity for IP(3) than the parental IP(3)R1 and specifically competed with the endogenous IP(3)R for binding to IP(3). Trapping IP(3) with the IP(3) sponge inhibited IP(3)-induced Ca(2+) release (IICR) from cerebellar microsomes in a dose-dependent manner. The IP(3) sponge expressed in HEK293 cells also inhibited IICR in response to stimulation with carbachol or ATP. Its inhibitory effects were dependent upon the level of its expression over the increased IP(3) contents. Moreover, the IP(3) sponge significantly reduced the carbachol-induced phosphorylation of cAMP-response element-binding protein in HEK293 cells, indicating that the activation of cAMP-response element-binding protein by Ca(2+)-dependent phosphorylation may be partly attributable to IICR.     

Inositol 1,4,5-trisphosphate IP(3) receptors and their role in neuronal cell function

Inositol 1,4,5-trisphosphate (IP(3)) receptor is a Ca(2+) release channel localized on the endoplasmic reticulum (ER) and plays an important role in neuronal function. IP(3) receptor was discovered as a developmentally regulated protein missing in the cerebellar mutant mice. Recent studies indicate that IP(3)Rs are involved in early development and neuronal plasticity. IP(3) works to release IRBIT from the IP(3) binding core in addition to release Ca(2+). IRBIT binds to and activates Na, Bicarbonate cotransporter. Electron microscopic study show the IP(3) receptor has allosteric property to change its form from square to windmill in the presence of Ca(2+). IP(3)R associates with ERp44, a redox sensor, Homer, other proteins and is transported as vesicular ER on microtubules. All these data suggests IP(3) receptor/CA(2+) channel works as a signaling center inside cells.     

Interaction of chromogranin B and the near N-terminal region of chromogranin B with an intraluminal loop peptide of the inositol 1,4, 5-trisphosphate receptor

Given the interaction of the inositol 1,4,5-trisphosphate receptor (IP(3)R) with chromogranins A (CGA) and B (CGB), two major Ca(2+) storage proteins of secretory granules that have been shown to be IP(3)-sensitive intracellular Ca(2+) store of neuroendocrine cells, we have investigated the potential interaction of the intraluminal loop regions of the IP(3)R with both intact CGB and the conserved near N-terminal region of CGB. The interaction studies carried out with CGB and glutathione S-transferase fusion proteins of intraluminal loop regions of bovine type 1 IP(3)R showed that CGB interacts with intraluminal loop 3-2 (the second loop formed between transmembrane regions 5 and 6) of the IP(3)R at both pH 5.5 and 7.5. Analytical ultracentrifugation studies also indicated that CGB interacts with the same intraluminal loop region of the IP(3)R and the interaction was much stronger than that between CGA and the loop. Moreover, the conserved near N-terminal region of CGB also interacted with the intraluminal loop region of the IP(3)R. The CGB interaction with the IP(3)R intraluminal loop peptide at pH 7.5 showed a DeltaG(0) value of -8.1 kcal/mol at 37 degrees C for a 1:1 stoichiometry, indicating a K(d) of approximately 1.9 micrometer. These results give insight into the molecular organization of the IP(3)-sensitive Ca(2+) store.     

ERK binds, phosphorylates InsP3 type 1 receptor and regulates intracellular calcium dynamics in DT40 cells

Modulation on the duration of intracellular Ca(2+) transients is essential for B-cell activation. We have previously shown that extracellular-signal-regulated kinase (ERK) can phosphorylate inositol 1,4,5-trisphosphate receptor type 1 (IP(3)R1) at serine 436 and regulate its calcium channel activity. Here we investigate the potential physiological interaction between ERK and IP(3)R1 using chicken DT40 B-cell line in which different mutants are expressed. The interaction between ERK and IP(3)R1 is confirmed by co-immunoprecipitation and fluorescence resonance energy transfer (FRET) assays. This constitutive interaction is independent of either ERK kinase activation or IP(3)R1 phosphorylation status. Back phosphorylation analysis further shows that type 1 IP(3)R (IP(3)R1) is phosphorylated by ERK in anti-IgM-activated DT40 cells. Finally, our data show that the phosphorylation of Ser 436 in the IP(3)-binding domain of IP(3)R1 leads to less Ca(2+) release from endoplasmic reticulum (ER) microsomes and accelerates the declining of calcium increase in DT40 cells in response to anti-IgM stimulation.     

Real time analysis of interaction between inositol 1,4, 5-trisphosphate receptor type I and its ligand.

Inositol 1,4,5-trisphosphate (IP(3)) is an important second messenger that releases intracellular Ca(2+) by binding to its specific receptor, inositol 1,4,5-trisphosphate receptor (IP(3)R), in a wide range of cellular processes. We report here large-scale expression and purification of N-terminal 604 amino acids of IP(3)R type 1 (T604) expressed in E. coli, which contains the ligand binding domain. Surface plasmon resonance biosensor studies showed that purified T604 could bind to its ligands with binding specificity identical to that of full-length native IP(3)R type 1. Kinetic parameters of T604 for IP(3) consisted of a fast association rate constant (K(ass) = 1.2 x 10(6) M(-1) s(-1)) and a rapid dissociation rate constant (k(diss) = 1 s(-1)), and the equilibrium dissociation constant was determined to be 336 nM, at 150 mM NaCl and pH 7.4. However, association and dissociation patterns depended on the pH level and ionic strength. These results pave the way toward detail analysis of structure-function analysis of the ligand binding domain of IP(3)R type 1 for its ligands.     

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.     

Regulation of the phosphorylation of the inositol 1,4,5-trisphosphate receptor by protein kinase C

The various inositol 1,4,5-trisphosphate receptor (IP(3)R) isoforms are potential substrates for several protein kinases. We compared the in vitro phosphorylation of purified IP(3)R1 and IP(3)R3 by the catalytic subunit of protein kinase C (PKC). Phosphorylation of IP(3)R1 by PKC was about eight times stronger than that of IP(3)R3 under identical conditions. Protein kinase A strongly stimulated the PKC-induced phosphorylation of IP(3)R1. In contrast, Ca(2+) inhibited its phosphorylation (IC(50)相似文献   

Protein kinase A and two phosphatases are components of the inositol 1,4,5-trisphosphate receptor macromolecular signaling complex

The inositol 1,4,5-trisphosphate receptor (IP3R) is a ubiquitously expressed intracellular calcium (Ca(2+)) release channel on the endoplasmic reticulum. IP3Rs play key roles in controlling Ca(2+) signals that activate numerous cellular functions including T cell activation, neurotransmitter release, oocyte fertilization and apoptosis. There are three forms of IP3R, all of which are ligand-gated channels activated by the second messenger inositol 1,4,5-trisphosphate. Channel function is modulated via cross-talk with other signaling pathways including those mediated by kinases and phosphatases. In particular IP3Rs are known to be regulated by cAMP-dependent protein kinase (PKA) phosphorylation. In the present study we show that PKA and the protein phosphatases PP1 and PP2A are components of the IP3R1 macromolecular signaling complex. PKA phosphorylation of IP3R1 increases channel activity in planar lipid bilayers. These studies indicate that regulation of IP3R1 function via PKA phosphorylation involves components of a macromolecular signaling complex.     

Human inositol 1,4,5-trisphosphate 3-kinase isoform B (IP3KB) is a nucleocytoplasmic shuttling protein specifically enriched at cortical actin filaments and at invaginations of the nuclear envelope

Recent studies have shown that inositol 1,4,5-trisphosphate 3-kinase isoform B (IP3KB) possesses important roles in the development of immune cells. IP3KB can be targeted to multiple cellular compartments, among them nuclear localization and binding in close proximity to the plasma membrane. The B isoform is the only IP3K that is almost ubiquitously expressed in mammalian cells. Detailed mechanisms of its targeting regulation will be important in understanding the role of Ins(1,4,5)P(3) phosphorylation on subcellular calcium signaling and compartment-specific initiation of pathways leading to regulatory active higher phosphorylated inositol phosphates. Here, we identified an exportin 1-dependent nuclear export signal ((134)LQRELQNVQV) and characterized the amino acids responsible for nuclear localization of IP3KB ((129)RKLR). These two targeting domains regulate the amount of nuclear IP3KB in cells. We also demonstrated that the localization of IP3KB at the plasma membrane is due to its binding to cortical actin structures. Intriguingly, all three of these targeting activities reside in one small polypeptide segment (amino acids 104-165), which acts as a multitargeting domain (MTD). Finally, a hitherto unknown subnuclear localization of IP3KB could be demonstrated in rapidly growing H1299 cells. IP3KB is specifically enriched at nuclear invaginations extending perpendicular between the apical and basal surface of the nucleus of these flat cells. Such nuclear invaginations are known to be involved in Ins(1,4,5)P(3)-mediated Ca(2+) signaling of the nucleus. Our findings indicate that IP3KB not only regulates cytoplasmic Ca(2+) signals by phosphorylation of subplasmalemmal and cytoplasmic Ins(1,4,5)P(3) but may also be involved in modulating nuclear Ca(2+) signals generated from these nuclear envelope invaginations.     

Mapping of the ATP-binding sites on inositol 1,4,5-trisphosphate receptor type 1 and type 3 homotetramers by controlled proteolysis and photoaffinity labeling

Submillimolar ATP concentrations strongly enhance the inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release, by binding specifically to ATP-binding sites on the IP(3) receptor (IP(3)R). To locate those ATP-binding sites on IP(3)R1 and IP(3)R3, both proteins were expressed in Sf9 insect cells and covalently labeled with 8-azido-[alpha-(32)P]ATP. IP(3)R1 and IP(3)R3 were then purified and subjected to a controlled proteolysis, and the labeled proteolytic fragments were identified by site-specific antibodies. Two fragments of IP(3)R1 were labeled, each containing one of the previously proposed ATP-binding sites with amino acid sequence GXGXXG (amino acids 1773-1780 and 2016-2021, respectively). In IP(3)R3, only one fragment was labeled. This fragment contained the GXGXXG sequence (amino acids 1920-1925), which is conserved in the three IP(3)R isoforms. The presence of multiple interaction sites for ATP was also evident from the IP(3)-induced Ca(2+) release in permeabilized A7r5 cells, which depended on ATP over a very broad concentration range from micromolar to millimolar.