Specific binding of tritium-labeled inositol 1,4,5-trisphosphate to human platelet membranes: ionic and GTP regulation.

Specific, saturable and reversible binding of tritium-labeled inositol 1,4,5-trisphosphate [( 3H]Ins(1,4,5)P3) to human platelet membranes is demonstrated. The Ins(1,4,5)P3-binding sites are abundant and display high selectivity for Ins(1,4,5)P3. Other inositol phosphates exhibit much lower affinity for this site. The specific [3H]Ins(1,4,5)P3 binding was found to be modulated by pH, monovalent and divalent cations, and GTP. A sharp increase in binding occurs at slightly alkaline pH. The monovalent cations, Na+, K+ and Li+ almost double the binding at 30 mM. Mg2+ inhibits the specific [3H]Ins(1,4,5)P3 binding. At low concentrations of Ca2+, the binding is inhibited, but at concentrations higher than 5 mM the binding is potentiated and increases by almost 5-fold at 100 mM. Similar pattern of the effects is also observed for Mn2+ and Sr2+. The specific [3H]Ins(1,4,5)P3 binding is specifically inhibited by GTP. Other nucleotides also inhibit the binding but at higher concentrations. From saturation binding studies, Ca2+ potentiation seems to be due to the conversion of the receptor from the low-affinity state to the high-affinity one. In the absence of Ca2+, the Scatchard plot is nonlinear and concave, and statistically can be fitted best with two equilibrium dissociation constants (Kd values), 0.19 +/- 0.11 and 13.2 +/- 18.1 nM, respectively, for high- and low-affinity binding sites. However, in the presence of 100 mM CaCl2, the Scatchard plot reveals only the high-affinity binding sites with a Kd value of 0.32 +/- 0.15 nM. The specific Ins(1,4,5)P3 receptor in human platelets could therefore exist in multiple conformational states to regulate the intracellular Ca2+ concentration.     

Modulation of inositol 1,4,5-trisphosphate binding to the recombinant ligand-binding site of the type-1 inositol 1,4, 5-trisphosphate receptor by Ca2+ and calmodulin

A recombinant protein (Lbs-1) containing the N-terminal 581 amino acids of the mouse type 1 inositol 1,4,5-trisphosphate receptor (IP3R-1), including the complete IP3-binding site, was expressed in the soluble fraction of E. coli. The characteristics of IP3 binding to this protein were similar as observed previously for the intact IP3R-1. Ca2+ dose-dependently inhibited IP3 binding to Lbs-1 with an IC50 of about 200 nM. This effect represented a decrease in the affinity of Lbs-1 for IP3, because the Kd increased from 115 +/- 15 nM in the absence to 196 +/- 18 nM in the presence of 5 microM Ca2+. The maximal effect of Ca2+ on Lbs-1 (5 microM Ca2+, 42.0 +/- 6.4% inhibition) was similar to the maximal inhibition observed for microsomes of insect Sf9 cells expressing full-length IP3R-1 (33.8 +/- 10.2%). Conceivably, the two contiguous Ca2+-binding sites (residues 304-450 of mouse IP3R-1) previously found by us (Sienaert, I., Missiaen, L., De Smedt, H., Parys, J.B., Sipma, H., and Casteels, R. (1997) J. Biol. Chem. 272, 25899-25906) mediate the effect of Ca2+ on IP3 binding to IP3R-1. Calmodulin also dose-dependently inhibited IP3 binding to Lbs-1 with an IC50 of about 3 microM. Maximal inhibition (10 microM calmodulin, 43.1 +/- 5.9%) was similar as observed for Sf9-IP3R-1 microsomes (35.8 +/- 8.7%). Inhibition by calmodulin occurred independently of Ca2+ and was additive to the inhibitory effect of 5 microM Ca2+ (together 74.5 +/- 5.1%). These results suggest that the N-terminal ligand-binding region of IP3R-1 contains a calmodulin-binding domain that binds calmodulin independently of Ca2+ and that mediates the inhibition of IP3 binding to IP3R-1.     

Hydrophobic modifications at 1-phosphate of inositol 1,4,5-trisphosphate analogues enhance receptor binding

Inositol 1,4,5-trisphosphate (IP(3)) analogues were synthesized in order to investigate the importance of the environment of 1-phosphate of IP(3) for strong binding to the IP(3) receptor. Our results show that hydrophobic modifications of the 1-phosphate moiety enhance the binding affinity, with considerable latitude of substituent structure.     

Characterization of a membrane protein from brain mediating the inhibition of inositol 1,4,5-trisphosphate receptor binding by calcium.

Inositol 1,4,5-trisphosphate (InsP3) is a component of the phosphoinositide second-messenger system which mobilizes Ca2+ from intracellular stores. Recently, an InsP3 receptor binding protein from rat cerebellar membranes was solubilized and purified to homogeneity. The potent inhibition by Ca2+ of [3H]InsP3 binding to the InsP3 receptor in cellular membranes is not apparent in the purified receptor. The Ca2+-dependent inhibition of [3H]InsP3 binding in the crude homogenate (concn. giving 50% inhibition = 300 nM) can be restored by addition of solubilized cerebellar membranes to the purified receptor. In the present study, we further characterize the protein in solubilized membranes which confers Ca2+-sensitivity to the receptor, and which we term 'calmedin'. Calmedin appears to be a neutral membrane protein with an estimated Mr of 300,000 by gel filtration in the presence of Triton X-100. Calmedin confers a Ca2+-sensitivity to InsP3 receptor binding, which can be completely reversed by 10 min incubation with EDTA and therefore does not represent Ca2+-dependent proteinase action. Calmedin effects on the purified InsP3 receptor depend on Ca2+ binding to the calmedin, although Ca2+ also binds directly to the InsP3 receptor. The regional distribution of calmedin differs from that of the InsP3 receptor in the brain, suggesting that it also mediates other Ca2+-dependent functions. Calmedin activity in peripheral tissues is much lower than in brain.     

Chronic muscarinic stimulation of SH-SY5Y neuroblastoma cells suppresses inositol 1,4,5-trisphosphate action. Parallel inhibition of inositol 1,4,5-trisphosphate-induced Ca2+ mobilization and inositol 1,4,5-trisphosphate binding.

The possibility that chronic activation of the phosphoinositide-mediated signaling pathway modifies the Ca(2+)-mobilizing action of inositol 1,4,5-trisphosphate (InsP3) was examined. SH-SY5Y human neuroblastoma cells were exposed to carbachol, permeabilized electrically, loaded with 45Ca2+, and 45Ca2+ mobilization in response to exogenous InsP3 was assessed. In control permeabilized cells, InsP3 released 65 +/- 2% of sequestered 45Ca2+ (EC50 = 0.32 +/- 0.05 microM). Pre-treatment with carbachol reduced both maximal InsP3-induced 45Ca2+ release (to 34 +/- 3%, with half-maximal and maximal inhibition at approximately 3 and 6 h, respectively) and the potency of InsP3 (EC50 = 0.92 +/- 0.13 microM). This inhibitory effect of carbachol was half-maximal at approximately 5 microM, was mediated by muscarinic receptors, and was reversible following withdrawal of agonist. Pretreatment with phorbol 12,13-dibutyrate did not alter the maximal effect of InsP3 but doubled its EC50. Evidence suggesting that the inhibitory effects of carbachol pretreatment resulted from altered Ca2+ homeostasis was not forthcoming; both 45Ca2+ uptake and release induced by ionomycin and thapsigargin were identical in control and pretreated permeabilized cells, as were the characteristics of reuptake of released Ca2+. In contrast, carbachol pretreatment, without altering the affinity of InsP3 (Kd = 64 +/- 7 nM), reduced the density of [32P]InsP3-binding sites from 2.0 +/- 0.1 to 1.0 +/- 0.1 pmol/mg protein with a time course essentially identical to that for the reduction in responsiveness to InsP3. This effect was not mimicked by pretreatment of cells with phorbol 12,13-dibutyrate. These data indicate that chronic activation of phosphoinositide hydrolysis can reduce the abundance of InsP3 receptors and that this causes a reduction in size of the InsP3-sensitive Ca2+ store. This modification, possibly in conjunction with a protein kinase C-mediated event, appears to account for the carbachol-induced suppression of InsP3 action. As intracellular InsP3 mass remained elevated above basal for at least 24 h after addition of carbachol, suppression of the Ca(2+)-mobilizing activity of InsP3 represents an important adaptive response to cell stimulation that can limit the extent to which intracellular Ca2+ is mobilized.     

Different localization of inositol 1,4,5-trisphosphate and ryanodine binding sites in rat liver.

The distribution of inositol 1,4,5-trisphosphate and ryanodine binding sites between plasma membrane, microsomal, and mitochondrial fractions of rat liver were compared. IP3 bound mostly to the plasma membrane fraction (Kd = 6 nM; Bmax = 802 fmol/mg protein). Some IP3 binding sites were also present in the microsomal and mitochondrial fractions (Kd = 2.5 and 2.9 nM; Bmax = 35 and 23 fmol/mg protein respectively). The possibility that these binding sites are due to contamination of the fractions with plasma membrane cannot be excluded. Binding of IP3 to the plasma membrane was inhibited by heparin but not by either caffeine or tetracaine. High-affinity ryanodine binding sites were present mostly in the microsomal fraction (Kd = 13 nM; Bmax = 301 fmol/mg protein). Lower affinity binding sites were also found to be present in the mitochondrial and plasma membrane fractions. Binding of ryanodine to the microsomal fraction was inhibited by both caffeine and tetracaine but not by heparin. These data demonstrate that IP3 and ryanodine binding sites are present in different cellular compartments in the liver. These differences in the localization of the binding sites might be indicative of their functional differences.     

D-[35S(U)]inositol 1,4,5-trisphosphorothioate, a novel radioligand for the inositol 1,4,5-trisphosphate receptor. Complex binding to rat cerebellar membranes.

D-[35S(U)]myo-inositol 1,4,5-trisphosphorothioate [( 35S]InsPS3), a synthetic, metabolically stable analogue of inositol 1,4,5-trisphosphate (InsP3), binds with high affinity (Kd 58.6 +/- 9.1 nM) to rat cerebellar membranes revealing a high density of specific binding sites (Bmax 21.5 +/- 2.1 pmol/mg of protein). Comparison with [3H]InsP3 binding reveals a higher density of sites labelled by [35S]InsPS3 and complex competition curves for displacement of specific [35S]InsPS3 by InsP3. The results suggest that [35S]InsPS3 labels two sites in rat cerebellar membranes with equal affinity: the InsP3 receptor and a site that displays low affinity for InsP3.     

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.     

The second messenger binding site of inositol 1,4,5-trisphosphate 3-kinase is centered in the catalytic domain and related to the inositol trisphosphate receptor site

A segment of inositol 1,4,5-trisphosphate 3-kinase responsible for inositol 1,4,5-trisphosphate (InsP(3)) binding was characterized and confirmed by three different approaches employing the fully active expressed catalytic domain of the enzyme. Part of this moiety was protected from limited tryptic proteolysis by InsP(3). Sequencing of two fragments of 16 and 21 kDa, generated in the absence or presence of InsP(3), respectively, identified segment Glu-271 to Arg-305 as being protected. 15 monoclonal antibodies, all binding to epitopes within this region, inhibited enzyme activity and interfered with inositol phosphate binding. Detailed enzyme kinetic parameters of 32 site-directed mutants revealed residues Arg-276 and Lys-303 in this segment and Arg-322, located nearby, as directly involved in and five other closely neighbored residues, all located within a segment of 73 amino acids, as also influencing InsP(3) binding. Part of this region is similar in sequence to an InsP(3) binding segment in InsP(3) receptors. Combined with the finding that mutants influencing only ATP binding all lie outside this region, these data indicate that an InsP(3) binding core domain is inserted between two segments acting together in ATP binding and phosphate transfer.     

Novel regulation of calcium inhibition of the inositol 1,4,5-trisphosphate receptor calcium-release channel

The inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R), a Ca2+-release channel localized to the endoplasmic reticulum, plays a critical role in generating complex cytoplasmic Ca2+ signals in many cell types. Three InsP3R isoforms are expressed in different subcellular locations, at variable relative levels with heteromultimer formation in different cell types. A proposed reason for this diversity of InsP3R expression is that the isoforms are differentially inhibited by high cytoplasmic free Ca2+ concentrations ([Ca2+]i), possibly due to their different interactions with calmodulin. Here, we have investigated the possible roles of calmodulin and bath [Ca2+] in mediating high [Ca2+]i inhibition of InsP3R gating by studying single endogenous type 1 InsP3R channels through patch clamp electrophysiology of the outer membrane of isolated Xenopus oocyte nuclei. Neither high concentrations of a calmodulin antagonist nor overexpression of a dominant-negative Ca2+-insensitive mutant calmodulin affected inhibition of gating by high [Ca2+]i. However, a novel, calmodulin-independent regulation of [Ca2+]i inhibition of gating was revealed: whereas channels recorded from nuclei kept in the regular bathing solution with [Ca2+] approximately 400 nM were inhibited by 290 muM [Ca2+]i, exposure of the isolated nuclei to a bath solution with ultra-low [Ca2+] (<5 nM, for approximately 300 s) before the patch-clamp experiments reversibly relieved Ca2+ inhibition, with channel activities observed in [Ca2+]i up to 1.5 mM. Although InsP3 activates gating by relieving high [Ca2+]i inhibition, it was nevertheless still required to activate channels that lacked high [Ca2+]i inhibition. Our observations suggest that high [Ca2+]i inhibition of InsP3R channel gating is not regulated by calmodulin, whereas it can be disrupted by environmental conditions experienced by the channel, raising the possibility that presence or absence of high [Ca2+]i inhibition may not be an immutable property of different InsP3R isoforms. Furthermore, these observations support an allosteric model in which Ca2+ inhibition of the InsP3R is mediated by two Ca2+ binding sites, only one of which is sensitive to InsP3.     

Isoform-specific regulation of the inositol 1,4,5-trisphosphate receptor by O-linked glycosylation

The inositol 1,4,5-trisphosphate receptor (InsP(3)R), an intracellular calcium channel, has three isoforms with >65% sequence homology, yet the isoforms differ in their function and regulation by post-translational modifications. We showed previously that InsP(3)R-1 is functionally modified by O-linked β-N-acetylglucosamine glycosylation (O-GlcNAcylation) (Rengifo, J., Gibson, C. J., Winkler, E., Collin, T., and Ehrlich, B. E. (2007) J. Neurosci. 27, 13813-13821). We now report the effect of O-GlcNAcylation on InsP(3)R-2 and InsP(3)R-3. Analysis of AR4-2J cells, a rat pancreatoma cell line expressing predominantly InsP(3)R-2, showed no detectable O-GlcNAcylation of InsP(3)R-2 and no significant functional changes despite the presence of the enzymes for addition (O-β-N-acetylglucosaminyltransferase) and removal (O-β-N-acetylglucosaminidase) of the monosaccharide. In contrast, InsP(3)R-3 in Mz-ChA-1 cells, a human cholangiocarcinoma cell line expressing predominantly InsP(3)R-3, was functionally modified by O-GlcNAcylation. Interestingly, the functional impact of O-GlcNAcylation on the InsP(3)R-3 channel was opposite the effect measured with InsP(3)R-1. Addition of O-GlcNAc by O-β-N-acetylglucosaminyltransferase increased InsP(3)R-3 single channel open probability. Incubation of Mz-ChA-1 cells in hyperglycemic medium caused an increase in the InsP(3)-dependent calcium release from the endoplasmic reticulum. The dynamic and inducible nature of O-GlcNAcylation and the InsP(3)R isoform specificity suggest that this form of modification of InsP(3)R and subsequent changes in intracellular calcium transients are important in physiological and pathophysiological processes.     

Regulation of the type 1 inositol 1,4,5-trisphosphate receptor by phosphorylation at tyrosine 353

The inositol 1,4,5-trisphosphate receptor (IP3R) plays an essential role in Ca2+ signaling during lymphocyte activation. Engagement of the T cell or B cell receptor by antigen initiates a signal transduction cascade that leads to tyrosine phosphorylation of IP3R by Src family nonreceptor protein tyrosine kinases, including Fyn. However, the effect of tyrosine phosphorylation on the IP3R and subsequent Ca2+ release is poorly understood. We have identified tyrosine 353 (Tyr353) in the IP3-binding domain of type 1 IP3R (IP3R1) as a phosphorylation site for Fyn both in vitro and in vivo. We have developed a phosphoepitope-specific antibody and shown that IP3R1-Y353 becomes phosphorylated during T cell and B cell activation. Furthermore, tyrosine phosphorylation of IP3R1 increased IP3 binding at low IP3 concentrations (<10 nm). Using wild-type IP3R1 or an IP3R1-Y353F mutant that cannot be tyrosine phosphorylated at Tyr353 or expressed in IP3R-deficient DT40 B cells, we demonstrated that tyrosine phosphorylation of Tyr353 permits prolonged intracellular Ca2+ release during B cell activation. Taken together, these data suggest that one function of tyrosine phosphorylation of IP3R1-Y353 is to enhance Ca2+ signaling in lymphocytes by increasing the sensitivity of IP3R1 to activation by low levels of IP3.     

Structure and expression of the rat inositol 1,4,5-trisphosphate receptor

The complete primary structure of the inositol 1,4,5-trisphosphate receptor from rat brain was elucidated using a series of overlapping cDNA clones. Two different sets of clones that either contain or lack a 45-nucleotide sequence in the amino-terminal third of the protein were isolated, suggesting a differential splicing event that results in the biosynthesis of either a 2734- or 2749-amino acid receptor protein. Hydrophobicity analysis demonstrates the presence of a cluster of hydrophobic sequences in the carboxyl-terminal third of the protein that probably comprise eight transmembrane regions and that may form the calcium channel intrinsic to the receptor. The receptor was universally expressed at low levels in all tissues and cultured cells tested. Transfection of a full-length expression construct of the inositol 1,4,5-trisphosphate receptor into COS cells resulted in the biosynthesis of a 260-kDa protein that bound inositol 1,4,5-trisphosphate and formed high molecular weight complexes similar to the native receptor as analyzed by sucrose gradient centrifugations. On the other hand, the protein product synthesized by a mutant receptor construct in which the amino-terminal 418 amino acids were deleted failed to bind inositol 1,4,5-trisphosphate. The mutant receptor still formed high molecular weight complexes, suggesting that it folded normally and that the amino-terminal sequences of the receptor are part of the ligand binding domain.     

Heterogeneity of [3H]inositol 1,4,5-trisphosphate binding sites in adrenal-cortical membranes. Characterization and validation of a radioreceptor assay.

1. The characterization of a radioreceptor assay for determining Ins(1,4,5)P3 concentration in tissue extracts is described which utilizes the binding of [3H]Ins(1,4,5)P3 to an adrenal-cortex membrane fraction. 2. Analysis of [3H]Ins(1,4,5)P3 binding by isotope dilution demonstrated an apparent single population of binding sites (KD 3.65 +/- 0.18 nM, Bmax. 872 +/- 70 fmol/mg of protein). Specific binding of [3H]Ins(1,4,5)P3 was enhanced at alkaline pH values (maximum at pH 8.5), with complete loss of specific binding at pH less than 6. These binding sites displayed strict stereo- and positional specificity for Ins(1,4,5)P3, with L-Ins(1,4,5)P3, Ins(1,3,4)P3 and DL-Ins(1,3,4,5)P4 causing 50% displacement of specific [3H]Ins(1,4,5)P3 binding (IC50 values) at concentrations of 14 +/- 3 microM, 3.0 +/- 0.3 microM and 0.53 +/- 0.03 microM respectively. 3. Kinetic analysis of binding data, however, revealed a high-affinity [3H]Ins(1,4,5)P3 binding site (KD 0.052 nM) in addition to the lower-affinity site (KD 2.53 nM) already demonstrated in displacement studies. 4. It is shown that the presence of the high-affinity site can be exploited to increase the sensitivity of the [3H]Ins(1,4,5)P3 radioreceptor assay, allowing accurate detection of 20 fmol of Ins(1,4,5)P3 in 300 microliters of tissue extract. 5. Further validation of the specificity of the above assay for Ins(1,4,5)P3 was provided by incubating tissue extracts with either a 5-phosphatase or 3-kinase preparation. It was shown that identical loss occurred of both Ins(1,4,5)P3 mass and [3H]Ins(1,4,5)P3, added to parallel incubations. 6. The ability of the assay to measure basal and agonist-stimulated increases in Ins(1,4,5)P3 concentration has been demonstrated with rat cerebral cortex and bovine tracheal smooth-muscle slices and a range of cultured and isolated cell preparations.     

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.     

Inhibition of the type 1 inositol 1,4,5-trisphosphate receptor by 2-aminoethoxydiphenylborate

2-Aminoethoxydiphenylborate (2-APB) inhibits the extent of inositol 1,4,5-trisphosphate (InsP(3))-induced Ca(2+) release from cerebellar microsomes with a potency that is dependent upon the InsP(3) concentration used. At high InsP(3) concentrations (10 microM), the concentration of 2-APB required to cause half-maximal InsP(3)-induced Ca(2+) release (IC(50)) was greater than 1 mM, while at 0.25 microM InsP(3) this reduced to 220 microM. The fact that the inhibition of the extent of InsP(3)-induced Ca(2+) release (IICR) by 2-APB was not restored to control levels by high concentrations of InsP(3), in addition to the fact 2-APB did not substantially inhibit [3H]InsP(3) binding to its receptor, indicates that the inhibition is not competitive in nature. Since the cooperativity of IICR as a function of InsP(3) was reduced in the presence of 2-APB (Hill coefficient changing from 1.9 in the absence of 2-APB to 1.4 in the presence of 1 mM 2-APB), this suggests that it is acting as an allosteric inhibitor. 2-APB also reduces the rate constants for IICR. In cerebellar microsomes this release process is biphasic in nature, with a fast and slow phase. 2-APB appears particularly to affect the fast-phase component. Although 2-APB does not inhibit the ryanodine receptor, it does inhibit the Ca(2+) ATPase activity as well store-operated Ca(2+) entry channels, which may limit its use as a specific membrane permeant InsP(3) receptor inhibitor.     

Stereospecific recognition of inositol 1,4,5-trisphosphate analogs by the phosphatase, kinase, and binding proteins

A series of DL-inositol 1,4,5-trisphosphate (IP3) analogs, with a bulky substitutent on the 2nd carbon of the inositol ring, has been synthesized. These compounds exert biological activities with only minor reduction in potency, in several assay systems (Hirata, M., Watanabe, Y., Ishimatsu, T., Ikebe, T., Kimura, Y., Yamaguchi, K., Ozaki, S., and Koga, T. (1989) J. Biol. Chem. 264, 20303-20308). Two analogs with aminocyclohexanecarbonyl (designated as analog 206) or aminobenzoyl group (analog 209) were separated into individual optical isomers and examined for stereospecificity in recognition by IP3-5-phosphatase, IP3-3-kinase and IP3 binding activity. IP3-5-phosphatase activity of erythrocyte ghosts was competitively inhibited by L-209 with a lower Ki value than D-IP3, but with a higher Ki value by L-206. D-Isomers of both analogs at 100 microM failed to inhibit the hydrolysis of D-[3H]IP3. On the other hand, D-isomers but not L-isomers of both analogs were as potent as D-IP3 in the recognition by IP3-3-kinase of rat brain cytosol and only the D-isomer of analog 206 could serve as substrate for the kinase. Also D-isomers of both analogs were equipotent to D-IP3 in displacing [3H]IP3 binding to rat cerebellum microsomes. These observations suggest that the IP3 analogs we synthesized are stereospecifically recognized by three IP3-recognizable proteins, but the phosphatase recognizes opposite isomers. Such being the case, the second hydroxyl group of D-IP3 may be involved in the recognition by IP3-5-phosphatase, but not by IP3-3-kinase and binding sites.     

Postnatal expression of the inositol 1,4,5-trisphosphate receptor in canine cerebellum.

1. Inositol 1,4,5-trisphosphate (IP3), an intracellular second messenger, has been shown to be the link between activation of several plasma membrane receptors and Ca2+ release from intracellular, membrane-bound compartments. In this study, the postnatal expression of the canine cerebellum IP3 receptor was investigated by biochemical, ligand binding and immunocytochemical methods. 2. Specific receptor sites for IP3 and the extent of IP3-induced Ca2+ release were quantitated in microsomal fractions isolated from cerebella of developing (0-28 day-old) and adult dogs. The IP3 receptor was detected in newborn animals and adult levels were attained within 3-4 weeks. 3. The time-course of IP3 receptor ontogeny paralleled both growth of Purkinje neurons, as indicated by immunofluorescence of cerebellum cortex cryosections with anti-IP3 receptor antibodies, and synaptogenesis, as judged by Western blotting of the microsomal fractions with anti-synaptophysin antibodies.