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1
MACKRILL, John J., Robert A. WILCOX, Atsushi MIYAWAKI, Katsuhiko MIKOSHIBA, Stefan R. NAHORSKI, and R. A. John CHALLISS. "Stable overexpression of the type-1 inositol 1,4,5-trisphosphate receptor in L fibroblasts: subcellular distribution and functional consequences." Biochemical Journal 318, no. 3 (September 15, 1996): 871–78. http://dx.doi.org/10.1042/bj3180871.
Full textAbstract:
InsP3 receptor (InsP3R)/Ca2+-release channels differ markedly in abundance in different tissues/cell types and InsP3R expression levels may be modulated in response to a variety of external cues. Cell lines overexpressing InsP3Rs will provide useful models for the study of the influence of receptor density and subtype on InsP3-mediated Ca2+ signalling. We have investigated the properties of InsP3Rs in mouse L fibroblast cell lines transfected with either type-1 InsP3R cDNA (L15) or vector control (Lvec). L15 cells express approximately eightfold higher levels of the type-1 InsP3R protein than Lvec cells, as assessed by radioligand binding and immunoblotting. Increased expression was stable since it did not alter over ten cell passages. Both L15 and Lvec cells express predominantly the type-1 InsP3R isoform, indicating that functional differences in the InsP3-mediated Ca2+ signalling in these cell lines are due to alteration in the levels of receptor rather than changes in the isoform expressed. Type-1 InsP3R in L15 cells is largely associated with subcellular membrane fractions bearing the sarco/endoplasmic reticulum Ca2+ ATPase pump, appropriate for rapidly exchanging Ca2+ pools. Functionally, there is an approximately fourfold increase in the sensitivity of permeabilized L15-cell Ca2+ mobilization in response to increasing concentrations of Ins(1,4,5)P3. This study indicates that L15/Lvec cells provide a suitable model for studying the effects of InsP3R expression level on InsP3-induced Ca2+ mobilization.
2
Mak, Don-On Daniel, Sean McBride, and J. Kevin Foskett. "Regulation by Ca2+ and Inositol 1,4,5-Trisphosphate (Insp3) of Single Recombinant Type 3 Insp3 Receptor Channels." Journal of General Physiology 117, no. 5 (April 30, 2001): 435–46. http://dx.doi.org/10.1085/jgp.117.5.435.
Full textAbstract:
The inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) is an endoplasmic reticulum–localized Ca2+-release channel that controls complex cytoplasmic Ca2+ signaling in many cell types. At least three InsP3Rs encoded by different genes have been identified in mammalian cells, with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. To examine regulation of channel gating of the type 3 isoform, recombinant rat type 3 InsP3R (r-InsP3R-3) was expressed in Xenopus oocytes, and single-channel recordings were obtained by patch-clamp electrophysiology of the outer nuclear membrane. Gating of the r-InsP3R-3 exhibited a biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). In the presence of 0.5 mM cytoplasmic free ATP, r-InsP3R-3 gating was inhibited by high [Ca2+]i with features similar to those of the endogenous Xenopus type 1 InsP3R (X-InsP3R-1). Ca2+ inhibition of channel gating had an inhibitory Hill coefficient of ∼3 and half-maximal inhibiting [Ca2+]i (Kinh) = 39 μM under saturating (10 μM) cytoplasmic InsP3 concentrations ([InsP3]). At [InsP3] < 100 nM, the r-InsP3R-3 became more sensitive to Ca2+ inhibition, with the InsP3 concentration dependence of Kinh described by a half-maximal [InsP3] of 55 nM and a Hill coefficient of ∼4. InsP3 activated the type 3 channel by tuning the efficacy of Ca2+ to inhibit it, by a mechanism similar to that observed for the type 1 isoform. In contrast, the r-InsP3R-3 channel was uniquely distinguished from the X-InsP3R-1 channel by its enhanced Ca2+ sensitivity of activation (half-maximal activating [Ca2+]i of 77 nM instead of 190 nM) and lack of cooperativity between Ca2+ activation sites (activating Hill coefficient of 1 instead of 2). These differences endow the InsP3R-3 with high gain InsP3–induced Ca2+ release and low gain Ca2+–induced Ca2+ release properties complementary to those of InsP3R-1. Thus, distinct Ca2+ signals may be conferred by complementary Ca2+ activation properties of different InsP3R isoforms.
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Yule, D. I., S. V. Straub, and J. I. E. Bruce. "Modulation of Ca2+ oscillations by phosphorylation of Ins(1,4,5)P3 receptors." Biochemical Society Transactions 31, no. 5 (October 1, 2003): 954–57. http://dx.doi.org/10.1042/bst0310954.
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Activation of InsP3Rs (InsP3 receptors) represents the major mechanism underlying intracellular calcium release in non-excitable cells such as hepatocytes and exocrine cells from the pancreas and salivary glands. Modulation of calcium release through InsP3Rs is therefore a major route whereby the temporal and spatial characteristics of calcium waves and oscillations can potentially be ‘shaped’. In this study, the functional consequences of phosphoregulation of InsP3Rs were investigated. Pancreatic and parotid acinar cells express all three types of InsP3R in differing abundance, and all are potential substrates for phosphoregulation. PKA (protein kinase A)-mediated phosphorylation of InsP3Rs in pancreatic acinar cells resulted in slowed kinetics of calcium release following photo-release of InsP3. In contrast, activation of PKA in parotid cells resulted in a marked potentiation of calcium release. In pancreatic acinar cells the predominant InsP3R isoform phosphorylated was the type 3 receptor, while the type 2 receptor was markedly phosphorylated in parotid acinar cells. In order to further decipher the effects of phosphorylation on individual InsP3R subtypes, DT-40 cell lines expressing homotetramers of a single isoform of InsP3R were utilized. These data demonstrate that phosphoregulation of InsP3Rs results in subtype-specific effects and may play a role in the specificity of calcium signals by ‘shaping’ the spatio-temporal profile of the response.
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Kaznacheyeva, Elena, Vitalie D. Lupu, and Ilya Bezprozvanny. "Single-Channel Properties of Inositol (1,4,5)-Trisphosphate Receptor Heterologously Expressed in HEK-293 Cells." Journal of General Physiology 111, no. 6 (June 1, 1998): 847–56. http://dx.doi.org/10.1085/jgp.111.6.847.
Full textAbstract:
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 (Kd = 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.
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DAVIS, Richard J., R. A. John CHALLISS, and Stefan R. NAHORSKI. "Enhanced purinoceptor-mediated Ca2+ signalling in L-fibroblasts overexpressing type 1 inositol 1,4,5-trisphosphate receptors." Biochemical Journal 341, no. 3 (July 26, 1999): 813–20. http://dx.doi.org/10.1042/bj3410813.
Full textAbstract:
Mouse L-fibroblast cells stably transfected with either type 1 Ins(1,4,5)P3 receptor (InsP3R) cDNA (L15) or the vector control (Lvec) have been used to investigate the functional consequences of increased InsP3R density on receptor-mediated Ca2+ signalling. L15 cells express approx. 8-fold higher levels of the type 1 InsP3R compared with Lvec cells, which endogenously express essentially only the type 1 InsP3R protein. Stimulation of Lvec and L15 cells with UTP or ATP increased cytosolic Ca2+ concentration to a greater extent in L15 cells at all agonist concentrations. UTP and ATP were equipotent, suggestive of the presence of endogenous cell-surface metabotropic P2Y2-purinoceptors. In both cell clones the purinoceptors were coupled via pertussis-toxin-insensitive G-protein(s) to phospholipase C activation, resulting in similar concentration-dependent accumulations of InsP3. Single-cell microfluorimetry revealed that overexpression of InsP3Rs reduced the threshold for purinoceptor-mediated Ca2+ signalling. L-fibroblasts also exhibited temporally complex sinusoidal cytosolic Ca2+ oscillations in response to submaximal agonist concentrations, with significant increases in oscillatory frequencies exhibited by cells overexpressing InsP3Rs. Sustainable oscillatory responses were dependent on Ca2+ entry and, at higher agonist concentrations, cytosolic Ca2+ oscillations were superseded by biphasic peak-and-plateau Ca2+ responses. Overexpression of InsP3Rs in L15 cells resulted in a 4-fold reduction in the threshold for this change in the temporal pattern of Ca2+ mobilization. These data provide the first direct evidence demonstrating that altering the expression of the type 1 InsP3R significantly affects receptor-mediated InsP3-induced Ca2+ mobilization.
6
Ashhad, Sufyan, Daniel Johnston, and Rishikesh Narayanan. "Activation of InsP3 receptors is sufficient for inducing graded intrinsic plasticity in rat hippocampal pyramidal neurons." Journal of Neurophysiology 113, no. 7 (April 2015): 2002–13. http://dx.doi.org/10.1152/jn.00833.2014.
Full textAbstract:
The synaptic plasticity literature has focused on establishing necessity and sufficiency as two essential and distinct features in causally relating a signaling molecule to plasticity induction, an approach that has been surprisingly lacking in the intrinsic plasticity literature. In this study, we complemented the recently established necessity of inositol trisphosphate (InsP3) receptors (InsP3R) in a form of intrinsic plasticity by asking if InsP3R activation was sufficient to induce intrinsic plasticity in hippocampal neurons. Specifically, incorporation of d-myo-InsP3 in the recording pipette reduced input resistance, maximal impedance amplitude, and temporal summation but increased resonance frequency, resonance strength, sag ratio, and impedance phase lead. Strikingly, the magnitude of plasticity in all these measurements was dependent on InsP3 concentration, emphasizing the graded dependence of such plasticity on InsP3R activation. Mechanistically, we found that this InsP3-induced plasticity depended on hyperpolarization-activated cyclic nucleotide-gated channels. Moreover, this calcium-dependent form of plasticity was critically reliant on the release of calcium through InsP3Rs, the influx of calcium through N-methyl-d-aspartate receptors and voltage-gated calcium channels, and on the protein kinase A pathway. Our results delineate a causal role for InsP3Rs in graded adaptation of neuronal response dynamics, revealing novel regulatory roles for the endoplasmic reticulum in neural coding and homeostasis.
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NOSYREVA, Elena, Tomoya MIYAKAWA, Zhengnan WANG, Lyuba GLOUCHANKOVA, Akiko MIZUSHIMA, Masamitsu IINO, and Ilya BEZPROZVANNY. "The high-affinity calcium–calmodulin-binding site does not play a role in the modulation of type 1 inositol 1,4,5-trisphosphate receptor function by calcium and calmodulin." Biochemical Journal 365, no. 3 (August 1, 2002): 659–67. http://dx.doi.org/10.1042/bj20011789.
Full textAbstract:
Modulation of the inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3R) by cytosolic calcium (Ca2+) plays an essential role in Ca2+ signalling, but structural determinants and mechanisms responsible for the InsP3R regulation by Ca2+ are poorly understood. In the present study, we expressed rat InsP3R type 1 (InsP3R1) in Spodoptera frugiperda cells using a baculovirus-expression system and reconstituted the recombinant InsP3R1 into planar lipid bilayers for functional analysis. We observed only minor effects of 0.5mM of calmodulin (CaM) antagonist W-7 on the Ca2+ dependence of InsP3R1. Based on a previous analysis of mouse InsP3R1 [Yamada, Miyawaki, Saito, Nakajima, Yamamoto-Hino, Ryo, Furuichi and Mikoshiba (1995) Biochem J. 308, 83–88], we generated the Trp1577→Ala (W1577A) mutant of rat InsP3R1 which lacks the high-affinity Ca2+—CaM-binding site. We found that the W1577A mutant displayed a bell-shaped Ca2+ dependence similar to the wild-type InsP3R1 in planar lipid bilayers. Activation of B cell receptors resulted in identical Ca2+ signals in intact DT40 cells lacking the endogenous InsP3R and transfected with the wild-type InsP3R1 or the W1577A mutant cDNA subcloned into a mammalian expression vector. In the planar lipid bilayer experiments, we showed that both wild-type InsP3R1 and W1577A mutant were equally sensitive to inhibition by exogenous CaM. From these results, we concluded that the interaction of CaM with the high-affinity Ca2+—CaM-binding site in the coupling domain of the InsP3R1 does not play a direct role in biphasic modulation of InsP3R1 by cytosolic Ca2+ or in InsP3R1 inhibition by CaM.
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Foskett, J. Kevin, Carl White, King-Ho Cheung, and Don-On Daniel Mak. "Inositol Trisphosphate Receptor Ca2+ Release Channels." Physiological Reviews 87, no. 2 (April 2007): 593–658. http://dx.doi.org/10.1152/physrev.00035.2006.
Full textAbstract:
The inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3Rs) are a family of Ca2+ release channels localized predominately in the endoplasmic reticulum of all cell types. They function to release Ca2+ into the cytoplasm in response to InsP3 produced by diverse stimuli, generating complex local and global Ca2+ signals that regulate numerous cell physiological processes ranging from gene transcription to secretion to learning and memory. The InsP3R is a calcium-selective cation channel whose gating is regulated not only by InsP3, but by other ligands as well, in particular cytoplasmic Ca2+. Over the last decade, detailed quantitative studies of InsP3R channel function and its regulation by ligands and interacting proteins have provided new insights into a remarkable richness of channel regulation and of the structural aspects that underlie signal transduction and permeation. Here, we focus on these developments and review and synthesize the literature regarding the structure and single-channel properties of the InsP3R.
9
Ramos-Franco, Josefina, Daniel Galvan, Gregory A. Mignery, and Michael Fill. "Location of the Permeation Pathway in the Recombinant Type 1 Inositol 1,4,5-Trisphosphate Receptor." Journal of General Physiology 114, no. 2 (August 1, 1999): 243–50. http://dx.doi.org/10.1085/jgp.114.2.243.
Full textAbstract:
The inositol 1,4,5-trisphosphate receptor (InsP3R) forms ligand-regulated intracellular Ca2+ release channels in the endoplasmic reticulum of all mammalian cells. The InsP3R has been suggested to have six transmembrane regions (TMRs) near its carboxyl terminus. A TMR-deletion mutation strategy was applied to define the location of the InsP3R pore. Mutant InsP3Rs were expressed in COS-1 cells and single channel function was defined in planar lipid bilayers. Mutants having the fifth and sixth TMR (and the interceding lumenal loop), but missing all other TMRs, formed channels with permeation properties similar to wild-type channels (gCs = 284; gCa = 60 pS; PCa/PCs = 6.3). These mutant channels bound InsP3, but ligand occupancy did not regulate the constitutively open pore (Po > 0.80). We propose that a region of 191 amino acids (including the fifth and sixth TMR, residues 2398–2589) near the COOH terminus of the protein forms the InsP3R pore. Further, we have produced a constitutively open InsP3R pore mutant that is ideal for future site-directed mutagenesis studies of the structure–function relationships that define Ca2+ permeation through the InsP3R channel.
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Mak, Don-On Daniel, Sean McBride, and J. Kevin Foskett. "Atp-Dependent Adenophostin Activation of Inositol 1,4,5-Trisphosphate Receptor Channel Gating." Journal of General Physiology 117, no. 4 (March 12, 2001): 299–314. http://dx.doi.org/10.1085/jgp.117.4.299.
Full textAbstract:
The inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) is a ligand-gated intracellular Ca2+ release channel that plays a central role in modulating cytoplasmic free Ca2+ concentration ([Ca2+]i). The fungal metabolite adenophostin A (AdA) is a potent agonist of the InsP3R that is structurally different from InsP3 and elicits distinct calcium signals in cells. We have investigated the effects of AdA and its analogues on single-channel activities of the InsP3R in the outer membrane of isolated Xenopus laevis oocyte nuclei. InsP3R activated by either AdA or InsP3 have identical channel conductance properties. Furthermore, AdA, like InsP3, activates the channel by tuning Ca2+ inhibition of gating. However, gating of the AdA-liganded InsP3R has a critical dependence on cytoplasmic ATP free acid concentration not observed for InsP3-liganded channels. Channel gating activated by AdA is indistinguishable from that elicited by InsP3 in the presence of 0.5 mM ATP, although the functional affinity of the channel is 60-fold higher for AdA. However, in the absence of ATP, gating kinetics of AdA-liganded InsP3R were very different. Channel open time was reduced by 50%, resulting in substantially lower maximum open probability than channels activated by AdA in the presence of ATP, or by InsP3 in the presence or absence of ATP. Also, the higher functional affinity of InsP3R for AdA than for InsP3 is nearly abolished in the absence of ATP. Low affinity AdA analogues furanophostin and ribophostin activated InsP3R channels with gating properties similar to those of AdA. These results provide novel insights for interpretations of observed effects of AdA on calcium signaling, including the mechanisms that determine the durations of elementary Ca2+ release events in cells. Comparisons of single-channel gating kinetics of the InsP3R activated by InsP3, AdA, and its analogues also identify molecular elements in InsP3R ligands that contribute to binding and activation of channel gating.
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Joseph, J. Donald, Yi Peng, Don-On Daniel Mak, King-Ho Cheung, Horia Vais, J. Kevin Foskett, and Huafeng Wei. "General Anesthetic Isoflurane Modulates Inositol 1,4,5-Trisphosphate Receptor Calcium Channel Opening." Anesthesiology 121, no. 3 (September 1, 2014): 528–37. http://dx.doi.org/10.1097/aln.0000000000000316.
Full textAbstract:
Abstract Background: Pharmacological evidence suggests that inhalational general anesthetics induce neurodegeneration in vitro and in vivo through overactivation of inositol trisphosphate receptor (InsP3R) Ca2+-release channels, but it is not clear whether these effects are due to direct modulation of channel activity by the anesthetics. Methods: Using single-channel patch clamp electrophysiology, the authors examined the gating of rat recombinant type 3 InsP3R (InsP3R-3) Ca2+-release channels in isolated nuclei (N = 3 to 15) from chicken lymphocytes modulated by isoflurane at clinically relevant concentrations in the absence and presence of physiological levels of the agonist inositol 1,4,5-trisphosphate (InsP3). The authors also examined the effects of isoflurane on InsP3R-mediated Ca2+ release from the endoplasmic reticulum and changes in intracellular Ca2+ concentration ([Ca2+]i). Results: Clinically relevant concentrations (approximately 1 minimal alveolar concentration) of the commonly used general anesthetic, isoflurane, activated InsP3R-3 channels with open probability similar to channels activated by 1 µM InsP3 (Po ≈ 0.2). This isoflurane modulation of InsP3R-3 Po depended biphasically on [Ca2+]i. Combination of isoflurane with subsaturating levels of InsP3 in patch pipettes resulted in at least two-fold augmentations of InsP3R-3 channel Po compared with InsP3 alone. These effects were not noted in the presence of saturating [InsP3]. Application of isoflurane to DT40 cells resulted in a 30% amplification of InsP3R-mediated [Ca2+]i oscillations, whereas InsP3-induced increase in [Ca2+]i and cleaved caspase-3 activity were enhanced by approximately 2.5-fold. Conclusion: These results suggest that the InsP3R may be a direct molecular target of isoflurane and plays a role in the mechanisms of anesthetic-mediated pharmacological or neurotoxic effects.
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Nathanson, M. H., A. F. O'Neill, and A. D. Burgstahler. "Primitive organization of cytosolic Ca(2+) signals in hepatocytes from the little skate Raja erinacea." Journal of Experimental Biology 202, no. 22 (November 15, 1999): 3049–56. http://dx.doi.org/10.1242/jeb.202.22.3049.
Full textAbstract:
Cytosolic Ca(2+) (Ca(i)(2+)) signals begin as polarized, inositol 1, 4,5-trisphosphate (InsP3)-mediated Ca(i)(2+) waves in mammalian epithelia, and this signaling pattern directs secretion together with other cell functions. To investigate whether Ca(i)(2+) signaling is similarly organized in elasmobranch epithelia, we examined Ca(i)(2+) signaling patterns and InsP3 receptor (InsP3R) expression in hepatocytes isolated from the little skate, Raja erinacea. Ca(i)(2+) signaling was examined by confocal microscopy, InsP3R expression by immunoblot, and the subcellular distribution of InsP3Rs by immunochemistry. ATP induced a rapid increase in Ca(i)(2+) in skate hepatocytes, as it does in mammalian hepatocytes. Unlike in mammalian hepatocytes, however, the Ca(i)(2+) increase in skate hepatocytes began randomly throughout the cell rather than in the apical region. In cells loaded with heparin ATP-induced Ca(i)(2+) signals were inhibited, but de-N-sulfated heparin was not inhibitory, suggesting that the increases in Ca(i)(2+) were mediated by InsP3. Immunoblot analysis showed that the type I but not the types II or III InsP3R was expressed in skate liver. Confocal immunofluorescence revealed that the InsP3R was distributed throughout the hepatocyte, rather than concentrated apically as in mammalian epithelia. These findings demonstrate that ATP-induced Ca(i)(2+) signals are mediated by InsP3 in skate hepatocytes, as they are in mammalian hepatocytes. However, in skate hepatocytes Ca(i)(2+) signals begin at loci throughout the cell rather than as an organized apical-to-basal Ca(i)(2+) wave, which is probably because the InsP3R is distributed throughout these cells. This primitive organization of Ca(i)(2+) signaling may in part explain the observation that Ca(2+)-mediated events such as secretion occur much less efficiently in elasmobranchs than in mammals.
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Ionescu, Lucian, Carl White, King-Ho Cheung, Jianwei Shuai, Ian Parker, John E. Pearson, J. Kevin Foskett, and Don-On Daniel Mak. "Mode Switching Is the Major Mechanism of Ligand Regulation of InsP3 Receptor Calcium Release Channels." Journal of General Physiology 130, no. 6 (November 12, 2007): 631–45. http://dx.doi.org/10.1085/jgp.200709859.
Full textAbstract:
The inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) plays a critical role in generation of complex Ca2+ signals in many cell types. In patch clamp recordings of isolated nuclei from insect Sf9 cells, InsP3R channels were consistently detected with regulation by cytoplasmic InsP3 and free Ca2+ concentrations ([Ca2+]i) very similar to that observed for vertebrate InsP3R. Long channel activity durations of the Sf9-InsP3R have now enabled identification of a novel aspect of InsP3R gating: modal gating. Using a novel algorithm to analyze channel modal gating kinetics, InsP3R gating can be separated into three distinct modes: a low activity mode, a fast kinetic mode, and a burst mode with channel open probability (Po) within each mode of 0.007 ± 0.002, 0.24 ± 0.03, and 0.85 ± 0.02, respectively. Channels reside in each mode for long periods (tens of opening and closing events), and transitions between modes can be discerned with high resolution (within two channel opening and closing events). Remarkably, regulation of channel gating by [Ca2+]i and [InsP3] does not substantially alter channel Po within a mode. Instead, [Ca2+]i and [InsP3] affect overall channel Po primarily by changing the relative probability of the channel being in each mode, especially the high and low Po modes. This novel observation therefore reveals modal switching as the major mechanism of physiological regulation of InsP3R channel activity, with implications for the kinetics of Ca2+ release events in cells.
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Lièvremont, J. P., A. M. Hill, M. Hilly, and J. P. Mauger. "The inositol 1,4,5-trisphosphate receptor is localized on specialized sub-regions of the endoplasmic reticulum in rat liver." Biochemical Journal 300, no. 2 (June 1, 1994): 419–27. http://dx.doi.org/10.1042/bj3000419.
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Inositol 1,4,5-trisphosphate (InsP3) is involved in the mobilization of Ca2+ from intracellular non-mitochondrial stores. In rat liver, it has been shown that the InsP3-binding site co-purifies with the plasma membrane. This suggests that in the liver the InsP3 receptor (InsP3R) associates with plasma membrane. We studied the subcellular distribution of the liver InsP3R by measuring the maximal binding capacity of [3H]InsP3 and using antibodies against the 14 C-terminal residues of the type 1 InsP3R. The antibodies recognized a large amount of an InsP3R protein of 260 kDa in a membrane fraction which is also enriched with [3H]InsP3-binding sites and with markers of the basal, the lateral and the bile-canalicular membrane and the plasma-membrane Ca2+ pump (PMCA). The fractions enriched in markers of the endoplasmic reticulum (ER) and the Ca2+ pump of the ER (SERCA2b) contained low levels of InsP3 receptors. The immunofluorescent labelling of cultured hepatocytes with anti-InsP3R antibodies indicated that the receptor is concentrated in the perinuclear area and in some regions near the plasma membrane. The fraction enriched with InsP3R is also contaminated with markers of the ER and with SERCA2b. It was exposed to alkaline medium (pH 10.5) to extract endogenous actin and membrane-associated proteins before being subfractionated by Percoll-gradient centrifugation. The alkaline treatment allowed partial separation of the markers of the ER from the markers of the plasma membrane. The InsP3R was recovered in the heavy subfraction, which was also enriched with markers for the ER and with the SERCA2b and contained low levels of markers of the plasma membrane. These data indicate that the InsP3R is neither localized on the plasma membrane itself nor homogeneously distributed on the ER membrane. This supports the view that part of the receptor is localized on a specialized sub-region of the ER which interacts with the plasma membrane.
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Mak, Don-On Daniel, Sean McBride, Viswanathan Raghuram, Yun Yue, Suresh K. Joseph, and J. Kevin Foskett. "Single-Channel Properties in Endoplasmic Reticulum Membrane of Recombinant Type 3 Inositol Trisphosphate Receptor." Journal of General Physiology 115, no. 3 (February 14, 2000): 241–56. http://dx.doi.org/10.1085/jgp.115.3.241.
Full textAbstract:
The inositol 1,4,5-trisphosphate receptor (InsP3R) is an intracellular Ca2+-release channel localized in endoplasmic reticulum (ER) with a central role in complex Ca2+ signaling in most cell types. A family of InsP3Rs encoded by several genes has been identified with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. This diversity suggests that cells require distinct InsP3Rs, but the functional correlates of this diversity are largely unknown. Lacking are single-channel recordings of the recombinant type 3 receptor (InsP3R-3), a widely expressed isoform also implicated in plasma membrane Ca2+ influx and apoptosis. Here, we describe functional expression and single-channel recording of recombinant rat InsP3R-3 in its native membrane environment. The approach we describe suggests a novel strategy for expression and recording of recombinant ER-localized ion channels in the ER membrane. Ion permeation and channel gating properties of the rat InsP3R-3 are strikingly similar to those of Xenopus type 1 InsP3R in the same membrane. Using two different two-electrode voltage clamp protocols to examine calcium store-operated calcium influx, no difference in the magnitude of calcium influx was observed in oocytes injected with rat InsP3R-3 cRNA compared with control oocytes. Our results suggest that if cellular expression of multiple InsP3R isoforms is a mechanism to modify the temporal and spatial features of [Ca2+]i signals, then it must be achieved by isoform-specific regulation or localization of various types of InsP3Rs that have relatively similar Ca2+ permeation properties.
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Vais, Horia, J. Kevin Foskett, and Don-On Daniel Mak. "Unitary Ca2+ current through recombinant type 3 InsP3 receptor channels under physiological ionic conditions." Journal of General Physiology 136, no. 6 (November 15, 2010): 687–700. http://dx.doi.org/10.1085/jgp.201010513.
Full textAbstract:
The ubiquitous inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) channel, localized primarily in the endoplasmic reticulum (ER) membrane, releases Ca2+ into the cytoplasm upon binding InsP3, generating and modulating intracellular Ca2+ signals that regulate numerous physiological processes. Together with the number of channels activated and the open probability of the active channels, the size of the unitary Ca2+ current (iCa) passing through an open InsP3R channel determines the amount of Ca2+ released from the ER store, and thus the amplitude and the spatial and temporal nature of Ca2+ signals generated in response to extracellular stimuli. Despite its significance, iCa for InsP3R channels in physiological ionic conditions has not been directly measured. Here, we report the first measurement of iCa through an InsP3R channel in its native membrane environment under physiological ionic conditions. Nuclear patch clamp electrophysiology with rapid perfusion solution exchanges was used to study the conductance properties of recombinant homotetrameric rat type 3 InsP3R channels. Within physiological ranges of free Ca2+ concentrations in the ER lumen ([Ca2+]ER), free cytoplasmic [Ca2+] ([Ca2+]i), and symmetric free [Mg2+] ([Mg2+]f), the iCa–[Ca2+]ER relation was linear, with no detectable dependence on [Mg2+]f. iCa was 0.15 ± 0.01 pA for a filled ER store with 500 µM [Ca2+]ER. The iCa–[Ca2+]ER relation suggests that Ca2+ released by an InsP3R channel raises [Ca2+]i near the open channel to ∼13–70 µM, depending on [Ca2+]ER. These measurements have implications for the activities of nearby InsP3-liganded InsP3R channels, and they confirm that Ca2+ released by an open InsP3R channel is sufficient to activate neighboring channels at appropriate distances away, promoting Ca2+-induced Ca2+ release.
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Bimboese, Patricia, Craig J. Gibson, Stefan Schmidt, Wanqing Xiang, and Barbara E. Ehrlich. "Isoform-specific Regulation of the Inositol 1,4,5-Trisphosphate Receptor by O-Linked Glycosylation." Journal of Biological Chemistry 286, no. 18 (March 7, 2011): 15688–97. http://dx.doi.org/10.1074/jbc.m110.206482.
Full textAbstract:
The inositol 1,4,5-trisphosphate receptor (InsP3R), 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 InsP3R-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 InsP3R-2 and InsP3R-3. Analysis of AR4-2J cells, a rat pancreatoma cell line expressing predominantly InsP3R-2, showed no detectable O-GlcNAcylation of InsP3R-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, InsP3R-3 in Mz-ChA-1 cells, a human cholangiocarcinoma cell line expressing predominantly InsP3R-3, was functionally modified by O-GlcNAcylation. Interestingly, the functional impact of O-GlcNAcylation on the InsP3R-3 channel was opposite the effect measured with InsP3R-1. Addition of O-GlcNAc by O-β-N-acetylglucosaminyltransferase increased InsP3R-3 single channel open probability. Incubation of Mz-ChA-1 cells in hyperglycemic medium caused an increase in the InsP3-dependent calcium release from the endoplasmic reticulum. The dynamic and inducible nature of O-GlcNAcylation and the InsP3R isoform specificity suggest that this form of modification of InsP3R and subsequent changes in intracellular calcium transients are important in physiological and pathophysiological processes.
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Chen, Rui, Ignacio Valencia, Fei Zhong, Karen S. McColl, H. Llewelyn Roderick, Martin D. Bootman, Michael J. Berridge, et al. "Bcl-2 functionally interacts with inositol 1,4,5-trisphosphate receptors to regulate calcium release from the ER in response to inositol 1,4,5-trisphosphate." Journal of Cell Biology 166, no. 2 (July 19, 2004): 193–203. http://dx.doi.org/10.1083/jcb.200309146.
Full textAbstract:
Inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3Rs) are channels responsible for calcium release from the endoplasmic reticulum (ER). We show that the anti-apoptotic protein Bcl-2 (either wild type or selectively localized to the ER) significantly inhibited InsP3-mediated calcium release and elevation of cytosolic calcium in WEHI7.2 T cells. This inhibition was due to an effect of Bcl-2 at the level of InsP3Rs because responses to both anti-CD3 antibody and a cell-permeant InsP3 ester were decreased. Bcl-2 inhibited the extent of calcium release from the ER of permeabilized WEHI7.2 cells, even at saturating concentrations of InsP3, without decreasing luminal calcium concentration. Furthermore, Bcl-2 reduced the open probability of purified InsP3Rs reconstituted into lipid bilayers. Bcl-2 and InsP3Rs were detected together in macromolecular complexes by coimmunoprecipitation and blue native gel electrophoresis. We suggest that this functional interaction of Bcl-2 with InsP3Rs inhibits InsP3R activation and thereby regulates InsP3-induced calcium release from the ER.
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Roderick, H. L., and M. D. Bootman. "Bi-directional signalling from the InsP3 receptor: regulation by calcium and accessory factors." Biochemical Society Transactions 31, no. 5 (October 1, 2003): 950–53. http://dx.doi.org/10.1042/bst0310950.
Full textAbstract:
Calcium is a pleiotropic messenger controlling a diverse array of intracellular events from fertilization to cell death. One of the main mechanisms by which intracellular calcium is elevated is through InsP3 [Ins(1,4,5)P3]-induced mobilization of calcium from its receptor on the endoplasmic reticulum calcium store. The activity of the InsP3R (InsP3 receptor) is subject to regulation by many factors other than InsP3, most notably calcium itself, which regulates the channel in a bell-shaped dependent manner. InsP3R sensitivity is also regulated by post-translational modifications such as phosphorylation and by binding of accessory proteins. Taken together it appears that the InsP3R can be regarded as a cellular sensor for many signalling pathways, qualitatively and quantitatively regulating intracellular calcium signals with consequences for downstream cellular physiology.
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Mak, Don-On Daniel, Sean McBride, and J. Kevin Foskett. "Atp Regulation of Recombinant Type 3 Inositol 1,4,5-Trisphosphate Receptor Gating." Journal of General Physiology 117, no. 5 (April 30, 2001): 447–56. http://dx.doi.org/10.1085/jgp.117.5.447.
Full textAbstract:
A family of inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) Ca2+ release channels plays a central role in Ca2+ signaling in most cells, but functional correlates of isoform diversity are unclear. Patch-clamp electrophysiology of endogenous type 1 (X-InsP3R-1) and recombinant rat type 3 InsP3R (r-InsP3R-3) channels in the outer membrane of isolated Xenopus oocyte nuclei indicated that enhanced affinity and reduced cooperativity of Ca2+ activation sites of the InsP3-liganded type 3 channel distinguished the two isoforms. Because Ca2+ activation of type 1 channel was the target of regulation by cytoplasmic ATP free acid concentration ([ATP]i), here we studied the effects of [ATP]i on the dependence of r-InsP3R-3 gating on cytoplasmic free Ca2+ concentration ([Ca2+]i). As [ATP]i was increased from 0 to 0.5 mM, maximum r-InsP3R-3 channel open probability (Po) remained unchanged, whereas the half-maximal activating [Ca2+]i and activation Hill coefficient both decreased continuously, from 800 to 77 nM and from 1.6 to 1, respectively, and the half-maximal inhibitory [Ca2+]i was reduced from 115 to 39 μM. These effects were largely due to effects of ATP on the mean closed channel duration. Whereas the r-InsP3R-3 had a substantially higher Po than X-InsP3R-1 in activating [Ca2+]i (<1 μM) and 0.5 mM ATP, the Ca2+ dependencies of channel gating of the two isoforms became remarkably similar in the absence of ATP. Our results suggest that ATP binding is responsible for conferring distinct gating properties on the two InsP3R channel isoforms. Possible molecular models to account for the distinct regulation by ATP of the Ca2+ activation properties of the two channel isoforms and the physiological implications of these results are discussed. Complex regulation by ATP of the types 1 and 3 InsP3R channel activities may enable cells to generate sophisticated patterns of Ca2+ signals with cytoplasmic ATP as one of the second messengers.
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Mak, Don-On Daniel, Sean M. J. McBride, Nataliya B. Petrenko, and J. Kevin Foskett. "Novel Regulation of Calcium Inhibition of the Inositol 1,4,5-trisphosphate Receptor Calcium-release Channel." Journal of General Physiology 122, no. 5 (October 27, 2003): 569–81. http://dx.doi.org/10.1085/jgp.200308808.
Full textAbstract:
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+] ∼400 nM were inhibited by 290 μM [Ca2+]i, exposure of the isolated nuclei to a bath solution with ultra-low [Ca2+] (<5 nM, for ∼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.
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Wagner, Larry E., Wen-Hong Li, Suresh K. Joseph, and David I. Yule. "Functional Consequences of Phosphomimetic Mutations at Key cAMP-dependent Protein Kinase Phosphorylation Sites in the Type 1 Inositol 1,4,5-Trisphosphate Receptor." Journal of Biological Chemistry 279, no. 44 (August 11, 2004): 46242–52. http://dx.doi.org/10.1074/jbc.m405849200.
Full textAbstract:
Regulation of Ca2+release through inositol 1,4,5-trisphosphate receptors (InsP3R) has important consequences for defining the particular spatio-temporal properties of intracellular Ca2+signals. In this study, regulation of Ca2+release by phosphorylation of type 1 InsP3R (InsP3R-1) was investigated by constructing “phosphomimetic” charge mutations in the functionally important phosphorylation sites of both the S2+ and S2- InsP3R-1 splice variants. Ca2+release was investigated following expression in Dt-40 3ko cells devoid of endogenous InsP3R. In cells expressing either the S1755E S2+ or S1589E/S1755E S2- InsP3R-1, InsP3-induced Ca2+release was markedly enhanced compared with nonphosphorylatable S2+ S1755A and S2- S1589A/S1755A mutants. Ca2+release through the S2- S1589E/S1755E InsP3R-1 was enhanced ∼8-fold over wild type and ∼50-fold when compared with the nonphosphorylatable S2- S1589A/S1755A mutant. In cells expressing S2- InsP3R-1 with single mutations in either S1589E or S1755E, the sensitivity of Ca2+release was enhanced ∼3-fold; sensitivity was midway between the wild type and the double glutamate mutation. Paradoxically, forskolin treatment of cells expressing either single Ser/Glu mutation failed to further enhance Ca2+release. The sensitivity of Ca2+release in cells expressing S2+ S1755E InsP3R-1 was comparable with the sensitivity of S2- S1589E/S1755E InsP3R-1. In contrast, mutation of S2+ S1589E InsP3R-1 resulted in a receptor with comparable sensitivity to wild type cells. Expression of S2- S1589E/S1755E InsP3R-1 resulted in robust Ca2+oscillations when cells were stimulated with concentrations of α-IgM antibody that were threshold for stimulation in S2- wild type InsP3R-1-expressing cells. However, at higher concentrations of α-IgM antibody, Ca2+oscillations of a similar period and magnitude were initiated in cells expressing either wild type or S2- phosphomimetic mutations. Thus, regulation by phosphorylation of the functional sensitivity of InsP3R-1 appears to define the threshold at which oscillations are initiated but not the frequency or amplitude of the signal when established.
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Vais, Horia, J. Kevin Foskett, Ghanim Ullah, John E. Pearson, and Don-On Daniel Mak. "Permeant calcium ion feed-through regulation of single inositol 1,4,5-trisphosphate receptor channel gating." Journal of General Physiology 140, no. 6 (November 12, 2012): 697–716. http://dx.doi.org/10.1085/jgp.201210804.
Full textAbstract:
The ubiquitous inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) Ca2+ release channel plays a central role in the generation and modulation of intracellular Ca2+ signals, and is intricately regulated by multiple mechanisms including cytoplasmic ligand (InsP3, free Ca2+, free ATP4−) binding, posttranslational modifications, and interactions with cytoplasmic and endoplasmic reticulum (ER) luminal proteins. However, regulation of InsP3R channel activity by free Ca2+ in the ER lumen ([Ca2+]ER) remains poorly understood because of limitations of Ca2+ flux measurements and imaging techniques. Here, we used nuclear patch-clamp experiments in excised luminal-side-out configuration with perfusion solution exchange to study the effects of [Ca2+]ER on homotetrameric rat type 3 InsP3R channel activity. In optimal [Ca2+]i and subsaturating [InsP3], jumps of [Ca2+]ER from 70 nM to 300 µM reduced channel activity significantly. This inhibition was abrogated by saturating InsP3 but restored when [Ca2+]ER was raised to 1.1 mM. In suboptimal [Ca2+]i, jumps of [Ca2+]ER (70 nM to 300 µM) enhanced channel activity. Thus, [Ca2+]ER effects on channel activity exhibited a biphasic dependence on [Ca2+]i. In addition, the effect of high [Ca2+]ER was attenuated when a voltage was applied to oppose Ca2+ flux through the channel. These observations can be accounted for by Ca2+ flux driven through the open InsP3R channel by [Ca2+]ER, raising local [Ca2+]i around the channel to regulate its activity through its cytoplasmic regulatory Ca2+-binding sites. Importantly, [Ca2+]ER regulation of InsP3R channel activity depended on cytoplasmic Ca2+-buffering conditions: it was more pronounced when [Ca2+]i was weakly buffered but completely abolished in strong Ca2+-buffering conditions. With strong cytoplasmic buffering and Ca2+ flux sufficiently reduced by applied voltage, both activation and inhibition of InsP3R channel gating by physiological levels of [Ca2+]ER were completely abolished. Collectively, these results rule out Ca2+ regulation of channel activity by direct binding to the luminal aspect of the channel.
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Ramos, Jorge, Wonyong Jung, Josefina Ramos-Franco, Gregory A. Mignery, and Michael Fill. "Single Channel Function of Inositol 1,4,5-trisphosphate Receptor Type-1 and -2 Isoform Domain-Swap Chimeras." Journal of General Physiology 121, no. 5 (April 14, 2003): 399–411. http://dx.doi.org/10.1085/jgp.200208718.
Full textAbstract:
The InsP3R proteins have three recognized domains, the InsP3-binding, regulatory/coupling, and channel domains (Mignery, G.A., and T.C. Südhof. 1990. EMBO J. 9:3893–3898). The InsP3 binding domain and the channel-forming domain are at opposite ends of the protein. Ligand regulation of the channel must involve communication between these different regions of the protein. This communication likely involves the interceding sequence (i.e., the regulatory/coupling domain). The single channel functional attributes of the full-length recombinant type-1, -2, and -3 InsP3R channels have been defined. Here, two type-1/type-2 InsP3R regulatory/coupling domain chimeras were created and their single channel function defined. One chimera (1-2-1) contained the type-2 regulatory/coupling domain in a type-1 backbone. The other chimera (2-1-2) contained the type-1 regulatory/coupling domain in a type-2 backbone. These chimeric proteins were expressed in COS cells, isolated, and then reconstituted in proteoliposomes. The proteoliposomes were incorporated into artificial planar lipid bilayers and the single-channel function of the chimeras defined. The chimeras had permeation properties like that of wild-type channels. The ligand regulatory properties of the chimeras were altered. The InsP3 and Ca2+ regulation had some unique features but also had features in common with wild-type channels. These results suggest that different independent structural determinants govern InsP3R permeation and ligand regulation. It also suggests that ligand regulation is a multideterminant process that involves several different regions of the protein. This study also demonstrates that a chimera approach can be applied to define InsP3R structure-function.
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Park, Hyung Seo, Matthew J. Betzenhauser, Yu Zhang, and David I. Yule. "Regulation of Ca2+ release through inositol 1,4,5-trisphosphate receptors by adenine nucleotides in parotid acinar cells." American Journal of Physiology-Gastrointestinal and Liver Physiology 302, no. 1 (January 2012): G97—G104. http://dx.doi.org/10.1152/ajpgi.00328.2011.
Full textAbstract:
Secretagogue-stimulated intracellular Ca2+ signals are fundamentally important for initiating the secretion of the fluid and ion component of saliva from parotid acinar cells. The Ca2+ signals have characteristic spatial and temporal characteristics, which are defined by the specific properties of Ca2+ release mediated by inositol 1,4,5-trisphosphate receptors (InsP3R). In this study we have investigated the role of adenine nucleotides in modulating Ca2+ release in mouse parotid acinar cells. In permeabilized cells, the Ca2+ release rate induced by submaximal [InsP3] was increased by 5 mM ATP. Enhanced Ca2+ release was not observed at saturating [InsP3]. The EC50 for the augmented Ca2+ release was ∼8 μM ATP. The effect was mimicked by nonhydrolysable ATP analogs. ADP and AMP also potentiated Ca2+ release but were less potent than ATP. In acini isolated from InsP3R-2-null transgenic animals, the rate of Ca2+ release was decreased under all conditions but now enhanced by ATP at all [InsP3]. In addition the EC50 for ATP potentiation increased to ∼500 μM. These characteristics are consistent with the properties of the InsP3R-2 dominating the overall features of InsP3R-induced Ca2+ release despite the expression of all isoforms. Finally, Ca2+ signals were measured in intact parotid lobules by multiphoton microscopy. Consistent with the release data, carbachol-stimulated Ca2+ signals were reduced in lobules exposed to experimental hypoxia compared with control lobules only at submaximal concentrations. Adenine nucleotide modulation of InsP3R in parotid acinar cells likely contributes to the properties of Ca2+ signals in physiological and pathological conditions.
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García, Kelly D., Tanvi Shah, and Jesús García. "Immunolocalization of type 2 inositol 1,4,5-trisphosphate receptors in cardiac myocytes from newborn mice." American Journal of Physiology-Cell Physiology 287, no. 4 (October 2004): C1048—C1057. http://dx.doi.org/10.1152/ajpcell.00004.2004.
Full textAbstract:
The precise localization and role of inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3Rs) in cardiac muscle cells are largely unknown. It is believed that waves and oscillations in cytosolic free calcium triggered by activation of InsP3Rs underlie modifications of cellular responses that lead to changes in gene expression in other cells. However, how changes in cytosolic calcium alter gene expression in cardiac cells is unknown. Moreover, it is unclear how changes in cytosolic calcium that alter gene expression do so independently of effects of calcium on other cellular functions, such as contraction. Here we show that InsP3R type 2 is the only isoform present in cardiac myocytes isolated from neonatal mouse ventricles. We also show that type 2 InsP3Rs are associated with the nucleus and that activation of type 2 InsP3Rs with endothelin-1 or phenylephrine selectively increases transcription of atrial natriuretic factor and skeletal α-actin. Type 2 InsP3Rs are also in striations. Activation of InsP3Rs with adenophostin A in permeabilized cells induced calcium release in the nuclear domain and other regions of the cell away from the nucleus. Agonist-induced increase in gene expression and calcium release were blocked by the InsP3R inhibitors 2-aminoethoxydiphenyl borate and xestospongin C. The spatial separation of type 2 InsP3Rs provides support for the concept that microdomains of calcium discretely alter various cell processes. Our experiments suggest that calcium released by InsP3Rs in the nuclear domain provides a direct mechanism for the control of gene expression, whereas release of calcium in the cytoplasm may modulate other processes, such as contraction.
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Valério, Patricia, C. C. P. Mendes, Marivalda Pereira, Alfredo Goes, and M. Fatima Leite. "Glutamate Release by Osteoblasts in the Presence of Ionic Products from Bioactive Glass 60S." Key Engineering Materials 284-286 (April 2005): 537–40. http://dx.doi.org/10.4028/www.scientific.net/kem.284-286.537.
Full textAbstract:
Osteoblasts constitutively release glutamate and this release appears to be regulated by calcium entry. In this work we investigated if the bioactive glass with 60% of silicon (BG60S) could alter glutamate release by osteoblasts. We demonstrated that osteoblasts incubated with medium containing ionic products from the dissolution of BG60S showed lower release of glutamate when compared to control. Since intracellular calcium (Cai 2+) increase is required for glutamate release we investigated the subcellular distribution of the calcium channel inositol triphosphate receptors (InsP3Rs) in the presence of BG60S compared to control. We found that the type-III InsP3R was not expressed in osteoblast, while the type-II InsP3R was expressed mainly in the cytosol. We also found that the expression of type-II InsP3R decreased in BG60S treated osteoblasts compared to control. On the other hand, we found that the type-I InsP3R was expressed mainly in the nucleus and its expression increased in the presence of the biomaterial.
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Bezprozvanny, I., and B. E. Ehrlich. "Inositol (1,4,5)-trisphosphate (InsP3)-gated Ca channels from cerebellum: conduction properties for divalent cations and regulation by intraluminal calcium." Journal of General Physiology 104, no. 5 (November 1, 1994): 821–56. http://dx.doi.org/10.1085/jgp.104.5.821.
Full textAbstract:
The conduction properties of inositol (1,4,5)-trisphosphate (InsP3)-gated calcium (Ca) channels (InsP3R) from canine cerebellum for divalent cations and the regulation of the channels by intraluminal Ca were studied using channels reconstituted into planar lipid bilayers. Analysis of single-channel recordings performed with different divalent cations present at 55 mM on the trans (intraluminal) side of the membrane revealed that the current amplitude at 0 mV and the single-channel slope conductance fell in the sequence: Ba (2.2 pA, 85 pS) > Sr (2.0 pA, 77 pS) > Ca (1.4 pA, 53 pS) > Mg (1.1 pA, 42 pS). The mean open time of the InsP3R recorded with Ca (2.9 ms) was significantly shorter than with other divalent cations (approximately 5.5 ms). The "anomalous mole fraction effect" was not observed in mixtures of divalent cations (Mg and Ba), suggesting that these channels are single-ion pores. Measurements of InsP3R activity at different intraluminal Ca levels demonstrated that Ca in the submillimolar range did not potentiate channel activity, and that very high levels of intraluminal Ca (> or = 10 mM) decreased channel open probability 5-10-fold. When InsP3R were measured with Ba as a current carrier in the presence of 110 mM cis potassium, a PBa/PK of 6.3 was estimated from the extrapolated value for the reversal potential. When the unitary current through the InsP3R at 0 mV was measured as a function of the permeant ion (Ba) concentration, the half-maximal current occurred at 10 mM trans Ba. The following conclusions are drawn from these data: (a) the conduction properties of InsP3R are similar to the properties of the ryanodine receptor, another intracellular Ca channel, and differ dramatically from the properties of voltage-gated Ca channels of the plasma membrane. (b) The estimated size of the Ca current through the InsP3R under physiological conditions is 0.5 pA, approximately four times less than the Ca current through the ryanodine receptor. (c) The potentiation of InsP3R by intraluminal Ca in the submillimolar range remains controversial. (d) A quantitative model that explains the inhibitory effects of high trans Ca on InsP3R activity was developed and the kinetic parameters of InsP3R gating were determined.
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Velmurugan, Gopal V., and Carl White. "Calcium homeostasis in vascular smooth muscle cells is altered in type 2 diabetes by Bcl-2 protein modulation of InsP3R calcium release channels." American Journal of Physiology-Heart and Circulatory Physiology 302, no. 1 (January 2012): H124—H134. http://dx.doi.org/10.1152/ajpheart.00218.2011.
Full textAbstract:
This study examines the extent to which the antiapoptotic Bcl-2 proteins Bcl-2 and Bcl-xLcontribute to diabetic Ca2+dysregulation and vessel contractility in vascular smooth muscle cells (VSMCs) through their interaction with inositol 1,4,5-trisphosphate receptor (InsP3R) intracellular Ca2+release channels. Measurements of intracellular ([Ca2+]i) and sarcoplasmic reticulum ([Ca2+]SR) calcium concentrations were made in primary cells isolated from diabetic ( db/db) and nondiabetic ( db/m) mice. In addition, [Ca2+]iand constriction were recorded simultaneously in isolated intact arteries. Protein expression levels of Bcl-xLbut not Bcl-2 were elevated in VSMCs isolated from db/db compared with db/m age-matched controls. In single cells, InsP3-evoked [Ca2+]isignaling was enhanced in VSMCs from db/db mice compared with db/m. This was attributed to alterations in the intrinsic properties of the InsP3R itself because there were no differences between db/db and db/m in the steady-state [Ca2+]SRor InsP3R expression levels. Moreover, in permeabilized cells the rate of InsP3R-dependent SR Ca2+release was increased in db/db compared with db/m VSMCs. The enhanced InsP3-dependent SR Ca2+release was attenuated by the Bcl-2 protein inhibitor ABT-737 only in diabetic cells. Application of ABT-737 similarly attenuated enhanced agonist-induced [Ca2+]isignaling only in intact aortic and mesenteric db/db vessels. In contrast, ABT-737 had no effect on agonist-evoked contractility in either db/db or db/m vessels. Taken together, the data suggest that in type 2 diabetes the mechanism for [Ca2+]idysregulation in VSMCs involves Bcl-2 protein-dependent increases in InsP3R excitability and that dysregulated [Ca2+]isignaling does not appear to contribute to increased vessel reactivity.
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TURNER, Helen, Andrea FLEIG, Alexander STOKES, Jean-Pierre KINET, and Reinhold PENNER. "Discrimination of intracellular calcium store subcompartments using TRPV1 (transient receptor potential channel, vanilloid subfamily member 1) release channel activity." Biochemical Journal 371, no. 2 (April 15, 2003): 341–50. http://dx.doi.org/10.1042/bj20021381.
Full textAbstract:
The store-operated calcium-release-activated calcium current, ICRAC, is a major mechanism for calcium entry into non-excitable cells. ICRAC refills calcium stores and permits sustained calcium signalling. The relationship between inositol 1,4,5-trisphosphate receptor (InsP3R)-containing stores and ICRAC is not understood. A model of global InsP3R store depletion coupling with ICRAC activation may be simplistic, since intracellular stores are heterogeneous in their release and refilling activities. Here we use a ligand-gated calcium channel, TRPV1 (transient receptor potential channel, vanilloid subfamily member 1), as a new tool to probe store heterogeneity and define intracellular calcium compartments in a mast cell line. TRPV1 has activity as an intracellular release channel but does not mediate global calcium store depletion and does not invade a store coupled with ICRAC. Intracellular TRPV1 localizes to a subset of the InsP3R-containing stores. TRPV1 sensitivity functionally subdivides the InsP3-sensitive store, as does heterogeneity in the sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase isoforms responsible for store refilling. These results provide unequivocal evidence that a specific ‘CRAC store’ exists within the InsP3-releasable calcium stores and describe a novel methodology for manipulation of intracellular free calcium.
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DARGAN, Sheila L., Edward J. A. LEA, and Alan P. DAWSON. "Modulation of type-1 Ins(1,4,5)P3 receptor channels by the FK506-binding protein, FKBP12." Biochemical Journal 361, no. 2 (January 8, 2002): 401–7. http://dx.doi.org/10.1042/bj3610401.
Full textAbstract:
FK506-binding protein (FKBP12) is highly expressed in neuronal tissue, where it is proposed to localize calcineurin to intracellular calcium-release channels, ryanodine receptors and Ins(1,4,5)P3 receptors (InsP3Rs). The effects of FKBP12 on ryanodine receptors have been well characterized but the nature and function of binding of FKBP12 to InsP3R is more controversial, with evidence for and against a tight interaction between these two proteins. To investigate this, we incorporated purified type-1 InsP3R from rat cerebellum into planar lipid bilayers to monitor the effects of exogenous recombinant FKBP12 on single-channel activity, using K+ as the current carrier. Here we report for the first time that FKBP12 causes a substantial change in single-channel properties of the type-1 InsP3R, specifically to increase the amount of time the channel spends in a fully open state. In the presence of ATP, FKBP12 can also induce co-ordinated gating with neighbouring receptors. The effects of FKBP12 were reversed by FK506. We also present data showing that rapamycin, at sub-optimal concentrations of Ins(2,4,5)P3, decreases the rate of calcium release from cerebellar microsomes. These results provide evidence for a direct functional interaction between FKBP12 and the type-1 InsP3R.
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Mak, Don-On Daniel, Sean M. J. McBride, and J. Kevin Foskett. "Spontaneous Channel Activity of the Inositol 1,4,5-Trisphosphate (InsP3) Receptor (InsP3R). Application of Allosteric Modeling to Calcium and InsP3 Regulation of InsP3R Single-channel Gating." Journal of General Physiology 122, no. 5 (October 27, 2003): 583–603. http://dx.doi.org/10.1085/jgp.200308809.
Full textAbstract:
The InsP3R Ca2+ release channel has a biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). InsP3 activates gating primarily by reducing the sensitivity of the channel to inhibition by high [Ca2+]i. To determine if relieving Ca2+ inhibition is sufficient for channel activation, we examined single-channel activities in low [Ca2+]i in the absence of InsP3, by patch clamping isolated Xenopus oocyte nuclei. For both endogenous Xenopus type 1 and recombinant rat type 3 InsP3R channels, spontaneous InsP3-independent channel activities with low open probability Po (∼0.03) were observed in [Ca2+]i < 5 nM with the same frequency as in the presence of InsP3, whereas no activities were observed in 25 nM Ca2+. These results establish the half-maximal inhibitory [Ca2+]i of the channel to be 1.2–4.0 nM in the absence of InsP3, and demonstrate that the channel can be active when all of its ligand-binding sites (including InsP3) are unoccupied. In the simplest allosteric model that fits all observations in nuclear patch-clamp studies of [Ca2+]i and InsP3 regulation of steady-state channel gating behavior of types 1 and 3 InsP3R isoforms, including spontaneous InsP3-independent channel activities, the tetrameric channel can adopt six different conformations, the equilibria among which are controlled by two inhibitory and one activating Ca2+-binding and one InsP3-binding sites in a manner outlined in the Monod-Wyman-Changeux model. InsP3 binding activates gating by affecting the Ca2+ affinities of the high-affinity inhibitory sites in different conformations, transforming it into an activating site. Ca2+ inhibition of InsP3-liganded channels is mediated by an InsP3-independent low-affinity inhibitory site. The model also suggests that besides the ligand-regulated gating mechanism, the channel has a ligand-independent gating mechanism responsible for maximum channel Po being less than unity. The validity of this model was established by its successful quantitative prediction of channel behavior after it had been exposed to ultra-low bath [Ca2+].
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Feriod, Colleen N., Lily Nguyen, Michael J. Jurczak, Emma A. Kruglov, Michael H. Nathanson, Gerald I. Shulman, Anton M. Bennett, and Barbara E. Ehrlich. "Inositol 1,4,5-trisphosphate receptor type II (InsP3R-II) is reduced in obese mice, but metabolic homeostasis is preserved in mice lacking InsP3R-II." American Journal of Physiology-Endocrinology and Metabolism 307, no. 11 (December 1, 2014): E1057—E1064. http://dx.doi.org/10.1152/ajpendo.00236.2014.
Full textAbstract:
Inositol 1,4,5-trisphosphate receptor type II (InsP3R-II) is the most prevalent isoform of the InsP3R in hepatocytes and is concentrated under the canalicular membrane, where it plays an important role in bile secretion. We hypothesized that altered calcium (Ca2+) signaling may be involved in metabolic dysfunction, as InsP3R-mediated Ca2+ signals have been implicated in the regulation of hepatic glucose homeostasis. Here, we find that InsP3R-II, but not InsP3R-I, is reduced in the livers of obese mice. In our investigation of the functional consequences of InsP3R-II deficiency, we found that organic anion secretion at the canalicular membrane and Ca2+ signals were impaired. However, mice lacking InsP3R-II showed no deficits in energy balance, glucose production, glucose tolerance, or susceptibility to hepatic steatosis. Thus, our results suggest that reduced InsP3R-II expression is not sufficient to account for any disruptions in metabolic homeostasis that are observed in mouse models of obesity. We conclude that metabolic homeostasis is maintained independently of InsP3R-II. Loss of InsP3R-II does impair secretion of bile components; therefore, we suggest that conditions of obesity would lead to a decrease in this Ca2+-sensitive process.
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MA, Hong-Tao, Kartik VENKATACHALAM, Krystyna E. RYS-SIKORA, Li-Ping HE, Fei ZHENG, and Donald L. GILL. "Modification of phospholipase C-γ-induced Ca2+ signal generation by 2-aminoethoxydiphenyl borate." Biochemical Journal 376, no. 3 (December 15, 2003): 667–76. http://dx.doi.org/10.1042/bj20031345.
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The mechanisms by which Ca2+-store-release channels and Ca2+-entry channels are coupled to receptor activation are poorly understood. Modification of Ca2+ signals by 2-aminoethoxydiphenyl borate (2-APB), suggests the agent may target entry channels or the machinery controlling their activation. In DT40 B-cells and Jurkat T-cells, complete Ca2+ store release was induced by 2-APB (EC50 10–20 µM). At 75 µM, 2-APB emptied stores completely in both lymphocyte lines, but had no such effect on other cells. In DT40 cells, 2-APB mimicked B-cell receptor (BCR) cross-linking, but no effect was observed in mutant DT40 lines devoid of inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3Rs) or phospholipase C-γ2 (PLC-γ2). Like the BCR, 2-APB activated transfected TRPC3 (canonical transient receptor potential) channels, which acted as sensors for PLC-γ2-generated diacylglycerol in DT40 cells. The action of 2-APB on InsP3Rs and TRPC3 channels was prevented by PLC-inhibition, and required PLC-γ2 catalytic activity. However, unlike BCR activation, no increased InsP3 level could be measured in response to 2-APB. Also, calyculin A-induced cytoskeletal reorganization prevented 2-APB-induced InsP3R and TRPC3-channel activation, but not that induced by the BCR. 2-APB still activated TRPC3 channels in DT40 cells with fully depleted Ca2+ stores, indicating its action was not via Ca2+ release. Significantly, 2-APB-induced InsP3R and TRPC3 activation was prevented in DT40 knockout cells devoid of the BCR- and PLC-γ2-coupled adaptor/kinases, Syk, Lyn, Btk or BLNK. The results suggest that 2-APB activates Ca2+ signals in lymphocytes by initiating and enhancing coupling between components of the BCR–PLC-γ2 complex and both Ca2+-entry and Ca2+-release channels.
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Betzenhauser, Matthew J., Larry E. Wagner, Hyung Seo Park, and David I. Yule. "ATP Regulation of Type-1 Inositol 1,4,5-Trisphosphate Receptor Activity Does Not Require Walker A-type ATP-binding Motifs." Journal of Biological Chemistry 284, no. 24 (April 22, 2009): 16156–63. http://dx.doi.org/10.1074/jbc.m109.006452.
Full textAbstract:
ATP is known to increase the activity of the type-1 inositol 1,4,5-trisphosphate receptor (InsP3R1). This effect is attributed to the binding of ATP to glycine rich Walker A-type motifs present in the regulatory domain of the receptor. Only two such motifs are present in neuronal S2+ splice variant of InsP3R1 and are designated the ATPA and ATPB sites. The ATPA site is unique to InsP3R1, and the ATPB site is conserved among all three InsP3R isoforms. Despite the fact that both the ATPA and ATPB sites are known to bind ATP, the relative contribution of these two sites to the enhancing effects of ATP on InsP3R1 function is not known. We report here a mutational analysis of the ATPA and ATPB sites and conclude neither of these sites is required for ATP modulation of InsP3R1. ATP augmented InsP3-induced Ca2+ release from permeabilized cells expressing wild type and ATP-binding site-deficient InsP3R1. Similarly, ATP increased the single channel open probability of the mutated InsP3R1 to the same extent as wild type. ATP likely exerts its effects on InsP3R1 channel function via a novel and as yet unidentified mechanism.
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Lee, B., PG Bradford, and SG Laychock. "Characterization of inositol 1,4,5-trisphosphate receptor isoform mRNA expression and regulation in rat pancreatic islets, RINm5F cells and betaHC9 cells." Journal of Molecular Endocrinology 21, no. 1 (August 1, 1998): 31–39. http://dx.doi.org/10.1677/jme.0.0210031.
Full textAbstract:
The inositol 1,4,5-trisphosphate receptor (InsP3R) is an intracellular Ca2+ channel that plays a role in the regulation of insulin secretion. In rat isolated pancreatic islets the expression of types I, II and III InsP3R mRNA was identified by reverse transcriptase-polymerase chain reaction and confirmed by cDNA cloning and sequencing. The islet ratios of types I, II and III InsP3R mRNA to beta-actin mRNA were 0.08 +/- 0.02, 0.08 +/- 0.03 and 0.25 +/- 0.04 respectively. Types I, II and III InsP3R mRNA were also expressed in rat (RINm5F) and mouse (betaHC9) pancreatic beta-cell lines, and rat cerebellum. Type III InsP3R mRNA was quantitatively the most abundant form in rat islets and RINm5F cells. In betaHC9 cells, types II and III InsP3R mRNA were expressed at similar levels, and in much greater abundance than type I mRNA. Type III was the least abundant InsP3R mRNA in cerebellum. Culture of betaHC9 cells for 5 days at 2.8 and 25 mM glucose, or RINm5F cells for 7 days at 5.5 and 20 mM glucose, resulted in significantly enhanced expression of type III, but not types I and II, InsP3R mRNA in the cells at the higher glucose concentrations. During short-term (0.5-2 h) incubations, betaHC9 cell type III InsP3R mRNA levels increased in response to glucose in a time- and concentration-dependent manner. Actinomycin D inhibited the glucose response. Alpha-ketoisocaproic acid also stimulated betaHC9 cell type III InsP3R mRNA expression in a concentration-dependent manner, whereas 2-deoxyglucose and 3-O-methylglucose were without effect. The different levels of expression of mRNA for three InsP3R isoforms in islets and insulinoma cells, and the influence of glucose and alpha-ketoisocaproic acid on the expression of type III mRNA, suggests that nutrient metabolism plays a role in the regulation of this gene and that the function of InsP3R subtypes may be unique with each playing a distinct role in beta-cell signal transduction and insulin secretion.
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Fujimoto, T., A. Miyawaki, and K. Mikoshiba. "Inositol 1,4,5-trisphosphate receptor-like protein in plasmalemmal caveolae is linked to actin filaments." Journal of Cell Science 108, no. 1 (January 1, 1995): 7–15. http://dx.doi.org/10.1242/jcs.108.1.7.
Full textAbstract:
We reported that a plasmalemmal inositol 1,4,5-trisphosphate receptor-like protein (PM InsP3R-L) is localized in caveolae of various non-neuronal cells in vivo (Fujimoto et al. (1992) J. Cell Biol. 119, 1507–1513). In the present study, we investigated the distribution of PM InsP3R-L in cultured cells. In mouse epidermal keratinocytes (Pam 212) cultured in standard Ca2+ (1.8 mM), PM InsP3R-L was distributed densely in the vicinity of cell-to-cell contacts. In contrast, when Pam cells were cultured in low Ca2+ (0.06 mM) without making cell-to-cell contacts, PM InsP3R-L was observed randomly; by restoring the Ca2+ concentration, the circumferential actin filaments became obvious and the density of PM InsP3R-L increased in the contact region. Treatment of Pam cells with cytochalasin D caused aggregation of caveolae where PM InsP3R-L as well as F-actin and fodrin were localized. In bovine aortic endothelial cells, PM InsP3R-L was aligned along actin filaments crossing the cytoplasm in various directions. PM InsP3R-L of Pam cells was hardly extracted by treatment with 0.5% Triton X-100 or 60 mM octyl-glucoside in a cytoskeleton-stabilizing buffer for 15 minutes at 4 degrees C. The results show that the distribution of caveolae bearing PM InsP3R-L changes when the actin cytoskeleton is modified. They also indicate that the association of PM InsP3R-L with actin filaments may mediate the redistribution of caveolae. Since caveolae are thought to be related to signal transduction, their location defined by the actin cytoskeleton may affect the site where cellular reaction is to occur in response to various stimuli.
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DeLisle, S., O. Blondel, F. J. Longo, W. E. Schnabel, G. I. Bell, and M. J. Welsh. "Expression of inositol 1,4,5-trisphosphate receptors changes the Ca2+ signal of Xenopus oocytes." American Journal of Physiology-Cell Physiology 270, no. 4 (April 1, 1996): C1255—C1261. http://dx.doi.org/10.1152/ajpcell.1996.270.4.c1255.
Full textAbstract:
The receptors for the second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] form a family of closely related proteins that play an important role in regulating the free intracellular Ca2+ concentration. To test the hypothesis that changing the expression level of Ins(1,4,5)P3 receptors could alter the Ins(1,4,5)P3-mediated Ca2+ signal, we overexpressed Ins(1,4,5)P3 receptor type 1 (InsP3R-1) or type 3 (InsP3R-3) in Xenopus laevis oocytes. Expression of InsP3R-1 increased the velocity of the propagating waves of intracellular Ca2+ release but did not affect the Ins(1,4,5)P3-induced entry of extracellular Ca2+ across the plasma membrane. In contrast, expression of intracellular Ca2+ but markedly increased the magnitude and duration of Ca2+ influx. Immunolocalization studied revealed InsP3R-3 at the endoplasmic reticulum, with a relatively stronger signal at or near the plasma membrane. The results suggest that changing the expression level of an InsP3R can alter the Ins(1,4,5)P3-mediated Ca2+ signal and that InsP3R-1 and InsP3R-3 may have different biological functions.
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Madesh, Muniswamy, Brian J. Hawkins, Tatyana Milovanova, Cunnigaiper D. Bhanumathy, Suresh K. Joseph, Satish P. RamachandraRao, Kumar Sharma, Tomohiro Kurosaki, and Aron B. Fisher. "Selective role for superoxide in InsP3 receptor–mediated mitochondrial dysfunction and endothelial apoptosis." Journal of Cell Biology 170, no. 7 (September 26, 2005): 1079–90. http://dx.doi.org/10.1083/jcb.200505022.
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Reactive oxygen species (ROS) play a divergent role in both cell survival and cell death during ischemia/reperfusion (I/R) injury and associated inflammation. In this study, ROS generation by activated macrophages evoked an intracellular Ca2+ ([Ca2+]i) transient in endothelial cells that was ablated by a combination of superoxide dismutase and an anion channel blocker. [Ca2+]i store depletion, but not extracellular Ca2+ chelation, prevented [Ca2+]i elevation in response to O2.− that was inositol 1,4,5-trisphosphate (InsP3) dependent, and cells lacking the three InsP3 receptor (InsP3R) isoforms failed to display the [Ca2+]i transient. Importantly, the O2.−-triggered Ca2+ mobilization preceded a loss in mitochondrial membrane potential that was independent of other oxidants and mitochondrially derived ROS. Activation of apoptosis occurred selectively in response to O2.− and could be prevented by [Ca2+]i buffering. This study provides evidence that O2.− facilitates an InsP3R-linked apoptotic cascade and may serve a critical function in I/R injury and inflammation.
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Striggow, F., and B. E. Ehrlich. "The inositol 1,4,5-trisphosphate receptor of cerebellum. Mn2+ permeability and regulation by cytosolic Mn2+." Journal of General Physiology 108, no. 2 (August 1, 1996): 115–24. http://dx.doi.org/10.1085/jgp.108.2.115.
Full textAbstract:
The inositol 1,4,5-trisphosphate receptor (InsP3R), an intracellular calcium release channel, is found in virtually all cells and is abundant in the cerebellum. We used Mn2+ as a tool to study two aspects of the cerebellar InsP3R. First, to investigate the structure of the ion pore, Mn2+ permeation through the channel was determined. We found that Mn2+ can pass through the InsP3R; the selectivity sequence for divalent cations is Ba2+ > Sr2+ > Ca2+ > Mg2+ > Mn2+. Second, to begin characterization of the cytosolic regulatory sites responsible for the Ca(2+)-dependent modulation of InsP3R function, the ability of Mn2+ to replace Ca2+ was investigated. We show that Mn2+, as Ca2+, modulates InsP3R activity with a bell-shaped dependence where the affinity of the activation site of the InsP3R is similar for both ions, but higher concentrations of Mn2+ were necessary to inhibit the channel. These results suggest that the two regulatory sites are structurally distinct. Our findings are also important for the understanding of cellular responses when Mn2+ is used to quench the intracellular fluorescence of Ca2+ indicator dyes.
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THROWER, C. Edwin, J. A. Edward LEA, and P. Alan DAWSON. "The effects of free [Ca2+] on the cytosolic face of the inositol (1,4,5)-trisphosphate receptor at the single channel level." Biochemical Journal 330, no. 1 (February 15, 1998): 559–64. http://dx.doi.org/10.1042/bj3300559.
Full textAbstract:
Cytosolic free Ca2+ has been shown to have both activating and inhibitory effects upon the inositol (1,4,5) trisphosphate receptor (InsP3R) during intracellular Ca2+ release. The effects of cytosolic free Ca2+ on the InsP3R have already been monitored using cerebellar microsomes (containing InsP3R) incorporated into planar lipid bilayers [Bezprozvanny, Watras and Ehrlich (1991) Nature (London) 351, 751-754]. In these experiments the open probability of the channel exhibited a ‘bell-shaped Ca2+ dependence’. However, this has only been seen when the receptor is in the presence of its native membrane (e.g. microsomal vesicles). Using solubilized, purified InsP3R incorporated into planar lipid bilayers using the ‘tip-dip’ technique, investigations were carried out to see if the same effect was seen in the absence of the native membrane. Channel activity was observed in the presence of 4 μM InsP3 and 200 nM free Ca2+. Mean single channel current was 2.69 pA and more than one population of lifetimes was observed. Two populations had mean open times of approx. 9 and 97 ms. Upon increasing the free [Ca2+] to 2 μM, the mean single channel current decreased slightly to 2.39 pA, and the lifetimes increased to 30 and 230 ms. Elevation of free [Ca2+] to 4 μM resulted in a further decrease in mean single channel current to 1.97 pA as well as a decrease in lifetime to approx. 8 and 194 ms. At 10 μM free [Ca2+] no channel activity was observed. Thus, with purified receptor in artificial bilayers, free [Ca2+] on the cytosolic face of the receptor has major effects on channel behaviour, particularly on channel closure, although inhibition of channel activity is not seen until very high free [Ca2+] is reached.
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COQUIL, Jean-François, Samantha BLAZQUEZ, Sabrina SOAVE, and Jean-Pierre MAUGER. "Regulation of the cerebellar inositol 1,4,5-trisphosphate receptor by univalent cations." Biochemical Journal 381, no. 2 (July 6, 2004): 423–28. http://dx.doi.org/10.1042/bj20031984.
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In the present study we investigated the effects of K+ and other univalent cations on [3H]InsP3 {[3H]Ins(1,4,5)P3} binding to sheep cerebellar microsomes. In equilibrium binding experiments performed over 4 s at pH 7.1 and 20 °C, the addition of K+ to the binding medium decreased the affinity and increased the total number of binding sites for InsP3 in a dose-dependent manner. At low InsP3 concentration (0.5 nM) these effects resulted in a biphasic dose–response curve, with maximal binding at about 75 mM K+. In contrast, the dose–response curve calculated for InsP3 at the physiological concentration of 5 μM, was linear up to 200 mM K+. Univalent inorganic cations stimulated [3H]InsP3 binding to various extents, with the following descending order of efficiency at 75 mM: Cs+≈Rb+≈K+>Na+>Li+. The effect of K+ on InsP3R affinity was rapidly reversed upon cation removal. We were therefore also able to demonstrate that K+ increased Bmax (maximal specific binding) by pre-treating microsomes with K+ before measuring [3H]InsP3 binding in the absence of that cation. The increase in Bmax was reversible, but this reversal occurred less rapidly than the change in affinity. These results are consistent with a process by which K+ reversibly converted very low-affinity sites into sites with higher affinity, making them detectable in competitive binding experiments. They suggest that interconversion between these two affinity states constitutes the basis of a K+-controlled regulatory mechanism for cerebellar InsP3R.
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LIÈVREMONT, Jean-Philippe, Haud LANCIEN, Mauricette HILLY, and Jean-Pierre MAUGER. "The properties of a subtype of the inositol 1,4,5-trisphosphate receptor resulting from alternative splicing of the mRNA in the ligand-binding domain." Biochemical Journal 317, no. 3 (August 1, 1996): 755–62. http://dx.doi.org/10.1042/bj3170755.
Full textAbstract:
Subtypes of the type-1 inositol 1,4,5-trisphosphate (InsP3) receptor differ at the mRNA level in two small variably spliced segments. One segment (SI) encodes for a sequence within the InsP3-binding domain, thus its presence or absence could affect the functions of the receptor. We have used anti-peptide antibodies to confirm the existence of different subtypes of the InsP3 receptor (InsP3R) protein. The antibody against residues 322–332, within the SI region, recognized a 260 kDa polypeptide in membranes prepared from rat cerebellum or cerebral cortex. The cerebellum contained a few percent of the InsP3R protein having the SI region, whereas the cerebral cortex contained a high proportion of receptors with the SI region. These two tissues were representative of both isoforms, SI- or SI+, and displayed the same [3H]InsP3-binding characteristics. Thus, the SI region was not involved in the basic properties of the receptor. Deletion of the peptide 316–352 containing the SI segment greatly reduced InsP3 binding [Miyawaki, Furuichi, Ryou, Yoshikawa, Nakagawa, Saitoh and Mikoshiba (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4911–4915]. The antibodies against the SI region or against residues 337–349 did not modify the binding of [3H]InsP3 in the cortical membranes rich in the SI+ isoform or in cerebellar membranes. These results suggested that the SI region was not part of the binding site. The subcellular distribution of these two isoforms was then investigated in rat liver. The two isoforms were identified in different membrane fractions and they followed the same subcellular distribution. We suggest that the domain with the SI region may be involved in a function other than InsP3-induced Ca2+ release.
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Vais, Horia, J. Kevin Foskett, and Don-On Daniel Mak. "InsP3R channel gating altered by clustering?" Nature 478, no. 7368 (October 12, 2011): E1—E2. http://dx.doi.org/10.1038/nature10493.
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Daniel Mak, Don-On, Horia Vais, Carl White, and J. Kevin Foskett. "Cytoplasmic [InsP3] Drop Induces Transient High-Open-Probability Gating Mode in Type 1 InsP3R Channel." Biophysical Journal 104, no. 2 (January 2013): 121a—122a. http://dx.doi.org/10.1016/j.bpj.2012.11.701.
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LEITE, M. Fatima, Jonathan A. DRANOFF, Ling GAO, and Michael H. NATHANSON. "Expression and subcellular localization of the ryanodine receptor in rat pancreatic acinar cells." Biochemical Journal 337, no. 2 (January 8, 1999): 305–9. http://dx.doi.org/10.1042/bj3370305.
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The ryanodine receptor (RyR) is the principal Ca2+-release channel in excitable cells, whereas the inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) is primarily responsible for Ca2+ release in non-excitable cells, including epithelia. RyR also is expressed in a number of non-excitable cell types, but is thought to serve as an auxiliary or alternative Ca2+-release pathway in those cells. Here we use reverse transcription PCR to show that a polarized epithelium, the pancreatic acinar cell, expresses the type 2, but not the type 1 or 3, isoform of RyR. We furthermore use immunochemistry to demonstrate that the type 2 RyR is distributed throughout the basolateral and, to a lesser extent, the apical region of the acinar cell, but is excluded from the trigger zone, where cytosolic Ca2+ signals originate in this cell type. Since propagation of Ca2+ waves in acinar cells is sensitive to ryanodine, caffeine and Ca2+, these findings suggest that Ca2+ waves in this cell type result from the co-ordinated release of Ca2+, first from InsP3Rs in the trigger zone, then from RyRs elsewhere in the cell. RyR may play a fundamental role in Ca2+ signalling in polarized epithelia, including for Ca2+ signals initiated by InsP3.
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Treves, Susan, Clara Franzini-Armstrong, Luca Moccagatta, Christophe Arnoult, Cristiano Grasso, Adam Schrum, Sylvie Ducreux, et al. "Junctate is a key element in calcium entry induced by activation of InsP3 receptors and/or calcium store depletion." Journal of Cell Biology 166, no. 4 (August 9, 2004): 537–48. http://dx.doi.org/10.1083/jcb.200404079.
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In many cell types agonist-receptor activation leads to a rapid and transient release of Ca2+ from intracellular stores via activation of inositol 1,4,5 trisphosphate (InsP3) receptors (InsP3Rs). Stimulated cells activate store- or receptor-operated calcium channels localized in the plasma membrane, allowing entry of extracellular calcium into the cytoplasm, and thus replenishment of intracellular calcium stores. Calcium entry must be finely regulated in order to prevent an excessive intracellular calcium increase. Junctate, an integral calcium binding protein of endo(sarco)plasmic reticulum membrane, (a) induces and/or stabilizes peripheral couplings between the ER and the plasma membrane, and (b) forms a supramolecular complex with the InsP3R and the canonical transient receptor potential protein (TRPC) 3 calcium entry channel. The full-length protein modulates both agonist-induced and store depletion–induced calcium entry, whereas its NH2 terminus affects receptor-activated calcium entry. RNA interference to deplete cells of endogenous junctate, knocked down both agonist-activated calcium release from intracellular stores and calcium entry via TRPC3. These results demonstrate that junctate is a new protein involved in calcium homeostasis in eukaryotic cells.
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Cruz, Pablo, Ulises Ahumada-Castro, Galdo Bustos, Jordi Molgó, Daniela Sauma, Alenka Lovy, and César Cárdenas. "Inhibition of InsP3R with Xestospongin B Reduces Mitochondrial Respiration and Induces Selective Cell Death in T Cell Acute Lymphoblastic Leukemia Cells." International Journal of Molecular Sciences 22, no. 2 (January 11, 2021): 651. http://dx.doi.org/10.3390/ijms22020651.
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T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy whose chemoresistance and relapse persist as a problem despite significant advances in its chemotherapeutic treatments. Mitochondrial metabolism has emerged as an interesting therapeutic target given its essential role in maintaining bioenergetic and metabolic homeostasis. T-ALL cells are characterized by high levels of mitochondrial respiration, making them suitable for this type of intervention. Mitochondrial function is sustained by a constitutive transfer of calcium from the endoplasmic reticulum to mitochondria through the inositol 1,4,5-trisphosphate receptor (InsP3R), making T-ALL cells vulnerable to its inhibition. Here, we determine the bioenergetic profile of the T-ALL cell lines CCRF-CEM and Jurkat and evaluate their sensitivity to InsP3R inhibition with the specific inhibitor, Xestospongin B (XeB). Our results show that T-ALL cell lines exhibit higher mitochondrial respiration than non-malignant cells, which is blunted by the inhibition of the InsP3R. Prolonged treatment with XeB causes T-ALL cell death without affecting the normal counterpart. Moreover, the combination of XeB and glucocorticoids significantly enhanced cell death in the CCRF-CEM cells. The inhibition of InsP3R with XeB rises as a potential therapeutic alternative for the treatment of T-ALL.
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Cruz, Pablo, Ulises Ahumada-Castro, Galdo Bustos, Jordi Molgó, Daniela Sauma, Alenka Lovy, and César Cárdenas. "Inhibition of InsP3R with Xestospongin B Reduces Mitochondrial Respiration and Induces Selective Cell Death in T Cell Acute Lymphoblastic Leukemia Cells." International Journal of Molecular Sciences 22, no. 2 (January 11, 2021): 651. http://dx.doi.org/10.3390/ijms22020651.
Full textAbstract:
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy whose chemoresistance and relapse persist as a problem despite significant advances in its chemotherapeutic treatments. Mitochondrial metabolism has emerged as an interesting therapeutic target given its essential role in maintaining bioenergetic and metabolic homeostasis. T-ALL cells are characterized by high levels of mitochondrial respiration, making them suitable for this type of intervention. Mitochondrial function is sustained by a constitutive transfer of calcium from the endoplasmic reticulum to mitochondria through the inositol 1,4,5-trisphosphate receptor (InsP3R), making T-ALL cells vulnerable to its inhibition. Here, we determine the bioenergetic profile of the T-ALL cell lines CCRF-CEM and Jurkat and evaluate their sensitivity to InsP3R inhibition with the specific inhibitor, Xestospongin B (XeB). Our results show that T-ALL cell lines exhibit higher mitochondrial respiration than non-malignant cells, which is blunted by the inhibition of the InsP3R. Prolonged treatment with XeB causes T-ALL cell death without affecting the normal counterpart. Moreover, the combination of XeB and glucocorticoids significantly enhanced cell death in the CCRF-CEM cells. The inhibition of InsP3R with XeB rises as a potential therapeutic alternative for the treatment of T-ALL.
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Won, Jong Hak, William J. Cottrell, Thomas H. Foster, and David I. Yule. "Ca2+ release dynamics in parotid and pancreatic exocrine acinar cells evoked by spatially limited flash photolysis." American Journal of Physiology-Gastrointestinal and Liver Physiology 293, no. 6 (December 2007): G1166—G1177. http://dx.doi.org/10.1152/ajpgi.00352.2007.
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Intracellular calcium concentration ([Ca2+]i) signals are central to the mechanisms underlying fluid and protein secretion in pancreatic and parotid acinar cells. Calcium release was studied in natively buffered cells following focal laser photolysis of caged molecules. Focal photolysis of caged-inositol 1,4,5 trisphosphate (InsP3) in the apical region resulted in Ca2+ release from the apical trigger zone and, after a latent period, the initiation of an apical-to-basal Ca2+ wave. The latency was longer and the wave speed significantly slower in pancreatic compared with parotid cells. Focal photolysis in basal regions evoked only limited Ca2+ release at the photolysis site and never resulted in a propagating wave. Instead, an apical-to-basal wave was initiated following a latent period. Again, the latent period was significantly longer under all conditions in pancreas than parotid. Although slower in pancreas than parotid, once initiated, the apical-to-basal wave speed was constant in a particular cell type. Photo release of caged-Ca2+ failed to evoke a propagating Ca2+ wave in either cell type. However, the kinetics of the Ca2+ signal evoked following photolysis of caged-InsP3 were significantly dampened by ryanodine in parotid but not pancreas, indicating a more prominent functional role for ryanodine receptor (RyR) following InsP3 receptor (InsP3R) activation. These data suggest that differing expression levels of InsP3R, RyR, and possibly cellular buffering capacity may contribute to the fast kinetics of Ca2+ signals in parotid compared with pancreas. These properties may represent a specialization of the cell type to effectively stimulate Ca2+-dependent effectors important for the differing primary physiological role of each gland.