Inhibition of the Phospholipase C-Linked Metabotropic Glutamate Receptor by 2-Amino-3-Phosphonopropionate Is Dependent on Extracellular Calcium

D,L-2-Amino-3-phosphonopropionate (AP-3), a proposed metabotropic receptor antagonist, produced a concentration-dependent increase in the formation of inositol 1,4,5-trisphosphate in rat hippocampal slices. The response was maximal at 1 mM and completely due to the L-isomer. D,L-AP-3 was half as efficacious as (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD), a selective agonist of this receptor. The response produced by maximally effective concentrations of L-AP-3 and 1S,3R-ACPD together for 5 min was not significantly different from that produced by 1S,3R-ACPD alone. However, pretreatment for 40 min with either 1 mM L-AP-3 or D,L-AP-3 completely inhibited the response to 1S,3R-ACPD. This inhibition was long-lasting (wash-resistant) and was reversed by reduction of the extracellular Ca2+ concentration. Also, pretreatment for 40 min with 1S,3R-ACPD reduced, but did not completely block, the response to readdition of 1S,3R-ACPD. L-AP-3 (1 mM) also produced a stereoselective 2.3-fold increase in the efflux of glutamate from the hippocampal slices. These data suggest that incubation of hippocampal slices with AP-3 induces a time-dependent desensitization of the metabotropic response by a mechanism that is dependent on extracellular Ca2+. The possible roles of receptor occupancy and inhibition of glutamate uptake by AP-3 are also discussed.     

Expression and signaling of group I metabotropic glutamate receptors in astrocytes and microglia

Stimulation of astrocytes with the excitatory neurotransmitter glutamate leads to the formation of inositol 1,4,5-trisphosphate and the subsequent increase of intracellular calcium content. Astrocytes express both ionotropic receptors and metabotropic glutamate (mGlu) receptors, of which mGlu5 receptors are probably involved in glutamate-induced calcium signaling. The mGlu5 receptor occurs as two splice variants, mGlu5a and mGlu5b, but it was hitherto unknown which splice variant is responsible for the glutamate-induced effects in astrocytes. We report here that both mRNAs encoding mGlu5 receptor splice variants are expressed by cultured astrocytes. The expression of mGlu5a receptor mRNA is much stronger than that of mGlu5b receptor mRNA in these cells. In situ hybridization experiments reveal neuronal expression of mGlu5b receptor mRNA in adult rat forebrain but a strong neuronal expression of mGlu5a mRNA only in olfactory bulb. Signals for mGlu5a receptor mRNA in the rest of the brain were diffuse and weak but consistently above background. Activation of mGlu5 receptors in astrocytes yields increases in inositol phosphate production and transient calcium responses. It is surprising that the rank order of agonist potency [quisqualate > (2S,1 'S,2'S)-2-(carboxycyclopropyl)glycine = trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (1S,3R-ACPD) > glutamate] differs from that reported for recombinantly expressed mGlu5a receptors. The expression of mGlu5a receptor mRNA and the occurrence of 1S,3R-ACPD-induced calcium signaling were found also in cultured microglia, indicating for the first time expression of mGlu5a receptors in these macrophage-like cells.     

Carbachol, but Not Norepinephrine, NMDA, lonomycin, Ouabain, or Phorbol Myristate Acetate, Increases Inositol 1,3,4,5-Tetrakisphosphate Accumulation in Rat Brain Cortical Slices

Abstract: lonomycin, a Ca²⁺ ionophore, stimulated phosphoinositide breakdown in rat brain cortical slices incubated in the presence of 1.2 m M Ca²⁺, but, unlike muscarinic cholinergic stimulation, it had little effect on inositol 1,3,4,5-tetrakisphosphate accumulation. However, at 2 min, the increase in inositol 1,4,5-trisphosphate due to 10 μ M ionomycin was equivalent to that seen with 1μ M carbachol. Phorbol 12-myristate 13-acetate or high K⁺ (30 μ M ) increased inositol 1,4,5-trisphosphate, but not inositol 1,3,4,5-tetrakisphosphate accumulation. The stimulation of inositol 1,4,5-trisphosphate accumulation due to ionomycin, unlike that seen with carbachol, was abolished in buffer containing 0.2 μ M Ca²⁺. The increase in inositol 1,3,4,5-tetrakisphosphate accumulation in brain slices due to 1 μ M carbachol ranged from 55 to 68% of that for inositol 1,4,5-trisphosphate. Norepinephrine, NMDA, veratridine, and ouabain also increased inositol 1,4,5-tris-phosphate, but had minimal effects on inositol 1,3,4,5-tetrakisphosphate accumulation. These results suggest that there is something unique about the stimulation of inositol 1,3,4,5-tetrakisphosphate accumulation by carbachol, which is also the only one of these agents that is able to activate phosphoinositidase Cβ, in isolated rat brain membranes.     

Evidence for the involvement of the Rho GTP-binding protein in egg activation of the ascidian Halocynthia roretzi

Eggs of the ascidian Halocynthia roretzi are activated by insemination and by treatment with calcium ionophore, leading to elevation of the vitelline coat. Here we describe the effects on egg activation of microinjection of guanosine 5'-(γ thio) triphosphate (GTPγS, a non-hydrolyzable GTP analog), heparin (an antagonist of the inositol 1,4,5-trisphosphate receptor) and a monoclonal antibody to the Rho GTP-binding protein. Microinjected GTPγS induced elevation of the vitelline coat, but not when it was co-injected with EGTA or heparin. Pre-injected heparin or the anti-Rho monoclonal antibody blocked subsequent sperm-induced elevation of the vitelline coat, but not calcium ionophore-induced elevation. We also demonstrated that the amount of cytosolic inositol 1,4,5-trisphosphate was increased by insemination. These results strongly suggest that the Rho GTP-binding protein functions prior to the heparin-blocked inositol 1,4,5-trisphosphate receptor-mediated Ca²⁺ release in the sperm induced activation process of H. roretzi eggs.     

Signaling mechanism for receptor-activated canonical transient receptor potential 3 (TRPC3) channels

Canonical transient receptor potential 3 (TRPC3) is a receptor-activated, calcium permeant, non-selective cation channel. TRPC3 has been shown to interact physically with the N-terminal domain of the inositol 1,4,5-trisphosphate receptor, consistent with a "conformational coupling" mechanism for its activation. Here we show that low concentrations of agonists that fail to produce levels of inositol 1,4,5-trisphosphate sufficient to induce Ca(2+) release from intracellular stores substantially activate TRPC3. By several experimental approaches, we demonstrate that neither inositol 1,4,5-trisphosphate nor G proteins are required for TRPC3 activation. However, diacylglycerols were sufficient to activate TRPC3 in a protein kinase C-independent manner. Surface receptor agonists and exogenously applied diacylglycerols were not additive in activating TRPC3. In addition, inhibition of metabolism of diacylglycerol slowed the reversal of receptor-dependent TRPC3 activation. We conclude that receptor-mediated activation of phospholipase C in intact cells activates TRPC3 via diacylglycerol production, independently of G proteins, protein kinase C, or inositol 1,4,5-trisphosphate.     

Solubilization, purification, and characterization of an inositol trisphosphate receptor

Inositol 1,4,5-trisphosphate is a second messenger of the phosphoinositide system which can mobilize calcium from intracellular stores. Rat cerebellum is an abundant source of a receptor for inositol 1,4,5-trisphosphate (Worley, P. F., Baraban, J. M., Supattapone, S., Wilson, V. S., and Snyder, S. H. (1987) J. Biol. Chem. 262, 12132-12136). In this study we have solubilized and purified this receptor to apparent homogeneity from rat cerebellum. Crude membrane, detergent-solubilized, and purified receptor preparations display similar selectivity for inositol 1,4,5-trisphosphate over other inositol phosphates. The purified receptor is globular with a Stokes' radius of approximately 10 nm. Electrophoretic analysis reveals one protein band with an Mr of 260,000. While binding is reversibly inhibited by 300 nM calcium in particulate fractions and detergent-solubilized membranes, the purified protein is not inhibited by calcium concentrations up to 1.5 mM. Inhibition by calcium is reconstituted by addition of detergent-solubilized cerebellar membranes, but not by the cytosolic fraction of cerebellum.     

Reassessment of the Ca2+ sensing property of a type I metabotropic glutamate receptor by simultaneous measurement of inositol 1,4,5-trisphosphate and Ca2+ in single cells

Transient transfection of Chinese hamster ovary or baby hamster kidney cells expressing the Group I metabotropic glutamate receptor mGlu1alpha with green fluorescent protein-tagged pleckstrin homology domain of phospholipase Cdelta1 allows real-time detection of inositol 1,4,5-trisphosphate. Loading with Fura-2 enables simultaneous measurement of intracellular Ca(2+) within the same cell. Using this technique we have studied the extracellular calcium sensing property of the mGlu1alpha receptor. Quisqualate, in extracellular medium containing 1.3 mm Ca(2+), increased inositol 1,4,5-trisphosphate in all cells. This followed a typical peak and plateau pattern and was paralleled by concurrent increases in intracellular Ca(2+) concentration. Under nominally Ca(2+)-free conditions similar initial peaks in inositol 1,4,5-trisphosphate and Ca(2+) concentration occurred with little change in either agonist potency or efficacy. However, sustained inositol 1,4,5-trisphosphate production was substantially reduced and the plateau in Ca(2+) concentration absent. Depletion of intracellular Ca(2+) stores using thapsigargin abolished quisqualate-induced increases in intracellular Ca(2+) and markedly reduced inositol 1,4,5-trisphosphate production. These data suggest that the mGlu1alpha receptor is not a calcium-sensing receptor because the initial response to agonist is not sensitive to extracellular Ca(2+) concentration. However, prolonged activation of phospholipase C requires extracellular Ca(2+), while the initial burst of activity is highly dependent on Ca(2+) mobilization from intracellular stores.     

Calcium modulation of inositol 1,4,5-trisphosphate-induced calcium release from neuroblastoma x glioma hybrid (NG108-15) microsomes

Subcellular fractions of neuroblastoma x glioma (NG108-15) hybrid cells were used to study the mechanism of inositol 1,4,5-trisphosphate-induced calcium release. A microsomal fraction, enriched in endoplasmic reticulum and plasma membranes and almost devoid of mitochondria, was the most active in inositol trisphosphate- or GTP-dependent release of calcium. Neither GTP nor inositol 1,4,5-trisphosphate affected the calcium efflux mediated by the other reagent, suggesting that inositol trisphosphate and GTP act on different calcium-sequestrating vesicles. The stimulation of calcium release by GTP was relatively slow (t1/2 = 90 s), dependent on polyethyleneglycol, and greater at 2 X 10(-5) M calcium (5 nmol X min-1 X mg-1) than at 10(-6) M calcium (0.8 nmol X min-1 X mg-1). The inositol trisphosphate-induced calcium efflux was not mimicked by inositol monophosphate; it was fast (t1/2 less than 10 s) and unaffected by 3% polyethyleneglycol. The amount of calcium released by inositol trisphosphate was greatest at 10(-6) M external calcium (1 nmol X min-1 X mg-1) and it was undetectable at 2 X 10(-5) M calcium. A feedback inhibition of the inositol trisphosphate-induced calcium release by cytoplasmic calcium provides a safety mechanism preventing deleterious effects of abnormally high calcium levels.     

4.1N binding regions of inositol 1,4,5-trisphosphate receptor type 1

Zhang et al. and Maximov et al. [S. Zhang, A. Mizutani, C. Hisatsune, T. Higo, H. Bannai, T. Nakayama, M. Hattori, and K. Mikoshiba, Protein 4.1N is required for translocation of inositol 1,4,5-trisphosphate receptor type 1 to the basolateral membrane domain in polarized Madin-Darby canine kidney cells, J. Biol. Chem. 278 (2003) 4048-4056; A. Maximov, T. S. Tang, and I. Bezprozvanny, Association of the type 1 inositol (1,4,5)-trisphosphate receptor with 4.1N protein in neurons, Mol. Cell. Neurosci. 22 (2003) 271-283.] reported that 4.1N is a binding partner of inositol 1,4,5-trisphosphate receptor type 1 (IP(3)R1), however the binding site of IP(3)R1 differed: the former determined the C-terminal 14 amino acids of the cytoplasmic tail (CTT14aa) as the binding site, while the latter assigned another segment, cytoplasmic tail middle 1 (CTM1). To solve this discrepancy, we performed immunoprecipitation and found that both the segments had binding activity to 4.1N. Both segments also interfered the 4.1N-regulated IP(3)R1 diffusion in neuronal dendrites. However, IP(3)R1 lacking the CTT14aa (IP(3)R1-DeltaCTT14aa) does not bind to 4.1N [S. Zhang, A. Mizutani, C. Hisatsune, T. Higo, H. Bannai, T. Nakayama, M. Hattori, and K. Mikoshiba, Protein 4.1N is required for translocation of inositol 1,4,5-trisphosphate receptor type 1 to the basolateral membrane domain in polarized Madin-Darby canine kidney cells, J. Biol. Chem. 278 (2003) 4048-4056.] and its diffusion constant is larger than that of IP(3)R1 full-length in neuronal dendrites [K. Fukatsu, H. Bannai, S. Zhang, H. Nakamura, T. Inoue, and K. Mikoshiba, Lateral diffusion of inositol 1,4,5-trisphosphate receptor type 1 is regulated by actin filaments and 4.1N in neuronal dendrites, J. Biol. Chem. 279 (2004) 48976-48982.]. We conclude that both the CTT14aa and CTM1 sequences can bind to 4.1N in peptide fragment forms. However, we propose that the responsible binding site for 4.1N binding in full-length tetramer form of IP(3)R1 is CTT14aa.     

Characterization of inositol trisphosphate receptor binding in brain. Regulation by pH and calcium

Inositol 1,4,5-trisphosphate is an intracellular second messenger, produced upon stimulation of the phosphoinositide system, capable of mobilizing calcium from intracellular stores. We have recently identified high levels of specific binding sites for inositol 1,4,5-trisphosphate in brain membranes (Worley, P. F., Baraban, J. M., Colvin, J. S., and Snyder, S. H. (1987) Nature 325, 159-161) and have now further characterized these sites. In cerebellar membranes, inositol 1,4,5-trisphosphate binding sites are abundant (20 pmol/mg protein) and display high affinity and selectivity for inositol 1,4,5-trisphosphate (KD approximately equal to 40 nM), whereas other inositol phosphates such as inositol 1,3,4,5-tetrakisphosphate (Ki approximately equal to 10 microM) and inositol 1,4-bisphosphate (Ki approximately equal to 10 microM) exhibit much lower affinity for this site. Submicromolar concentrations of calcium strongly inhibit inositol 1,4,5-trisphosphate binding (IC50 approximately equal to 300 nM). A sharp increase in binding occurs at slightly alkaline pH. These results suggest that actions of inositol 1,4,5-trisphosphate are regulated by physiological alterations in intracellular pH and calcium concentrations.     

Protein kinase C differently regulates quisqualate- and 1S,3R-trans aminocyclopentane dicarboxylate-induced phosphoinositide hydrolysis during in vitro development of hippocampal neurons

Transient peaks of quisqualate (QA)-, but not 1S,3R-1-amino-3-cyclopentane dicarboxylate (1S,3R-ACPD)- and carbachol-induced inositol phosphate formation occur between 2 and 5 days in vitro (DIV) in hippocampal neurons in culture. In order to elucidate the putative origin of such developmental activity differences, the effect of PKC on metabotropic glutamate receptor (mGluR) and muscarinic receptor responses was investigated at 3 and 10 DIV. (i) Stimulation of PKC by phorbol-12,13-dibutyrate inhibited QA, IS,3R-ACPD and carbachol responses at 3 DIV. At 10 DIV, only 1S,3R-ACPD response was still inhibited by phorbol esters. (ii) Inhibition of PKC by staurosporine at 3 DIV potentiated 1S,3R-ACPD-induced inositol phosphate formation, but had no effect on QA and carbachol responses. At 10 DIV, all responses were potentiated by staurosporine. These data strongly suggest that PKC differently modulates 1S,3R-ACPD- and QA-induced inositol phosphate accumulations during in vitro development. The specific activity of mGluRs during development, vs that of muscarinic receptor, and the peculiar modes of regulation by PKC of these two mG1uR activities further suggest their particular involvement in the maturation of neuronal culture.     

Inositol 1,4,5-trisphosphate 3-kinase activity in frog skeletal muscle

Frog skeletal muscle contains a kinase activity that phosphorylates inositol 1,4,5-trisphosphate to inositol 1,3,4,5-tetrakisphosphate. The inositol 1,4,5-trisphosphate 3-kinase activity was mainly recovered in the soluble fraction, where it presented a marked dependency on free calcium concentration in the physiological range in the presence of endogenous calmodulin. At pCa 5, where the activity was highest, the soluble 3-kinase activity displayed a K_m for inositol 1,4,5-trisphosphate of 1.6 μM and a V_max value of 25.1 pmol mg⁻¹ min⁻¹. The removal rates of inositol 1,4,5-trisphosphate by 3-kinase and 5-phosphatase activities of the total homogenate under physiological ionic conditions were very similar, suggesting that both routes are equally important in metabolizing inositol 1,4,5-trisphosphate in frog skeletal muscle.     

Phosphatidylethanol Affects Inositol 1,4,5-Trisphosphate Levels in NG108-15 Neuroblastoma × Glioma Hybrid Cells

Abstract: Phosphatidylethanol is formed by phospholipase D in animal cells exposed to ethanol. Previous reports have demonstrated that the degradation of phosphatidylethanol is slow, indicating that this lipid may be present in the cells after ethanol itself has disappeared. Accumulation of an abnormal alcohol metabolite may influence cellular functions. In the present study, cultivation of NG108–15 neuroblastoma × glioma hybrid cells in the presence of ethanol resulted in an accumulation of phosphatidylethanol and a simultaneous increase in basal inositol 1,4,5-trisphosphate levels. The direct effects of phosphatidylethanol on the phosphoinositide signal transduction system were examined through incorporation of exogenous phosphatidylethanol into membranes of ethanol-naive cells. An incorporation amounting to 2.8% of cellular phospholipids was achieved after a 5-h incubation with 30 μ M phosphatidylethanol. Phosphatidylethanol was found to cause a time-and dose-dependent increase in the basal levels of inositol 1,4,5-trisphosphate. The effects on inositol 1,4,5-trisphosphate levels of exogenously added phosphatidylethanol and ethanol exposure for 2 days were not additive. No effect on bradykinin-stimulated inositol 1,4,5-trisphosphate production could be detected. However, the increase in basal inositol 1,4,5-trisphosphate levels indicates that phosphatidylethanol affects inositol 1,4,5-trisphosphate turnover and emphasizes the importance of considering phosphatidylethanol as a possible mediator of ethanol-induced effects on cellular processes.     

Excitatory Amino Acid-Induced Formation of Inositol Phosphates in Guinea-Pig Cerebral Cortical Slices: Involvement of Ionotropic or Metabotropic Receptors?

In cerebral cortical slices from the guinea-pig, quinoxalinedione derivatives antagonised the generation of 3H-inositol phosphates evoked by the excitatory amino acids quisqualate and DL-alpha-amino-3-hydroxy-5-methyl-4-isoxalone propionic acid but were without effect on the trans-DL-1-amino-1,3-cyclopentanedicarboxylic acid and L-glutamate responses. Omission of calcium from the medium reduced the accumulation of 3H-inositol phosphates induced by incubation with trans-DL-1-amino-1,3-cyclopentanedicarboxylic acid (incubation for 45 min) by greater than 50%, whereas the responses to L-glutamate and the two other amino acid analogues were reduced by approximately 20%. Generation of inositol 1,4,5-trisphosphate over a 30-s period by treatment with quisqualate, trans-DL-1-amino-1,3-cyclopentane-dicarboxylic acid, KCl, and carbachol was abolished in the presence of nominally calcium-free medium. L-Glutamate induced a large, rapid increase in inositol 1,4,5-trisphosphate mass (more than three-fold), which was, however, unaffected by omission of calcium from the medium. These results indicate that of the excitatory amino acids tested, only L-glutamate may be classed as a metabotropic receptor agonist in guinea-pig cerebral cortical slices with respect to generation of inositol phosphates. The other agents appear to stimulate accumulation of inositol phosphates, at least in part through some mechanism requiring the presence of extracellular Ca2+, presumably Ca2+ entry.     

2-Aminoethyl diphenyl borinate (2-APB) inhibits TRPM7 channels through an intracellular acidification mechanism

2-APB is a widely used compound in ion channel research. It affects numerous channels including inositol 1,4,5-trisphosphate receptors, store-operated calcium channels and TRP channels, TRPV3 and TRPM7 among them. A characteristic property of TRPM7 channels is their sensitivity to intracellular Mg²⁺ and pH. Using patch clamp electrophysiology we find that in Jurkat T lymphocytes, 100–300 µM extracellular 2-APB reversibly inhibits TRPM7 channels when internal HEPES concentration is low (1 mM). Increasing the concentration to 140 mM abolishes the 2-APB effect. Using single-cell fluorescence pH video imaging, we show that at concentrations of 100 µM and higher, 2-APB potently acidifies the cytoplasm. We conclude that TRPM7 sensitivity to 2-APB is not direct but rather, can be explained by cytoplasmic acidification and a resulting channel inhibition.     

Interconversion of inositol (1,4,5)-trisphosphate to inositol (1,3,4,5)-tetrakisphosphate and (1,3,4)-trisphosphate in permeabilized adrenal glomerulosa cells is calcium-sensitive and ATP-dependent

The metabolism of [3H]inositol (1,4,5)-trisphosphate was followed in permeabilized bovine adrenal glomerulosa cells. At low Ca++ concentration (pCa = 7.2), more than 90% of [3H]inositol (1,4,5)-trisphosphate had disappeared within 2 min, while two other metabolites, [3H]inositol (1,3,4)-trisphosphate and [3H]inositol (1,3,4,5)-tetrakisphosphate appeared progressively. At higher Ca++ concentrations (pCa = 5.7 and 4.8), the formation of these two metabolites was markedly increased, but completely abolished if the medium was ATP-depleted. The peak levels for the generation of [3H]inositol (1,3,4,5)-tetrakisphosphate (1 min) preceded those of [3H]inositol (1,3,4)-trisphosphate and were closely correlated. These results suggest that, in adrenal glomerulosa cells, the isomer inositol (1,3,4)-trisphosphate is generated from inositol (1,4,5)-trisphosphate via a calcium-sensitive and ATP-dependent phosphorylation/dephosphorylation pathway involving the formation of inositol (1,3,4,5)-tetrakisphosphate.     

Regulation of the type III InsP(3) receptor by InsP(3) and calcium

It has been proposed that the inositol 1,4,5-trisphosphate receptor (InsP(3)R) type III acts as a trigger for InsP(3)-mediated calcium (Ca(2+)) signaling, because this InsP(3) isoform lacks feedback inhibition by cytosolic Ca(2+). We tested this hypothesis in RIN-m5F cells, which express predominantly the type III receptor. Extracellular ATP increases Ca(2+) in these cells, and we found that this effect is independent of extracellular Ca(2+) but is blocked by the InsP(3)R antagonist heparin. There was a dose-dependent increase in the number of cells responding to ATP and two-photon flash photolysis of caged-Ca(2+) heightened the sensitivity of RIN-m5F cells to this increase. These findings provide evidence that Ca(2+) increases the sensitivity of the InsP(3)R type III in intact cells and supports the idea that this isoform can act as a trigger for hormone-induced Ca(2+) signaling.     

Bridging the gaps in 3D structure of the inositol 1,4,5-trisphosphate receptor-binding core

Calcium concentration is strictly regulated in all cells. The inositol 1,4,5-trisphosphate receptor (IP(3)R), which forms a homotetrameric Ca2+ release channel in the endoplasmic reticulum, is one of the key molecules responsible for this regulation. The opening of this channel requires binding of two intracellular messengers, which are inositol 1,4,5-trisphosphate (IP(3)) and Ca2+. To promote the Ca2+-channel gating and release from the endoplasmic reticulum, IP(3) binds to the amino-terminal region of IP(3)R. Recently, the crystal structure of IP(3)R-binding core in complex with its ligand was presented [I. Bosanac, J.R. Alattia, T.K. Mai, J. Chan, S. Talarico, F.K. Tong, K.I. Tong, F. Yoshikawa, T. Furuichi, M. Iwai, T. Michikawa, K. Mikoshiba, M. Ikura, Structure of the inositol 1,4,5-trisphosphate receptor binding core in complex with its ligand, Nature 420 (2002) 696-700; I. Bosanac, H. Yamazaki, T. Matsu-ura, T. Michikawa, K. Mikoshiba, M. Ikura, Crystal structure of the ligand-binding suppressor domain of type 1 inositol 1,4,5-trisphosphate receptor, Mol. Cell 17 (2005) 193-203]. The space positions of residues 289-301 (segment A), 320-350 (segment B), 373-386 (segment C), and 529-545 (segment D) were not determined by the X-ray crystallography. To bridge these gaps, the computer modeling of physiologically meaningful low-energy 3D structures of the segments A-D of the inositol 1,4,5-trisphosphate receptor has been carried out by using a hierarchical conformational search algorithm combining two approaches: knowledge-based homology modeling and ab initio conformational search strategy. The structure analysis suggests a Ca2+-binding site of high affinity formed by residues 296-335, several low-energy regular secondary structure units within the segment B, and a number of hinge regions within the segments A-D, important for the receptor functioning.     

Metabotropic receptor activation,desensitization and sequestration-II: modelling the dynamics of the pleckstrin homology domain

Recent observations have been made regarding the generation of inositol 1,4,5-trisphosphate (IP(3)), using chimeras of green fluorescent protein and the pleckstrin homology domain of phospholipase C-delta. In this paper a model is presented giving the quantitative relations between the green fluorescent protein-pleckstrin homology domain (GFP-PHD) construct and membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) levels as well as the concentration of IP(3), the product of hydrolysis of PIP(2). The model can correctly reproduce the dependence of cytosolic GFP-PHD fluorescence on IP(3) concentration. This model extends a previous one (Metabotropic receptor activation, desensitization and sequestration-I: modelling calcium and inositol 1,4,5-trisphosphate dynamics following receptor activation, in this issue) dealing with the processes governing the production of IP(3) and the subsequent calcium (Ca2+) changes in cells following activation of metabotropic receptors. This model is applied to the case of purinergic P(2)Y(2) receptor activation in Madin-Darby Canine Kidney (MDCK) cells with adenosine triphosphate (ATP) (Science 284 (1999) 1527). It is shown that it can correctly reproduce the dependence of GFP-PHD fluorescence on the concentration of P(2)Y(2) receptor ligand, as well as the temporal changes of GFP-PHD fluorescence following application of ligand.     

Metabolism of inositol 1,4,5-trisphosphate in guinea-pig hepatocytes.

Metabolism of inositol 1,4,5-trisphosphate was investigated in permeabilized guinea-pig hepatocytes. The conversion of [3H]inositol 1,4,5-trisphosphate to a more polar 3H-labelled compound occurred rapidly and was detected as early as 5 s. This material co-eluted from h.p.l.c. with inositol 1,3,4,5 tetrakis[32P]phosphate and is presumably an inositol tetrakisphosphate. A significant increase in the 3H-labelled material co-eluting from h.p.l.c. with inositol 1,3,4-trisphosphate occurred only after a definite lag period. Incubation of permeabilized hepatocytes with inositol 1,3,4,5-tetrakis[32P]phosphate resulted in the formation of 32P-labelled material that co-eluted with inositol 1,3,4-trisphosphate; no inositol 1,4,5-tris[32P]phosphate was produced, suggesting the action of a 5-phosphomonoesterase. The half-time of hydrolysis of inositol 1,3,4,5-tetrakis[32P]phosphate of approx. 1 min was increased to 3 min by 2,3-bisphosphoglyceric acid. Similarly, the rate of production of material tentatively designed as inositol 1,3,4-tris[32P]phosphate from the tetrakisphosphate was reduced by 10 mM-2,3-bisphosphoglyceric acid. In the absence of ATP there was no conversion of [3H]inositol 1,4,5-trisphosphate to [3H]inositol tetrakisphosphate or to [3H]inositol 1,3,4-trisphosphate, which suggests that the 1,3,4 isomer does not result from isomerization of inositol 1,4,5-trisphosphate. The results of this study suggest that the origin of the 1,3,4 isomer of inositol trisphosphate in isolated hepatocytes is inositol 1,3,4,5-tetrakisphosphate and that inositol 1,4,5-trisphosphate is rapidly converted to this tetrakisphosphate. The ability of 2,3-bisphosphoglyceric acid, an inhibitor of 5-phosphomonoesterase of red blood cell membrane, to inhibit the breakdown of the tetrakisphosphate suggests that the enzyme which removes the 5-phosphate from inositol 1,4,5-trisphosphate may also act to convert the tetrakisphosphate to inositol 1,3,4-trisphosphate. It is not known if the role of inositol 1,4,5-trisphosphate kinase is to inactivate inositol 1,4,5-trisphosphate or whether the tetrakisphosphate product may have a messenger function in the cell.