InsP3R Type Research Articles

Characterization of NCS1–InsP3R1 interaction and its functional significance

Inositol 1,4,5-trisphosphate receptors (InsP3Rs) are endoplasmic reticulum-localized channels that mediate Ca2+ release from the endoplasmic reticulum into the cytoplasm. We previously reported that an EF-hand Ca2+-binding protein, neuronal calcium sensor 1 (NCS1), binds to the InsP3R and thereby increases channel open probability, an event associated with chemotherapy-induced peripheral neuropathy. However, the exact NCS1-binding site on InsP3R remains unknown. Using protein docking, co-immunoprecipitation, and blocking peptides, we mapped the NCS1-binding site to residues 66-110 on the suppressor domain of InsP3R type 1 (InsP3R1). We also identified Leu-89, a residue in the hydrophobic pocket of NCS1, as being critical for facilitating the NCS1-InsP3R1 interaction. Overexpression of WT NCS1 in MDA-MB231 breast cancer cells increased Ca2+ signaling and survival, whereas overexpression of Leu-89 NCS1 variants decreased Ca2+ signaling and survival, further suggesting the importance of this residue in the NCS1-InsP3R1 interaction. In conclusion, we show that NCS1-InsP3R1 interaction enhances intracellular Ca2+ signaling in cells and can be modulated by altering or occluding the hydrophobic pocket of NCS1. This improved understanding of the NCS1-InsP3R1 interaction may facilitate the development of management strategies for diseases resulting from aberrant NCS1 expression.

Regulation Of Inositol 1,4,5-Trisphosphate Receptor Isoforms By O-Linked Glycosylation

The inositiol 1,4,5 trisphospate receptor (InsP3R), an intracellular calcium channel, is a family of three isoforms. All three isoforms display a significant level of sequence identity yet they differ in expression level, localization and many functional aspects. We previously showed that InsP3R type 1 is modified by O-linked β-N-acetylglucosamine glycosylation (O-GlcNAcylated). Through this dynamic and inducible modification a single monosaccharide is covalently attached to serine and threonine residues of the protein backbone, providing protein regulation similar to O-phosphorylation. We also reported that increased O-GlcNAcylation of the InsP3R type 1 reduced the percent of cells that responded to addition of extracellular agonists and those that did respond had a decreased InsP3 dependent calcium release from the endoplasmic reticulum (ER). We now report that the InsP3R type 3 is also O-GlcNAcylated. Interestingly, the functional impact of O-GlcNAcylation on InsP3R type 3 channel is opposite to the effect measured with the InsP3R type 1. Human cholangiocytoma cells (MzChA-1) contain >90% InsP3R type 3. When these cells were incubated in hyperglycemic media there was an increase in the percent cells responding to InsP3 generating stimuli and there was an increase in the InsP3 dependent calcium release from the ER. A difference in functional response between InsP3R isoforms was reported previously for phosphorylation by cyclic AMP dependent protein kinase (PKA). In contrast, the InsP3R type 2 showed no detectable O-GlcNAc glycosylation and no significant functional changes even though the enzymes necessary for both the addition and removal of the monosccharide are present in all cell types tested. The dynamic and inducible nature of O-GlcNAcylation and the isoform specificity suggests that this form of modification of the InsP3R and subsequent changes in intracellular calcium transients will be important in physiological and pathophysiological processes.

Abstract 1371: NCS-1 is a Potential Therapeutic Target for Avoiding Bradycardia and Optimizing Cancer Chemotherapy with Paclitaxel

We hypothesize that the inositol 1,4,5-trisphosphate receptor (InsP3R) signaling pathway is crucial for the development of cardiac arrhythmia caused by paclitaxel treatment. Paclitaxel, a well established drug used for cancer chemotherapy, constrains tumor growth through a microtubule stabilizing mechanism. However, use of paclitaxel is limited by serious side effects including bradycardia, which cannot be explained by its effect on microtubules. Recently, a new binding partner of paclitaxel was described - neuronal calcium sensor-1 (NCS-1), a protein that modulates the function of the InsP3R and that we found to be expressed in cardiomoycytes. After treating neonatal ventricular cardiomyocytes from rats with a therapeutic dose of paclitaxel for several hours, NCS-1 levels were markedly elevated. The same effect was also detectable in vivo, in hearts extracted from adult mice chronically injected with paclitaxel. Using live cell imaging, we observed an increase in the peak of the intracellular calcium signal and a significant decrease in the spontaneous calcium oscillation frequency in paclitaxel treated cardiomyocytes compared to control. The changes could not be explained by the function of ryanodine receptors (RyR) because there is no functional interaction between NCS-1 and RyR, as previously shown in bilayer experiments, and no paclitaxel-dependent change in RyR expression. However, co-immunoprecipitation confirmed a direct interaction between NCS-1 and InsP3R type 2, the predominant isoform in cardiomyocytes. To test whether this interaction is functionally relevant, we stimulated cardiomyocytes with endothelin, an activator of the InsP3 signaling cascade and found a difference in calcium transients between paclitaxel-treated and untreated cardiomyocytes. NCS-1 knockdown in cardiomyocytes by shRNA validated our findings. Taken together, our results suggest that paclitaxel-induced cardiac arrhythmia in patients is mediated by the interaction of NCS-1 and the InsP3R. This gives us a new understanding of the pathomechanism and provides a new approach for reducing the side effects of paclitaxel treatment and optimizing cancer chemotherapy.

Elevated Testosterone Induces Apoptosis in Neuronal Cells

Testosterone plays a crucial role in neuronal function, but elevated concentrations can have deleterious effects. Here we show that supraphysiological levels of testosterone (micromolar range) initiate the apoptotic cascade. We used three criteria, annexin V labeling, caspase activity, and DNA fragmentation, to determine that apoptotic pathways were activated by testosterone. Micromolar, but not nanomolar, testosterone concentrations increased the response in all three assays of apoptosis. In addition, testosterone induced different concentration-dependent Ca2+ signaling patterns: at low concentrations of testosterone (100 nm), Ca2+ oscillations were produced, whereas high concentrations (1-10 microm) induced a sustained Ca2+ increase. Elevated testosterone concentrations increase cell death, and this effect was abolished in the presence of either inhibitors of caspases or the inositol 1,4,5-trisphosphate receptor (InsP3R)-mediated Ca2+ release. Knockdown of InsP3R type 1 with specific small interfering RNA also abolished the testosterone-induced cell death and the prolonged Ca2+ signals. In contrast, knockdown of InsP3R type 3 modified neither the apoptotic response nor the Ca2+ signals. These results support our hypothesis that elevated testosterone alters InsP3R type 1-mediated intracellular Ca2+ signaling and that the prolonged Ca2+ signals lead to apoptotic cell death. These effects of testosterone on neurons will have long term effects on brain function.

Neuronal calcium sensor-1 enhancement of InsP3 receptor activity is inhibited by therapeutic levels of lithium

Regulation and dysregulation of intracellular calcium (Ca2+) signaling via the inositol 1,4,5-trisphosphate receptor (InsP3R) has been linked to many cellular processes and pathological conditions. In the present study, addition of neuronal calcium sensor-1 (NCS-1), a high-affinity, low-capacity, calcium-binding protein, to purified InsP3R type 1 (InsP3R1) increased the channel activity in both a calcium-dependent and -independent manner. In intact cells, enhanced expression of NCS-1 resulted in increased intracellular calcium release upon stimulation of the phosphoinositide signaling pathway. To determine whether InsP3R1/NCS-1 interaction could be functionally relevant in bipolar disorders, conditions in which NCS-1 is highly expressed, we tested the effect of lithium, a salt widely used for treatment of bipolar disorders. Lithium inhibited the enhancing effect of NCS-1 on InsP3R1 function, suggesting that InsP3R1/NCS-1 interaction is an essential component of the pathomechanism of bipolar disorder.

The inositol 1,4,5-trisphosphate receptors

The inositol (1,4,5)-trisphosphate receptors (InsP 3R) are the intracellular calcium (Ca 2+) release channels that play a key role in Ca 2+ signaling in cells. Three InsP 3R isoforms—InsP 3R type 1 (InsP 3R1), InsP 3R type 2 (InsP 3R2), and InsP 3R type 3 (InsP 3R3) are expressed in mammals. A single InsP 3R isoform is expressed in Drosophila melanogaster (DmInsP 3R) and Caenorhabditis elegans (CeInsP 3R). The progress made during last decade towards understanding the function and the properties of the InsP 3R is briefly reviewed in this chapter. The main emphasis is on studies that revealed structural determinants responsible for the ligand recognition by the InsP 3R, ion permeability of the InsP 3R, modulation of the InsP 3R by cytosolic Ca 2+, ATP and PKA phosphorylation and on the recently identified InsP 3R-binding partners. The main focus is on the InsP 3R1, but the recent information about properties of other InsP 3R isoforms is also discussed.

Functional Characterization of Mammalian Inositol 1,4,5-Trisphosphate Receptor Isoforms

Inositol 1,4,5-trisphosphate receptors (InsP3R) play a key role in intracellular calcium (Ca2+) signaling. Three mammalian InsP3R isoforms—InsP3R type 1 (InsP3R1), InsP3R type 2 (InsP3R2), and InsP3R type 3 (InsP3R3) are expressed in mammals, but the functional differences between the three mammalian InsP3R isoforms are poorly understood. Here we compared single-channel behavior of the recombinant rat InsP3R1, InsP3R2, and InsP3R3 expressed in Sf9 cells, reconstituted into planar lipid bilayers and recorded with 50mM Ba2+ as a current carrier. We found that: 1), for all three mammalian InsP3R isoforms the size of the unitary current is 1.9 pA and single-channel conductance is 74–80 pS; 2), in optimal recording conditions the maximal single-channel open probability for all three mammalian InsP3R isoforms is in the range 30–40%; 3), in optimal recording conditions the mean open dwell time for all three mammalian InsP3R isoforms is 7–8ms, the mean closed dwell time is ∼10ms; 4), InsP3R2 has the highest apparent affinity for InsP3 (0.10μM), followed by InsP3R1 (0.27μM), and then by InsP3R3 (0.40μM); 5), InsP3R1 has a high-affinity (0.13mM) ATP modulatory site, InsP3R2 gating is ATP independent, and InsP3R3 has a low-affinity (2mM) ATP modulatory site; 6), ATP modulates InsP3R1 gating in a noncooperative manner (nHill=1.3); 7), ATP modulates InsP3R3 gating in a highly cooperative manner (nHill=4.1). Obtained results provide novel information about functional properties of mammalian InsP3R isoforms.

Presence of InsP3R type 2 in the endoplasmic reticulum, but not in secretory granules, in rat adrenal chromaffin cells

Presence of InsP3R type 2 in the endoplasmic reticulum, but not in secretory granules, in rat adrenal chromaffin cells

Immunolocalization of type 2 inositol 1,4,5-trisphosphate receptors in cardiac myocytes from newborn mice.

The precise localization and role of inositol 1,4,5-trisphosphate (InsP(3)) receptors (InsP(3)Rs) in cardiac muscle cells are largely unknown. It is believed that waves and oscillations in cytosolic free calcium triggered by activation of InsP(3)Rs 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 InsP(3)R type 2 is the only isoform present in cardiac myocytes isolated from neonatal mouse ventricles. We also show that type 2 InsP(3)Rs are associated with the nucleus and that activation of type 2 InsP(3)Rs with endothelin-1 or phenylephrine selectively increases transcription of atrial natriuretic factor and skeletal alpha-actin. Type 2 InsP(3)Rs are also in striations. Activation of InsP(3)Rs 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 InsP(3)R inhibitors 2-aminoethoxydiphenyl borate and xestospongin C. The spatial separation of type 2 InsP(3)Rs provides support for the concept that microdomains of calcium discretely alter various cell processes. Our experiments suggest that calcium released by InsP(3)Rs 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.

A Functional Interaction between Chromogranin B and the Inositol 1,4,5-Trisphosphate Receptor/Ca2+ Channel

Chromogranins A and B (CGA and CGB) are high capacity, low affinity calcium (Ca2+) storage proteins found in many cell types most often associated with secretory granules of secretory cells but also with the endoplasmic reticulum (ER) lumen of these cells. Both CGA and CGB associate with inositol 1,4,5-trisphosphate receptor (InsP3R) in a pH-dependent manner. At an intraluminal pH of 5.5, as found in secretory vesicles, both CGA and CGB bind to the InsP3R. When the intraluminal pH is 7.5, as found in the ER, CGA totally dissociates from InsP3R, whereas CGB only partially dissociates. To investigate the functional consequences of the interaction between the InsP3R and CGB monomers or CGA/CGB heteromers, purified mouse InsP3R type I were fused to planar lipid bilayers and activated by 2 microM InsP3. In the presence of luminal CGB monomers or CGA/CGB heteromers the InsP3R/Ca2+ channel open probability and mean open time increased significantly. The channel activity remained elevated when the pH was changed to 7.5, a reflection of CGB binding to the InsP3R even at pH 7.5. These results suggest that CGB may play an important modulatory role in the control of Ca2+ release from the ER. Furthermore, the difference in the ability of CGA and CGB to regulate the InsP3R/Ca2+ channel and the variability of CGA/CGB ratios could influence the pattern of InsP3-mediated Ca2+ release.

Characterization of Ca 2+ signaling pathways in human mesenchymal stem cells

Human mesenchymal stem cells (HMSC) have the potential to differentiate into many cell types. The physiological properties of HMSCs including their Ca 2+ signaling pathways, however, are not well understood. We investigated Ca 2+ influx and release functions in HMSCs. In Ca 2+ imaging experiments, spontaneous Ca 2+ oscillations were observed in 36 of 50 HMSCs. The Ca 2+ oscillations were completely blocked by the application of 10 μM cyclopiazonic acid (CPA) or 1 μM thapsigargin (TG). A brief application of 1 μM acetylcholine (ACh) induced a transient increase of [Ca 2+] i but the application of caffeine (10 mM) did not induce any Ca 2+ transient. When the stores were depleted with Ca 2+-ATPase blockers (CPA or TG) or muscarinic agonists (ACh), store-operated Ca 2+ (SOC) entry was observed. Using the patch-clamp technique, store-operated Ca 2+ currents ( I SOC) could be recorded in cells treated with ACh or CPA, but voltage-operated Ca 2+ currents (VOCCs) were not elicited in most of the cells (17/20), but in 15% of cells examined, small dihydropyridine (DHP)-sensitive Ca 2+ currents were recorded. Using RT–PCR, mRNAs were detected for inositol 1,4,5-trisphosphate receptor (InsP 3R) type I, II, and III and DHP receptors α1A and α1H were detected, but mRNA was not detected for ryanodine receptor (RyR) or N-type Ca 2+ channels. These results suggest that in undifferentiated HMSCs, Ca 2+ release is mediated by InsP 3Rs and Ca 2+ entry through plasma membrane is mainly mediated by the SOCs channels with a little contribution of VOCCs.

Distinct intracellular calcium transients in neurites and somata integrate neuronal signals.

Intracellular calcium signals have distinct temporal and spatial patterns in neurons in which signal initiation and repetitive spiking occurs predominantly in the neurite. We investigated the functional implications of the coexpression of different isoforms of ryanodine receptors (RyR) and inositol 1,4,5-trisphosphate receptors (InsP3Rs) using immunocytochemistry, Western blotting, and calcium imaging in neuronally differentiated PC12 cells. InsP3R type III, an isoform that has been shown to be upregulated in neuronal apoptosis, is exclusively expressed in the soma, serving as a gatekeeper for high-magnitude calcium surges. InsP3R type I is expressed throughout the cell and can be related to signal initiation and repetitive spiking in the neurite. RyR types 2 and 3 are distributed throughout the cell. In the soma, they serve as amplifying molecular switches, facilitating recruitment of the InsP3R type III-dependent pool. In the neurite, they decrease the probability of repetitive spiking. Use of a cell-permeant analog of InsP3 suggested that regional specificity in InsP3 production and surface-to-volume effects play minor roles in determining temporal and spatial calcium signaling patterns in neurons. Our findings suggest that additional modulatory processes acting on the intracellular channels are necessary to generate spatially specific calcium signaling.

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

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

Type 1 Inositol 1,4,5-Trisphosphate Receptor Is Required for Induction of Long-Term Depression in Cerebellar Purkinje Neurons

The inositol 1,4,5-trisphosphate receptor (InsP3R) is an intracellular Ca2+ channel that releases Ca2+ from internal Ca2+ stores in response to InsP3. Although InsP3R is highly expressed in various regions of the mammalian brain, the functional role of this receptor has not been clarified. We show here that cerebellar slices prepared from mice with a disrupted InsP3R type 1 gene, which is predominantly expressed in Purkinje cells, completely lack long-term depression (LTD), a model of synaptic plasticity in the cerebellum. Moreover, a specific antibody against InsP3R1, introduced into wild-type Purkinje cells through patch pipettes, blocked the induction of LTD. These data indicate that, in addition to Ca2+ influx through Ca2+ channels on the plasma membrane, Ca2+ release from InsP3R plays an essential role in the induction of LTD, suggesting a physiological importance for InsP3R in Purkinje cells.

Induction of inositol 1,4,5 trisphosphate receptor genes by ionizing radiation.

We used differential display, a method designed to amplify partial cDNA sequences from subsets of mRNAs, to identify mRNAs induced by ionizing radiation in human Epstein Barr Virus (EBV)-transformed lymphoblastoid cells. Increased expression of a cDNA corresponding to the inositol 1,4,5 trisphosphate receptor (InsP3R) type 1 was observed after exposure of cells to 3Gy gamma-rays. This was confirmed by Northern blot analysis. The increase in mRNA for InsP3R type 1 was accompanied by a corresponding increase in the level of InsP3R type 1 protein as determined by Western blotting. Exposure of cells from patients with the human genetic disorder ataxia-telangiectasia (A-T), characterized by hypersensitivity to ionizing radiation, failed to change the levels of InsP3R type 1 mRNA and, as expected, there was no increase in InsP3R type 1 protein in A-T cells in response to radiation exposure. Protein levels for two other InsP3Rs, types 2 and 3, were observed to increase in control and A-T cells after exposure to ionizing radiation. The induction of the InsP3R type 1, which is primarily located in the endoplasmic reticulum, may play an important role in radiation signal transduction.