Stereospecific mobilization of intracellular Ca2+ by inositol 1,4,5-triphosphate. Comparison with inositol 1,4,5-trisphosphorothioate and inositol 1,3,4-trisphosphate.
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ABSTRACT:
SUBMITTER: Strupish J
PROVIDER: S-EPMC1149387 | biostudies-other | 1988 Aug
REPOSITORIES: biostudies-other
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Project description:Cholesterol depletion reversibly abolishes carbachol-evoked Ca(2+) release from inositol (1,4,5)-trisphosphate (IP3)-sensitive stores, without affecting the distribution of IP3 receptors (IP3R) or endoplasmic reticulum, IP3 formation or responses to photolysis of caged IP3. Receptors that stimulate cAMP formation do not alone evoke Ca(2+) signals, but they potentiate those evoked by carbachol. We show that these potentiated signals are entirely unaffected by cholesterol depletion and that, within individual cells, different IP3-sensitive Ca(2+) stores are released by carbachol alone and by carbachol combined with receptors that stimulate cAMP formation. We suggest that muscarinic acetylcholine receptors in lipid rafts deliver IP3 at high concentration to associated IP3R, stimulating them to release Ca(2+). Muscarinic receptors outside rafts are less closely associated with IP3R and provide insufficient local IP3 to activate IP3R directly. These IP3R, probably type 2 IP3R within a discrete Ca(2+) store, are activated only when their sensitivity is increased by cAMP. Sensitization of IP3R by cAMP extends the effective range of signalling by phospholipase C, allowing muscarinic receptors that are otherwise ineffective to recruit additional IP3-sensitive Ca(2+) stores.
| S-EPMC3672942 | biostudies-literature
Project description:Inositol (1,4,5)-trisphosphate receptors (IP(3)Rs) release intracellular Ca(2+) as localized Ca(2+) signals (Ca(2+) puffs) that represent the activity of small numbers of clustered IP(3)Rs spaced throughout the endoplasmic reticulum. Although much emphasis has been placed on estimating the number of active Ca(2+) release channels supporting Ca(2+) puffs, less attention has been placed on understanding the role of cluster microarchitecture. This is important as recent data underscores the dynamic nature of IP(3)R transitions between heterogeneous cellular architectures and the differential behavior of IP(3)Rs socialized into clusters. Here, we applied a high-resolution model incorporating stochastically gating IP(3)Rs within a three-dimensional cytoplasmic space to demonstrate: 1), Ca(2+) puffs are supported by a broad range of clustered IP(3)R microarchitectures; 2), cluster ultrastructure shapes Ca(2+) puff characteristics; and 3), loosely corralled IP(3)R clusters (>200 nm interchannel separation) fail to coordinate Ca(2+) puffs, owing to inefficient triggering and impaired coupling due to reduced Ca(2+)-induced Ca(2+) release microwave velocity (<10 nm/s) throughout the channel array. Dynamic microarchitectural considerations may therefore influence Ca(2+) puff occurrence/properties in intact cells, contrasting with a more minimal role for channel number over the same simulated conditions in shaping local Ca(2+) dynamics.
| S-EPMC3037600 | biostudies-literature
Project description:We have augmented our previous studies [Storey, Shears, Kirk & Michell (1984) Nature (London) 312, 374-376] on the subcellular location and properties of Ins(1,4,5)P3 (inositol 1,4,5-trisphosphate) phosphatases in rat liver and human erythrocytes. We also investigate Ins(1,3,4)P3 (inositol 1,3,4-trisphosphate) metabolism by rat liver. Membrane-bound and cytosolic Ins(1,4,5)P3 phosphatases both attack the 5-phosphate. The membrane-bound enzyme is located on the inner face of the plasma membrane, and there is little or no activity associated with Golgi apparatus. Cytosolic Ins(1,4,5)P3 5-phosphatase (Mr 77,000) was separated by gel filtration from Ins(1,4)P2 (inositol 1,4-bisphosphate) and inositol 1-phosphate phosphatases (Mr 54,000). Ins(1,4,5)P3 5-phosphatase activity in hepatocytes was unaffected by treatment of the cells with insulin, vasopressin, glucagon or dibutyryl cyclic AMP. Ins(1,4,5)P3 5-phosphatase activity in cell homogenates was unaffected by changes in [Ca2+] from 0.1 to 2 microM. After centrifugation of a liver homogenate at 100,000 g, Ins(1,3,4)P3 phosphatase activity was largely confined to the supernatant. The sum of the activities in the supernatant and the pellet exceeded that in the original homogenate. When these fractions were recombined, Ins(1,3,4)P3 phosphatase activity was restored to that observed in unfractionated homogenate. Ins(1,3,4)P3 was produced from Ins(1,3,4,5)P4 (inositol 1,3,4,5-tetrakisphosphate) and was metabolized to a novel InsP2 that was the 3,4-isomer. Ins(1,3,4)P3 phosphatase activity was not changed by 50 mM-Li+ or 0.07 mM-Ins(1,4)P2 alone, but when added together these agents inhibited Ins(1,3,4)P3 metabolism. In Li+-treated and vasopressin-stimulated hepatocytes, Ins(1,4)P2 may reach concentrations sufficient to inhibit Ins(1,3,4)P3 metabolism, with little effect on Ins(1,4,5)P3 hydrolysis.
| S-EPMC1147718 | biostudies-other
Project description:Autosomal dominant polycystic kidney disease is characterized by the loss-of-function of a signaling complex involving polycystin-1 and polycystin-2 (TRPP2, an ion channel of the TRP superfamily), resulting in a disturbance in intracellular Ca(2+) signaling. Here, we identified the molecular determinants of the interaction between TRPP2 and the inositol 1,4,5-trisphosphate receptor (IP(3)R), an intracellular Ca(2+) channel in the endoplasmic reticulum. Glutathione S-transferase pulldown experiments combined with mutational analysis led to the identification of an acidic cluster in the C-terminal cytoplasmic tail of TRPP2 and a cluster of positively charged residues in the N-terminal ligand-binding domain of the IP(3)R as directly responsible for the interaction. To investigate the functional relevance of TRPP2 in the endoplasmic reticulum, we re-introduced the protein in TRPP2(-/-) mouse renal epithelial cells using an adenoviral expression system. The presence of TRPP2 resulted in an increased agonist-induced intracellular Ca(2+) release in intact cells and IP(3)-induced Ca(2+) release in permeabilized cells. Using pathological mutants of TRPP2, R740X and D509V, and competing peptides, we demonstrated that TRPP2 amplified the Ca(2+) signal by a local Ca(2+)-induced Ca(2+)-release mechanism, which only occurred in the presence of the TRPP2-IP(3)R interaction, and not via altered IP(3)R channel activity. Moreover, our results indicate that this interaction was instrumental in the formation of Ca(2+) microdomains necessary for initiating Ca(2+)-induced Ca(2+) release. The data strongly suggest that defects in this mechanism may account for the altered Ca(2+) signaling associated with pathological TRPP2 mutations and therefore contribute to the development of autosomal dominant polycystic kidney disease.
| S-EPMC2881802 | biostudies-literature
Project description:Activation of TRPC3 channels is concurrent with inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R)-mediated intracellular Ca(2+) release and associated with phosphatidylinositol 4,5-bisphosphate hydrolysis and recruitment to the plasma membrane. Here we report that interaction of TRPC3 with receptor for activated C-kinase-1 (RACK1) not only determines plasma membrane localization of the channel but also the interaction of IP(3)R with RACK1 and IP(3)-dependent intracellular Ca(2+) release. We show that TRPC3 interacts with RACK1 via N-terminal residues Glu-232, Asp-233, Glu-240, and Glu-244. Carbachol (CCh) stimulation of HEK293 cells expressing wild type TRPC3 induced recruitment of a ternary TRPC3-RACK1-IP(3)R complex and increased surface expression of TRPC3 and Ca(2+) entry. Mutation of the putative RACK1 binding sequence in TRPC3 disrupted plasma membrane localization of the channel. CCh-stimulated recruitment of TRPC3-RACK1-IP(3)R complex as well as increased surface expression of TRPC3 and receptor-operated Ca(2+) entry were also attenuated. Importantly, CCh-induced intracellular Ca(2+) release was significantly reduced as was RACK1-IP(3)R association without any change in thapsigargin-stimulated Ca(2+) release and entry. Knockdown of endogenous TRPC3 also decreased RACK1-IP(3)R association and decreased CCh-stimulated Ca(2+) entry. Furthermore, an oscillatory pattern of CCh-stimulated intracellular Ca(2+) release was seen in these cells compared with the more sustained pattern seen in control cells. Similar oscillatory pattern of Ca(2+) release was seen after CCh stimulation of cells expressing the TRPC3 mutant. Together these data demonstrate a novel role for TRPC3 in regulation of IP(3)R function. We suggest TRPC3 controls agonist-stimulated intracellular Ca(2+) release by mediating interaction between IP(3)R and RACK1.
| S-EPMC2583283 | biostudies-literature
Project description:The inositol trisphosphate liberated on stimulation of guinea-pig hepatocytes, pancreatic acinar cells and dimethyl sulphoxide-differentiated human myelomonocytic HL-60 leukaemia cells is composed of two isomers, the 1,4,5-trisphosphate and the 1,3,4-trisphosphate. Inositol 1,4,5-trisphosphate was released rapidly, with no measurable latency on hormone stimulation, and, consistent with its proposed role as an intracellular messenger for Ca2+ mobilization, there was good temporal correlation between its formation and Ca2+-mediated events in these tissues. There was a definite latency before an increase in the formation of inositol 1,3,4-trisphosphate could be detected. In all of these tissues, however, it formed a substantial proportion of the total inositol trisphosphate by 1 min of stimulation. In guinea-pig hepatocytes, where inositol trisphosphate increases for at least 30 min after hormone application, inositol 1,3,4-trisphosphate made up about 90% of the total inositol trisphosphate by 5-10 min. In pancreatic acinar cells, pretreatment with 20 mM-Li+ caused an increase in hormone-induced inositol trisphosphate accumulation. This increase was accounted for by a rise in inositol 1,3,4-trisphosphate; inositol 1,4,5-trisphosphate was unaffected. This finding is consistent with the observation that Li+ has no effect on Ca2+-mediated responses in these cells. The role, if any, of inositol 1,3,4-trisphosphate in cellular function is unknown.
| S-EPMC1152864 | biostudies-other
Project description:AIMS:Phospholamban (PLB) is the key regulator of the cardiac Ca2+ pump (SERCA2a)-mediated sarcoplasmic reticulum Ca2+ stores. We recently reported that PLB is highly concentrated in the nuclear envelope (NE) from where it can modulate perinuclear Ca2+ handling of the cardiomyocytes (CMs). Since inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) mediates nuclear Ca2+ release, we examined whether the nuclear pool of PLB regulates IP3-induced nuclear Ca2+ handling. METHODS AND RESULTS:Fluo-4 based confocal Ca2+ imaging was performed to measure Ca2+ dynamics across both nucleus and cytosol in saponin-permeabilized CMs isolated from wild-type (WT) or PLB-knockout (PLB-KO) mice. At diastolic intracellular Ca2+ ([Ca2+]i?=?100?nM), the Fab fragment of the monoclonal PLB antibody (anti-PLB Fab) facilitated the formation and increased the length of spontaneous Ca2+ waves (SCWs) originating from the nuclear region in CMs from WT but not from PLB-KO mice. We next examined nuclear Ca2+ activities at basal condition and after sequential addition of IP3, anti-PLB Fab, and the IP3R inhibitor 2-aminoethoxydiphenyl borate (2-APB) at a series of [Ca2+]i. In WT mice, at 10?nM [Ca2+]i where ryanodine receptor (RyR2) based spontaneous Ca2+ sparks rarely occurred, IP3 increased fluorescence amplitude (F/F0) of overall nuclear region to 1.19?±?0.02. Subsequent addition of anti-PLB Fab significantly decreased F/F0 to 1.09?±?0.02. At 50?nM [Ca2+]i, anti-PLB Fab not only decreased the overall nuclear F/F0 previously elevated by IP3, but also increased the amplitude and duration of spark-like nuclear Ca2+ release events. These nuclear Ca2+ releases were blocked by 2-APB. At 100?nM [Ca2+]i, IP3 induced short SCWs originating from nucleus. Anti-PLB Fab transformed those short waves into long SCWs with propagation from the nucleus into the cytosol. In contrast, neither nuclear nor cytosolic Ca2+ dynamics was affected by anti-PLB Fab in CMs from PLB-KO mice in all these conditions. Furthermore, in WT CMs pretreated with RyR2 blocker tetracaine, IP3 and anti-PLB Fab still increased the magnitude of nuclear Ca2+ release but failed to regenerate SCWs. Finally, anti-PLB Fab increased low Ca2+ affinity mag-fluo 4 fluorescence intensity in the lumen of NE of nuclei isolated from WT but not in PLB-KO mice. CONCLUSION:PLB regulates nuclear Ca2+ handling. By increasing Ca2+ uptake into lumen of the NE and perhaps other perinuclear membranes, the acute reversal of PLB inhibition decreases global Ca2+ concentration at rest in the nucleoplasm, and increases Ca2+ release into the nucleus, through mechanisms involving IP3R and RyR2 in the vicinity.
| S-EPMC6205300 | biostudies-literature
Project description:FSH binds to its receptor (FSHR) on target cells in the ovary and testis, to regulate oogenesis and spermatogenesis, respectively. The signaling cascades activated after ligand binding are extremely complex and have been shown to include protein kinase A, mitogen-activated protein kinase, phosphatidylinositol 3-kinase/protein kinase B, and inositol 1,4,5-trisphosphate-mediated calcium signaling pathways. The adapter protein APPL1 (Adapter protein containing Pleckstrin homology domain, Phosphotyrosine binding domain and Leucine zipper motif), which has been linked to an assortment of other signaling proteins, was previously identified as an interacting protein with FSHR. Thus, alanine substitution mutations in the first intracellular loop of FSHR were generated to determine which residues are essential for FSHR-APPL1 interaction. Three amino acids were essential; when any one of them was altered, APPL1 association with FSHR mutants was abrogated. Two of the mutants (L377A and F382A) that displayed poor cell-surface expression were not studied further. Substitution of FSHR-K376A did not affect FSH binding or agonist-stimulated cAMP production in either transiently transfected human embryonic kidney cells or virally transduced human granulosa cells (KGN). In the KGN line, as well as primary cultures of rat granulosa cells transduced with wild type or mutant receptor, FSH-mediated progesterone or estradiol production was not affected by the mutation. However, in human embryonic kidney cells inositol 1,4,5-trisphosphate production was curtailed and KGN cells transduced with FSHR-K376A evidenced reduced Ca(2+) mobilization from intracellular stores after FSH treatment.
| S-EPMC3060640 | biostudies-literature
Project description:Fertilization induces a species-specific Ca(2+) transient with specialized spatial and temporal dynamics, which are essential to temporally encode egg activation events such as the block to polyspermy and resumption of meiosis. Eggs acquire the competence to produce the fertilization-specific Ca(2+) transient during oocyte maturation, which encompasses dramatic potentiation of inositol 1,4,5-trisphosphate (IP(3))-dependent Ca(2+) release. Here we show that increased IP(3) receptor (IP(3)R) sensitivity is initiated at the germinal vesicle breakdown stage of maturation, which correlates with maturation promoting factor (MPF) activation. Extensive phosphopeptide mapping of the IP(3)R resulted in approximately 70% coverage and identified three residues, Thr-931, Thr-1136, and Ser-114, which are specifically phosphorylated during maturation. Phospho-specific antibody analyses show that Thr-1136 phosphorylation requires MPF activation. Activation of either MPF or the mitogen-activated protein kinase cascade independently, functionally sensitizes IP(3)-dependent Ca(2+) release. Collectively, these data argue that the kinase cascades driving meiotic maturation potentiates IP(3)-dependent Ca(2+) release, possibly trough direct phosphorylation of the IP(3)R.
| S-EPMC2740445 | biostudies-literature
Project description:Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are tetrameric intracellular Ca(2+)-release channels with each subunit containing a binding site for IP3in the amino terminus. We provide evidence that four IP3molecules are required to activate the channel under diverse conditions. Comparing the concentration-response relationship for binding and Ca(2+)release suggested that IP3Rs are maximally occupied by IP3before substantial Ca(2+)release occurs. We showed that ligand binding-deficient subunits acted in a dominant-negative manner when coexpressed with wild-type monomers in the chicken immune cell line DT40-3KO, which lacks all three genes encoding IP3R subunits, and confirmed the same effect in an IP3R-null human cell line (HEK-3KO) generated by CRISPR/Cas9 technology. Using dimeric and tetrameric concatenated IP3Rs with increasing numbers of binding-deficient subunits, we addressed the obligate ligand stoichiometry. The concatenated IP3Rs with four ligand-binding sites exhibited Ca(2+)release and electrophysiological properties of native IP3Rs. However, IP3failed to activate IP3Rs assembled from concatenated dimers consisting of one binding-competent and one binding-deficient mutant subunit. Similarly, IP3Rs containing two monomers of IP3R2short, an IP3binding-deficient splice variant, were nonfunctional. Concatenated tetramers containing only three binding-competent ligand-binding sites were nonfunctional under a wide range of activating conditions. These data provide definitive evidence that IP3-induced Ca(2+)release only occurs when each IP3R monomer within the tetramer is occupied by IP3, thereby ensuring fidelity of Ca(2+)release.
| S-EPMC4850551 | biostudies-literature