Identification of a new membrane-permeable inhibitor against inositol-1,4,5-trisphosphate-3-kinase A

Ectopic expression of the neuron-specific inositol-1,4,5-trisphosphate-3-kinase A (ITPKA) in lung cancer cells increases their metastatic potential because the protein exhibits two actin regulating activities; it bundles actin filaments and regulates inositol-1,4,5-trisphosphate (InsP₃)-mediated calcium signals by phosphorylating InsP₃. Thus, in order to inhibit the metastasis-promoting activity of ITPKA, both its actin bundling and its InsP₃kinase activity has to be blocked. In this study, we performed a high throughput screen in order to identify specific and membrane-permeable substances against the InsP₃kinase activity. Among 341,44 small molecules, 237 compounds (0.7%) were identified as potential InsP₃kinase inhibitors. After determination of IC₅₀-values, the three compounds with highest specificity and highest hydrophobicity (EPPC-3, BAMB-4, MEPTT-3) were further characterized. Only BAMB-4 was nearly completely taken up by H1299 cells and remained stable after cellular uptake, thus exhibiting a robust stability and a high membrane permeability. Determination of the inhibitor type revealed that BAMB-4 belongs to the group of mixed type inhibitors. Taken together, for the first time we identified a highly membrane-permeable inhibitor against the InsP₃kinase activity of ITPKA providing the possibility to partly inhibit the metastasis-promoting effect of ITPKA in lung tumor cells.     

Effects of ginsenosides on carbachol-stimulated formation of inositol phosphates in rat cortical cell cultures

We examined the effect of ginseng total saponins (GTS) on phosphoinositide metabolism stimulated by activation of muscarinic receptor using rat cortical cultures. Carbachol stimulated formation of [³H]inositol phosphates ([³H]InsPs) by 3.3-fold over basal level in [³H]inositol-prelabeled cells. Pretreatment of GTS inhibited formation of [³H]InsPs evoked by carbachol by 70%–90%. Addition of GTS alone had no effect on the basal formation of [³H]InsPs. The inhibitory effect of the GTS on carbachol-stimulated formation of [³H]InsPs was dose- and time-dependent. IC₅₀ was 6.0 ± 2.8 g/ml. We also examined the effect of GTS on [³H]InsP₁, [³H]InsP₂, or [³H]InsP₃ formation evoked by carbachol. Although GTS had no effect on the basal [³H]InsP₁, [³H]InsP₂, or [³H]InsP₃ formation, pretreatment of GTS inhibited [³H]InsP₁, [³H]InsP₂, or [³H]InsP₃ formation evoked by carbachol, respectively. Addition of individual ginsenosides such as ginsenoside Rb₁, Rc, Rd, Re, or Rg₂ had no effect on the basal formation of [³H]InsPs, whereas pretreatment of ginsenoside Rb₂, Rc, Rd, Re, Rf, Rg₁ or Rg₂ inhibited formation of [³H]InsPs evoked by carbachol by 79%–89%. The results suggest that the inhibitory effect of GTS and its individual ginsenosides on carbachol-stimulated formation of [³H]InsPs in cortical neurons could be one pharmacological action of Panax ginseng.     

Calcium-dependent Clustering of Inositol 1,4,5-Trisphosphate Receptors

Rat basophilic leukemia (RBL-2H3) cells predominantly express the type II receptor for inositol 1,4,5-trisphosphate (InsP₃), which operates as an InsP₃-gated calcium channel. In these cells, cross-linking the high-affinity immunoglobulin E receptor (FcεR1) leads to activation of phospholipase C γ isoforms via tyrosine kinase- and phosphatidylinositol 3-kinase-dependent pathways, release of InsP₃-sensitive intracellular Ca²⁺ stores, and a sustained phase of Ca²⁺ influx. These events are accompanied by a redistribution of type II InsP₃ receptors within the endoplasmic reticulum and nuclear envelope, from a diffuse pattern with a few small aggregates in resting cells to large isolated clusters after antigen stimulation. Redistribution of type II InsP₃ receptors is also seen after treatment of RBL-2H3 cells with ionomycin or thapsigargin. InsP₃ receptor clustering occurs within 5–10 min of stimulus and persists for up to 1 h in the presence of antigen. Receptor clustering is independent of endoplasmic reticulum vesiculation, which occurs only at ionomycin concentrations >1 μM, and maximal clustering responses are dependent on the presence of extracellular calcium. InsP₃ receptor aggregation may be a characteristic cellular response to Ca²⁺-mobilizing ligands, because similar results are seen after activation of phospholipase C-linked G-protein-coupled receptors; cholecystokinin causes type II receptor redistribution in rat pancreatoma AR4–2J cells, and carbachol causes type III receptor redistribution in muscarinic receptor-expressing hamster lung fibroblast E36^M3R cells. Stimulation of these three cell types leads to a reduction in InsP₃ receptor levels only in AR4–2J cells, indicating that receptor clustering does not correlate with receptor down-regulation. The calcium-dependent aggregation of InsP₃ receptors may contribute to the previously observed changes in affinity for InsP₃ in the presence of elevated Ca²⁺ and/or may establish discrete regions within refilled stores with varying capacity to release Ca²⁺ when a subsequent stimulus results in production of InsP₃.     

Mitochondrial Ca2+ Uptake Increases Ca2+ Release from Inositol 1,4,5-Trisphosphate Receptor Clusters in Smooth Muscle Cells

Smooth muscle activities are regulated by inositol 1,4,5-trisphosphate (InsP₃)-mediated increases in cytosolic Ca²⁺ concentration ([Ca²⁺]_c). Local Ca²⁺ release from an InsP₃ receptor (InsP₃R) cluster present on the sarcoplasmic reticulum is termed a Ca²⁺ puff. Ca²⁺ released via InsP₃R may diffuse to adjacent clusters to trigger further release and generate a cell-wide (global) Ca²⁺ rise. In smooth muscle, mitochondrial Ca²⁺ uptake maintains global InsP₃-mediated Ca²⁺ release by preventing a negative feedback effect of high [Ca²⁺] on InsP₃R. Mitochondria may regulate InsP₃-mediated Ca²⁺ signals by operating between or within InsP₃R clusters. In the former mitochondria could regulate only global Ca²⁺ signals, whereas in the latter both local and global signals would be affected. Here whether mitochondria maintain InsP₃-mediated Ca²⁺ release by operating within (local) or between (global) InsP₃R clusters has been addressed. Ca²⁺ puffs evoked by localized photolysis of InsP₃ in single voltage-clamped colonic smooth muscle cells had amplitudes of 0.5–4.0 F/F₀, durations of ∼112 ms at half-maximum amplitude, and were abolished by the InsP₃R inhibitor 2-aminoethoxydiphenyl borate. The protonophore carbonyl cyanide 3-chloropheylhydrazone and complex I inhibitor rotenone each depolarized ΔΨ_M to prevent mitochondrial Ca²⁺ uptake and attenuated Ca²⁺ puffs by ∼66 or ∼60%, respectively. The mitochondrial uniporter inhibitor, RU360, attenuated Ca²⁺ puffs by ∼62%. The “fast” Ca²⁺ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acted like mitochondria to prolong InsP₃-mediated Ca²⁺ release suggesting that mitochondrial influence is via their Ca²⁺ uptake facility. These results indicate Ca²⁺ uptake occurs quickly enough to influence InsP₃R communication at the intra-cluster level and that mitochondria regulate both local and global InsP₃-mediated Ca²⁺ signals.     

The Local Control of Cytosolic Ca2+ as a Propagator of CNS Communication—Integration of Mitochondrial Transport Mechanisms and Cellular Responses

Ca²⁺ signals propagate in wave form along individual cells of the central nervous system(CNS) and through networks of connected cells of neuronal and multiple glial cell types. Inorder for wave fronts to convey information, signaling mechanisms are required that allowwaves to propagate reproducibly and without decrement in signal strength over long distances.CNS Ca²⁺ waves are under specific integrated local control, made possible by interactions atlocal subcellular microdomains between endoplasmic reticulum and mitochondria. Activemitochondria located near the mouth of inositol trisphosphate receptor (InsP₃R) channel clustersin glia take up Ca²⁺, which may prevent a buildup of Ca²⁺ around the InsP₃R channel, therebydecreasing the rate of Ca²⁺-induced receptor inactivation, and prolonging channel open time.Mitochondria may amplify InsP;i3-dependent Ca2;pl signals by a transient permeability transitionin response to Ca²⁺ uptake into the mitochondrion. Other evidence suggests privileged accessinto mitochondria for Ca²⁺ entering neurons by glutamatergic receptor channels. This enablesspecific signal modulation as the Ca²⁺ wave is propagated into neurons, such that mitochondrialocated close to glutamate channels can prolong the neuronal cytosolic response time bysuccessive uptake and release of Ca²⁺. Disruption of mitochondrial function deregulates theability of CNS-derived cells to undergo normal Ca²⁺ signaling and wave propagation.     

Post-translational Regulation of the Type III Inositol 1,4,5-Trisphosphate Receptor by miRNA-506

The type III isoform of the inositol 1,4,5-trisphosphate receptor (InsP₃R3) is apically localized and triggers Ca²⁺ waves and secretion in a number of polarized epithelia. However, nothing is known about epigenetic regulation of this InsP3R isoform. We investigated miRNA regulation of InsP₃R3 in primary bile duct epithelia (cholangiocytes) and in the H69 cholangiocyte cell line, because the role of InsP₃R3 in cholangiocyte Ca²⁺ signaling and secretion is well established and because loss of InsP₃R3 from cholangiocytes is responsible for the impairment in bile secretion that occurs in a number of liver diseases. Analysis of the 3′-UTR of human InsP₃R3 mRNA revealed two highly conserved binding sites for miR-506. Transfection of miR-506 mimics into cell lines expressing InsP₃R3–3′UTR-luciferase led to decreased reporter activity, whereas co-transfection with miR-506 inhibitors led to enhanced activity. Reporter activity was abrogated in isolated mutant proximal or distal miR-506 constructs in miR-506-transfected HEK293 cells. InsP₃R3 protein levels were decreased by miR-506 mimics and increased by inhibitors, and InsP₃R3 expression was markedly decreased in H69 cells stably transfected with miR-506 relative to control cells. miR-506-H69 cells exhibited a fibrotic signature. In situ hybridization revealed elevated miR-506 expression in vivo in human-diseased cholangiocytes. Histamine-induced, InsP₃-mediated Ca²⁺ signals were decreased by 50% in stable miR-506 cells compared with controls. Finally, InsP₃R3-mediated fluid secretion was significantly decreased in isolated bile duct units transfected with miR-506, relative to control IBDU. Together, these data identify miR-506 as a regulator of InsP₃R3 expression and InsP₃R3-mediated Ca²⁺ signaling and secretion.     

Distribution of inositol 1,4,5-trisphosphate receptor isoforms, SERCA isoforms and Ca binding proteins in RBLm2H3 rat basophilic leukemia cells

RBL-2H3 rat basophilic leukemia cells were homogenized and fractionated. A fraction F3 obtained by differential centrifugation was 6-fold enriched in [3H]-inositol 1,4,5-trisphosphate (InsP3) binding activity, while the NADH-cytochrome c oxidoreductase and sulphatase-C activities were only 3.8- and 2.9-fold enriched, respectively. Furthermore, the three InsP3 receptor (InsP3R) isoforms, two sarco/endoplasmic reticulum Ca ²⁺ ATPase (SERCA) isoforms (2b and 3) as well as four Ca ²⁺ binding proteins (calreticulin, calnexin, protein disulfide isomerase (PDI) and BiP), were present in this fraction. Fraction F3 was, therefore, further purified on a discontinuous sucrose density gradient, and the 3 resulting fractions were analyzed. The InsP₃ binding sites were distributed over the gradient and did not co-migrate with the RNA. We examined the relative content of the three InsP3R isoforms, of both SERCA2b and 3, as well as that of the four Ca ²⁺ binding proteins in fraction F3 and the sucrose density gradient fractions. Ins P₃R-1 and InsP3R-2 showed a similar distribution, with the highest level in the light and intermediate density fractions. InsP₃R-3 distributed differently, with the highest level in the intermediate density fraction. Both SERCA isoforms distributed similarly to InsP₃R-1 and InsP₃R-2. SERCA3 was present at a very low level in the high density fraction. Calreticulin and BiP showed a pattern similar to that of InsP₃R-1 and InsP₃R-2 and the SERCs. PDI was clearly enriched in the light density fraction while calnexin was broadly distributed. These results indicate a heterogeneous distribution of the three InsP₃R isoforms, the two SERCA isoforms and the four Ca²⁺ binding proteins investigated. This heterogeneity may underlie specialization of the Ca²⁺ stores and the subsequent initiation of intracellular Ca²⁺ signals.     

Gold(I)-NHC complexes of antitumoral diarylimidazoles: structures, cellular uptake routes and anticancer activities

Five new heterocyclic gold carbene complexes were prepared, four chlorido-[1,3-dimethyl-4,5-diarylimidazol-2-ylidene]gold complexes 6a-d and a chlorido-[1,3-dibenzylimidazol-2-ylidene]gold complex 11, and three of them were characterised by X-ray single crystal analyses. They were tested for cytotoxicity against a panel of four human cancer cell lines and non-malignant fibroblasts, for tubulin interaction, and for the pathways of their uptake into 518A2 melanoma cells. All complexes showed cytotoxic activity in the micromolar IC₅₀ range with distinct selectivities for certain cell lines. In stark contrast to related metal-free 1-methyl-4,5-diarylimidazoles, the complexes 6 and 11 did not noticeably inhibit the polymerisation of tubulin to give microtubules. The cellular uptake of complexes 6 occurred mainly via the copper transporter (Ctr1) and the organic cation transporters (OCT-1/2). Complex 11 was accumulated preferentially via the organic cation transporters and by Na⁺/K⁺-dependent endocytosis. The new gold carbene complexes seem to operate by a mechanism different from that of the parent 1-methylimidazolium ligands.     

Ca<Superscript>2+</Superscript> Dynamics in the Secretory Vesicles of Neurosecretory PC12 and INS1 Cells

We have investigated the dynamics of the free [Ca²⁺] inside the secretory granules of neurosecretory PC12 and INS1 cells using a low-Ca²⁺-affinity aequorin chimera fused to synaptobrevin-2. The steady-state secretory granule [Ca²⁺] ([Ca²⁺]_SG] was around 20–40 μM in both cell types, about half the values previously found in chromaffin cells. Inhibition of SERCA-type Ca²⁺ pumps with thapsigargin largely blocked Ca²⁺ uptake by the granules in Ca²⁺-depleted permeabilized cells, and the same effect was obtained when the perfusion medium lacked ATP. Consistently, the SERCA-type Ca²⁺ pump inhibitor benzohydroquinone induced a rapid release of Ca²⁺ from the granules both in intact and permeabilized cells, suggesting that the continuous activity of SERCA-type Ca²⁺ pumps is essential to maintain the steady-state [Ca²⁺]_SG. Both inositol 1,4,5-trisphosphate (InsP₃) and caffeine produced a rapid Ca²⁺ release from the granules, suggesting the presence of InsP₃ and ryanodine receptors in the granules. The response to high-K⁺ depolarization was different in both cell types, a decrease in [Ca²⁺]_SG in PC12 cells and an increase in [Ca²⁺]_SG in INS1 cells. The difference may rely on the heterogeneous response of different vesicle populations in each cell type. Finally, increasing the glucose concentration triggered a decrease in [Ca²⁺]_SG in INS1 cells. In conclusion, our data show that the secretory granules of PC12 and INS1 cells take up Ca²⁺ through SERCA-type Ca²⁺ pumps and can release it through InsP₃ and ryanodine receptors, supporting the hypothesis that secretory granule Ca²⁺ may be released during cell stimulation and contribute to secretion.     

Regulation of Ca2+ Release by InsP3 in Single Guinea Pig Hepatocytes and Rat Purkinje Neurons

The repetitive spiking of free cytosolic [Ca²⁺] ([Ca²⁺]_i) during hormonal activation of hepatocytes depends on the activation and subsequent inactivation of InsP₃-evoked Ca²⁺ release. The kinetics of both processes were studied with flash photolytic release of InsP₃ and time resolved measurements of [Ca²⁺]_i in single cells. InsP₃ evoked Ca²⁺ flux into the cytosol was measured as d[Ca²⁺]_i/dt, and the kinetics of Ca²⁺ release compared between hepatocytes and cerebellar Purkinje neurons. In hepatocytes release occurs at InsP₃ concentrations greater than 0.1–0.2 μM. A comparison with photolytic release of metabolically stable 5-thio-InsP₃ suggests that metabolism of InsP₃ is important in determining the minimal concentration needed to produce Ca²⁺ release. A distinct latency or delay of several hundred milliseconds after release of low InsP₃ concentrations decreased to a minimum of 20–30 ms at high concentrations and is reduced to zero by prior increase of [Ca²⁺]_i, suggesting a cooperative action of Ca²⁺ in InsP₃ receptor activation. InsP₃-evoked flux and peak [Ca²⁺]_i increased with InsP₃ concentration up to 5–10 μM, with large variation from cell to cell at each InsP₃ concentration. The duration of InsP₃-evoked flux, measured as 10–90% risetime, showed a good reciprocal correlation with d[Ca²⁺]_i/dt and much less cell to cell variation than the dependence of flux on InsP₃ concentration, suggesting that the rate of termination of the Ca²⁺ flux depends on the free Ca²⁺ flux itself. Comparing this data between hepatocytes and Purkinje neurons shows a similar reciprocal correlation for both, in hepatocytes in the range of low Ca²⁺ flux, up to 50 μM · s⁻¹ and in Purkinje neurons at high flux up to 1,400 μM · s⁻¹. Experiments in which [Ca²⁺]_i was controlled at resting or elevated levels support a mechanism in which InsP₃-evoked Ca²⁺ flux is inhibited by Ca²⁺ inactivation of closed receptor/channels due to Ca²⁺ accumulation local to the release sites. Hepatocytes have a much smaller, more prolonged InsP₃-evoked Ca²⁺ flux than Purkinje neurons. Evidence suggests that these differences in kinetics can be explained by the much lower InsP₃ receptor density in hepatocytes than Purkinje neurons, rather than differences in receptor isoform, and, more generally, that high InsP₃ receptor density promotes fast rising, rapidly inactivating InsP₃-evoked [Ca²⁺]_i transients.     

Inositol trisphosphate mediates the action of hypertrehalosemic hormone on fat body of the American cockroach, Periplaneta americana

The rate of synthesis of inositol trisphosphate (InsP₃) in trophocytes derived from disaggregated cockroach (Periplaneta americana) fat body increases following treatment of the cells with hypertrehalosemic hormone I or II (HTH-I, -II) in vitro. Trophocytes preloaded with [³H]inositol display a significant increase in InsP₃ synthesis as early as 15 s after addition of the hormone. When the trophocytes are pre-incubated with LiCl and subsequently incubated with HTH the [³H] content of the InsP₃ fraction is greater than that found with HTH alone. This is taken as evidence that inositol monophosphate phosphatase is part of the mechanism for clearing InsP₃ from the cytosol. In contrast to HTH, octopamine, which is also capable of exerting a hypertrehalosemic effect in the cockroach, does not increase the synthesis of InsP₃. 1-Octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH₃), a potent and selective inhibitor of phosphatidylinositol phospholipase C, blocks the activation of phosphorylase by HTH-I as well as the hypertrehalosemic effect induced by the hormone.     

Characterisation and Distribution of Inositol Polyphosphate and Ryanodine Receptors in the Rat Brain

Abstract: The regional distribution of inositol 1,4,5-trisphosphate (InsP₃), inositol 1,3,4,5-tetrakisphosphate (InsP₄), and ryanodine binding sites has been characterised and compared in the rat brain using radioligand binding assays. Cortical [³H]InsP₃ binding indicated similar positional and stereospecificity as observed in other tissues, with 100-fold selectivity for lnsP₃ over InsP₄. Similarly, high-affinity [³²P]InsP₄ binding also showed a high degree of positional specificity, with a 1,000-fold selectivity for InsP₄ over InsP₃. Initial characterisation of [³H]ryanodine binding to cortical membranes demonstrated that specific binding was highly dependent on high salt and micromolar Ca²⁺ concentrations and inhibited by Ca²⁺ levels of >1 mM. [³H]-Ryanodine binding was also enhanced by β,γ-methylene-adenosine 5′-trisphosphate and caffeine and inhibited by magnesium and ruthenium red (K_i= 0.81 μM). However, dantrolene (300 μM) was ineffective on the binding. Therefore, although the results indicate a greater similarity to the binding properties of the Ca²⁺-induced Ca²⁺ release channel isoform present in skeletal, rather than cardiac, muscle, it does not appear to be identical. Detailed binding analysis of ryanodine and polyphosphate sites, with the exception of ruthenium red, indicated no interaction between binding sites. Ruthenium red markedly enhanced the binding of both [³H]InsP₃ and [³²P]InsP₄, an effect most probably due to nonspecific complex formation. Regional binding of InP₃, InsP₄, and ryanodine in the rat brain was of similar affinity for each ligand in each area, but the density profile for each ligand was clearly different. The highest density of InsP₃ sites was in the cerebellum, whereas the highest density of ryanodine sites was in the hippocampus. High-affinity InsP₄ sites showed less regional diversity, with highest densities in the cerebellum, cortex, and hippocampus. However, in each area studied the density of sites followed the order InsP₃ > InsP₄ > ryanodine.     

Temperature-dependence and conformational basis of inositol 1,4,5-trisphosphate receptor regulated by Ca2+

The inositol 1,4,5-trisphosphate (InsP₃) receptor was purified from bovine cerebellum and reconstituted in liposomes composed of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) (1:1) successfully. No effect of Ca²⁺ concentration on [³H]-InsP₃ binding to unreconstituted InsP₃ receptor could be observed either at 4°C or at 25°C, whereas the effect of [Ca²⁺] on reconstituted InsP₃ receptor depended on the temperature. The Ca²⁺ concentration outside the proteolipsome ([Ca²⁺]_o) had no detectable effect on InsP₃ binding to InsP₃ receptor at 4°C. In contrast, with increase of [Ca²⁺]_o from 0 to 100 nmol/L at 25°C, the InsP₃ binding activity increased gradually. Then the InsP₃ binding activity was decreased drastically at higher [Ca²⁺]_o and inhibited entirely at 50 μmol/L [Ca²⁺]_o. Conformational studies on intrinsic fluorescence of the reconstituted InsP₃ receptor and its quenching by KI and HB indicated that the global conformation of reconstituted InsP₃ receptor could not be affected by [Ca²⁺]_o at 4°C. While at 25°C, the effects of 10 μmol/L [Ca²⁺]_o on global, membrane and cytoplasmic conformation of the reconstituted InsP₃ receptor were different significantly from that of 100 nmol/L [Ca²⁺]_o.     

Potentiometric and P NMR studies on inositol phosphates and their interaction with iron(III) ions

Potentiometric, conductometric and ³¹P NMR titrations have been applied to study interactions between myo-inositol hexakisphosphate (phytic acid), (±)-myo-inositol 1,2,3,5-tetrakisphosphate and (±)-myo-inositol 1,2,3-trisphosphate with iron(III) ions. Potentiometric and conductometric titrations of myo-inositol phosphates show that addition of iron increases acidity and consumption of hydroxide titrant. By increasing the Fe(III)/InsP₆ ratio (from 0.5 to 4) 3 mol of protons are released per 2 mol of iron(III). At first, phytates coordinate iron octahedrally between P2 and P1,3. The second coordination site represents P5 and neighbouring P4,6 phosphate groups. Complexation is accompanied with the deprotonation of P1,3 and P4,6 phosphate oxygens. At higher concentration of iron(III) intermolecular P–O–Fe–O–P bonds trigger formation of a polymeric network and precipitation of the amorphous Fe(III)–InsP₆ aggregates. ³¹P NMR titration data complement the above results and display the largest chemical shift changes at pD values between 5 and 10 in agreement with strong interactions between iron and myo-inositol phosphates. The differences in T₁ relaxation times of phosphorous atoms have shown that phosphate groups at positions 1, 2 and 3 are complexated with iron(III). The interactions between iron(III) ions and inositol phosphates depend significantly on the metal to ligand ratio and an attempt to coordinate more than two irons per InsP₆ molecule results in an unstable heterogeneous system.     

Ca2+ dependence of inositol 1,4,5-trisphosphate-induced Ca2+ release in renal epithelial LLC-PK1 cells

We have studied arginine vasopressin (AVP)-, thapsigargin- and inositol 1,4,5-trisphosphate (InsP₃)-mediated Ca²⁺ release in renal epithelial LLC-PK₁ cells. AVP-induced changes in the intracellular free calcium concentration ([Ca²⁺]_i) were studied in indo-1 loaded single cells by confocal laser cytometry. AVP-mediated Ca²⁺ mobilization was also observed in the absence of extracellular Ca²⁺, but was completely abolished after depletion of the intracellular Ca²⁺ stores by 2 μM thapsigargin. Using ⁴⁵Ca²⁺ fluxes in saponin-permeabilized cell monolayers, we have analysed how InsP₃ affected the Ca²⁺ content of nonmitochondrial Ca²⁺ pools in different loading and release conditions. Less than 10% of the Ca²⁺ was taken up in a thapsigargin-insensitive pool when loading was performed in a medium containing 0.1 μM Ca²⁺. The thapsigargin-insensitive compartment amounted to 35% in the presence of 110 μM Ca²⁺, but Ca²⁺ sequestered in this pool could not be released by InsP₃. The thapsigargin-sensitive Ca²⁺ pool, in contrast, was nearly completely InsP₃ sensitive. A submaximal [InsP₃], however, released only a fraction of the sequestered Ca²⁺. This fraction was dependent on the cytosolic as well as on the luminal [Ca²⁺]. The cytosolic free [Ca²⁺] affected the InsP₃-induced Ca²⁺ release in a biphasic way. Maximal sensitivity toward InsP₃ was found at a free cytosolic [Ca²⁺] between 0.1 and 0.5 μM, whereas higher cytosolic [Ca²⁺] decreased the InsP₃ sensitivity. Other divalent cations or La³⁺ did not provoke similar inhibitory effects on InsP₃-induced Ca²⁺ release. The luminal free [Ca²⁺] was manipulated by varying the time of incubation of Ca²⁺ -loaded cells in an EGTA-containing medium. Reduction of the Ca²⁺ content to one-third of its initial value resulted in a fivefold decrease in the InsP₃ sensitivity of the Ca²⁺ release. © 1993 Wiley-Liss, Inc.     

1H, 15N, and 13C chemical shift assignments of calcium-bound calcium-binding protein 1 (CaBP1)

Calcium-binding protein 1 (CaBP1) regulates inositol 1,4,5-trisphosphate receptors (InsP₃Rs) and a variety of voltage-gated Ca²⁺ channels in the brain. We report complete NMR chemical shift assignments of Ca²⁺-bound CaBP1 (residues 1–167, BMRB no. 15623).     

Metabolism of inositol 1,4,5-trisphosphate in squid photoreceptors

Summary Inositol 1,4,5-trisphosphate (InsP₃) is rapidly formed in squid photoreceptors in response to light, where it is converted sequenctially into inositol bisphosphate (InsP₂) and inositol monophosphate (InsP₁). All of the InsP₃ appears to be degraded to inositol 1,4-bisphosphate via an InsP₃-phosphatase, which is characterized in this study. The enzyme is water-soluble and present in the light-transducing distal segments of squid photoreceptors. It has a K_m of 50 M for InsP₃, requires Mg⁺⁺ for its activity, is maximally active at neutral pH, specifically hydrolyses the 5-phosphate and is inhibited by 2,3-diphosphoglycerate. In these respects, InsP₃-phosphatase of squid is very similar to the enzymes of other cells. Since no InsP₄ or more highly phosphorylated inositols are found in squid photoreceptors, the InsP₃-phosphatase may be important in the regulation of InsP₃ concentration within these cells.Abbreviations InsP ₁ , InsP ₂ , InsP ₃ , InsP ₄ , InsP ₆ inositol monobis-, tris-, tetrakis-, hexakisphosphate, respectively - 2,3-DPG 2,3-diphosphoglycerate - EDTA ethylene diamine tetraacetic acid - DTT dithiothreitol - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - PMSF phenylmethylsulfonyl fluoride     

Ins(1,4,5)P3 receptor type 1 associates with AKAP9 (AKAP450 variant) and protein kinase A type IIβ in the Golgi apparatus in cerebellar granule cells

Background information. Interconnections between the Ca²⁺ and cAMP signalling pathways can determine the specificity and diversity of the cellular effects mediated by these second messengers. Most cAMP effects are mediated by PKA (protein kinase A), which is anchored close to its membranous substrates by AKAPs (A kinase‐anchoring proteins). In many cell types, the activation of InsP₃R (inositol 1,4,5‐trisphosphate receptor), an endoplasmic reticulum Ca²⁺ channel, is a key event of Ca²⁺ signalling. The phosphorylation of InsP₃R1 by PKA stimulates Ca²⁺ mobilization. This control is thought to be tight, involving the association of PKA with InsP₃R1. The InsP₃R1 isoform predominates in central nervous tissue and its concentration is highest in the cerebellar microsomes. We investigated the complex formed by InsP₃R1 and PKA in this fraction, vith a view to identifying its components and determining its distribution in the cerebellar cortex. Results. Immunoprecipitation experiments showed that InsP₃R1 associated with PKA type IIβ and AKAP450, the longer variant of AKAP9, in sheep cerebellar microsomes. The co‐purification of AKAP450 with InsP₃R1 on heparin‐agarose provided further evidence of the association of these proteins. Immunohistofluorescence experiments on slices of cerebellar cortex showed that AKAP450 was colocalized with InsP₃R1 and RIIβ (regulatory subunit of PKA IIβ) in granule cells, but not in Purkinje cells. AKAP450 was localized in the Golgi apparatus of these two cell types whereas InsP₃R1 was detected in this organelle only in granule cells. Conclusions. Taken together these results suggest that InsP₃R1 forms a complex with AKAP450 and PKAIIβ, localized in the Golgi apparatus of cerebellar granule cells. In contrast, the association of InsP₃R1 with PKA in Purkinje cells would require a different macromolecular complex.     

Single-Channel Properties of Inositol (1,4,5)-Trisphosphate Receptor Heterologously Expressed in HEK-293 Cells

The inositol (1,4,5)-trisphosphate receptor (InsP₃R) mediates Ca²⁺ release from intracellular stores in response to generation of second messenger InsP₃. InsP₃R was biochemically purified and cloned, and functional properties of native InsP₃-gated Ca²⁺ channels were extensively studied. However, further studies of InsP₃R are obstructed by the lack of a convenient functional assay of expressed InsP₃R activity. To establish a functional assay of recombinant InsP₃R activity, transient heterologous expression of neuronal rat InsP₃R cDNA (InsP₃R-I, SI− SII+ splice variant) in HEK-293 cells was combined with the planar lipid bilayer reconstitution experiments. Recombinant InsP₃R retained specific InsP₃ binding properties (K _d = 60 nM InsP₃) and were specifically recognized by anti–InsP₃R-I rabbit polyclonal antibody. Density of expressed InsP₃R-I was at least 20-fold above endogenous InsP₃R background and only 2–3-fold lower than InsP₃R density in rat cerebellar microsomes. When incorporated into planar lipid bilayers, the recombinant InsP₃R formed a functional InsP₃-gated Ca²⁺ channel with 80 pS conductance using 50 mM Ba²⁺ as a current carrier. Mean open time of recombinant InsP₃-gated channels was 3.0 ms; closed dwell time distribution was double exponential and characterized by short (18 ms) and long (130 ms) time constants. Overall, gating and conductance properties of recombinant neuronal rat InsP₃R-I were very similar to properties of native rat cerebellar InsP₃R recorded in identical experimental conditions. Recombinant InsP₃R also retained bell-shaped dependence on cytosolic Ca²⁺ concentration and allosteric modulation by ATP, similar to native cerebellar InsP₃R. The following conclusions are drawn from these results. (a) Rat neuronal InsP₃R-I cDNA encodes a protein that is either sufficient to produce InsP₃-gated channel with functional properties identical to the properties of native rat cerebellar InsP₃R, or it is able to form a functional InsP₃-gated channel by forming a complex with proteins endogenously expressed in HEK-293 cells. (b) Successful functional expression of InsP₃R in a heterologous expression system provides an opportunity for future detailed structure–function characterization of this vital protein.     

Spatially Defined InsP3-Mediated Signaling in Embryonic Stem Cell-Derived Cardiomyocytes

The functional role of inositol 1,4,5-trisphosphate (InsP₃) signaling in cardiomyocytes is not entirely understood but it was linked to an increased propensity for triggered activity. The aim of this study was to determine how InsP₃ receptors can translate Ca²⁺ release into a depolarization of the plasma membrane and consequently arrhythmic activity. We used embryonic stem cell-derived cardiomyocytes (ESdCs) as a model system since their spontaneous electrical activity depends on InsP₃-mediated Ca²⁺ release. [InsP₃]_i was monitored with the FRET-based InsP₃-biosensor FIRE-1 (Fluorescent InsP₃ Responsive Element) and heterogeneity in sub-cellular [InsP₃]_i was achieved by targeted expression of FIRE-1 in the nucleus (FIRE-1nuc) or expression of InsP₃ 5-phosphatase (m43) localized to the plasma membrane. Spontaneous activity of ESdCs was monitored simultaneously as cytosolic Ca²⁺ transients (Fluo-4/AM) and action potentials (current clamp). During diastole, the diastolic depolarization was paralleled by an increase of [Ca²⁺]_i and spontaneous activity was modulated by [InsP₃]_i. A 3.7% and 1.7% increase of FIRE-1 FRET ratio and 3.0 and 1.5 fold increase in beating frequency was recorded upon stimulation with endothelin-1 (ET-1, 100 nmol/L) or phenylephrine (PE, 10 µmol/L), respectively. Buffering of InsP₃ by FIRE-1nuc had no effect on the basal frequency while attenuation of InsP₃ signaling throughout the cell (FIRE-1), or at the plasma membrane (m43) resulted in a 53.7% and 54.0% decrease in beating frequency. In m43 expressing cells the response to ET-1 was completely suppressed. Ca²⁺ released from InsP₃Rs is more effective than Ca²⁺ released from RyRs to enhance I_NCX. The results support the hypothesis that in ESdCs InsP₃Rs form a functional signaling domain with NCX that translates Ca²⁺ release efficiently into a depolarization of the membrane potential.