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1.
Functional characterization of the P1059L mutation in the inositol 1,4,5-trisphosphate receptor type 1 identified in a Japanese SCA15 family 总被引:1,自引:0,他引:1
Yamazaki H Nozaki H Onodera O Michikawa T Nishizawa M Mikoshiba K 《Biochemical and biophysical research communications》2011,(4):754-758
Spinocerebellar ataxia type 15 (SCA15) is a group of human neurodegenerative disorders characterized by a slowly progressing pure cerebellar ataxia. The inositol 1,4,5-trisphosphate (IP3) receptor type 1 (IP3R1) is an intracellular IP3-induced Ca2+ release channel that was recently identified as a causative gene for SCA15. In most case studies, a heterozygous deletion of the IP3R1 gene was identified. However, one Japanese SCA15 family was found to have a Pro to Leu (P1059L) substitution in IP3R1. To investigate the effect of the P1059L mutation, we analyzed the channel properties of the mutant human IP3R1 by expressing it in an IP3R-deficient B lymphocyte cell line. The P1059L mutant was a functional Ca2+ release channel with a twofold higher IP3 binding affinity compared to wild-type IP3R1. The cooperative dependence of the Ca2+ release activity of the mutant on IP3 concentration was reduced, but both wild-type and mutant receptors produced similar B cell receptor-induced Ca2+ signals. These results demonstrate that the Ca2+ release properties of IP3R1 are largely unaffected by the P1059L mutation. 相似文献
2.
There is substantial evidence that crosstalk between the proliferation and Ca2+-signaling pathways plays a critical role in the regulation of normal physiological functions as well as in the pathogenesis of a variety of abnormal processes. In non-excitable cells, intracellular Ca2+ is mobilized through inositol 1,4,5-trisphosphate sensitive Ca2+ channels (IP3R) expressed on the endoplasmic reticulum. Here we report that mTOR, a point of convergence for signals from mitogenic growth factors, nutrients and cellular energy levels, phosphorylates the IP3R-2, the predominant isoform of IP3R in AR4-2J cells. Pretreatment with the mTOR inhibitor rapamycin, decreased carbachol-induced Ca2+ release in AR4-2J cells. Rapamycin also decreased IP3-induced Ca2+ release in permeabilized AR4-2J cells. We also showed that IGF-1 potentiates carbachol-induced Ca2+ release in AR4-2J cells, an effect that was prevented by rapamycin. Rapamycin also decreased carbachol-induced Ca2+ release in HEK 293A cells in which IP3R-1 and IP3R-3 had been knocked down. These results suggest that mTOR potentiates the activity of IP3R-2 by a phosphorylation mechanism. This conclusion supports the concept of crosstalk between Ca2+ signaling and proliferation pathways and thus provides another way by which intracellular Ca2+ signals are finely encoded. 相似文献
3.
Oscillatory fluctuations in the cytosolic concentration of free calcium ions (Ca2+) are considered a ubiquitous mechanism for controlling multiple cellular processes. Inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) are intracellular Ca2+ release channels that mediate Ca2+ release from endoplasmic reticulum (ER) Ca2+ stores. The three IP3R subtypes described so far exhibit differential structural, biophysical, and biochemical properties. Subtype specific regulation
of IP3R by the endogenous modulators IP3, Ca2+, protein kinases and associated proteins have been thoroughly examined. In this article we will review the contribution of
each IP3R subtype in shaping cytosolic Ca2+ oscillations. 相似文献
4.
Yun Li Netty G. Santoso Shengqiang Yu Owen M. Woodward Feng Qian William B. Guggino 《The Journal of biological chemistry》2009,284(52):36431-36441
The PKD1 or PKD2 genes encode polycystins (PC) 1 and 2, which are associated with polycystic kidney disease. Previously we demonstrated that PC2 interacts with the inositol 1,4,5-trisphosphate receptor (IP3R) to modulate Ca2+ signaling. Here, we investigate whether PC1 also regulates IP3R. We generated a fragment encoding the last six transmembrane (TM) domains of PC1 and the C-terminal tail (QIF38), a section with the highest homology to PC2. Using a Xenopus oocyte Ca2+ imaging system, we observed that expression of QIF38 significantly reduced the initial amplitude of IP3-induced Ca2+ transients, whereas a mutation lacking the C-terminal tail did not. Thus, the C terminus is essential to QIF38 function. Co-immunoprecipitation assays demonstrated that through its C terminus, QIF38 associates with the IP3-binding domain of IP3R. A shorter PC1 fragment spanning only the last TM and the C-terminal tail also reduced IP3-induced Ca2+ release, whereas another C-terminal fragment lacking any TM domain did not. Thus, only endoplasmic reticulum-localized PC1 can modulate IP3R. Finally, we show that in the polarized Madin-Darby canine kidney cells, heterologous expression of full-length PC1 resulted in a smaller IP3-induced Ca2+ response. Overexpression of the IP3-binding domain of IP3R reversed the inhibitory effect of PC1, suggesting interaction of full-length PC1 (or its cleavage forms) with endogenous IP3R in Madin-Darby canine kidney cells. These results indicate that the behavior of full-length PC1 in mammalian cells is congruent with that of PC1 C-terminal fragments in the oocyte system. These data demonstrate that PC1 inhibits Ca2+ release, perhaps opposing the effect of PC2, which facilitates Ca2+ release through the IP3R. 相似文献
5.
Fujimoto T Machida T Tsunoda T Doi K Ota T Kuroki M Shirasawa S 《Biochemical and biophysical research communications》2011,(2):214-217
KRAS-induced actin-interacting protein (KRAP) was originally characterized as a filamentous- actin-interacting protein. We have recently found that KRAP is an associated molecule with inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and is responsible for the proper subcellular localization of IP3R. Since it remains unknown whether KRAP regulates the IP3R-mediated Ca2+ signaling, we herein examined the effects of KRAP on the IP3R-mediated Ca2+ release by Ca2+ imagings in the cultured HEK293 or MCF7 cells. Reduction of KRAP protein by KRAP-specific siRNA diminishes ATP-induced Ca2+ release and the ATP-induced Ca2+ release is completely quenched by the pretreatment with the IP3R inhibitor but not with the ryanodine receptor inhibitor, indicating that KRAP regulates IP3R-mediated Ca2+ release. To further reveal mechanistic insights into the regulation of IP3R-mediated Ca2+ release by KRAP, we examined the effects of the KRAP-knockdown on the releasable Ca2+ content of intracellular Ca2+ stores. Consequently, reduction of KRAP does not affect the amount of ionophore- or Ca2+-ATPase inhibitor-induced Ca2+ release in the HEK293 cells, indicating that releasable Ca2+ content of intracellular Ca2+ stores is not altered by KRAP. Thus, KRAP is involved in the proper regulation of IP3R-mediated Ca2+ release. 相似文献
6.
7.
The inositol 1,4,5-trisphosphate receptor (IP3R) is one of two Ca2+ channels that gates Ca2+ release from ER-stores. The ligand IP3, generated upon specific G-protein coupled receptor activation, binds to IP3R to release Ca2+ into the cytosol. IP3R also mediates ER-store Ca2+ release into the mitochondria, under basal as well as stimulatory conditions; an activity that influences cellular bioenergetics and thus, cellular growth and proliferation. In Drosophila neuroendocrine cells expressing a hypomorphic mutant of IP3R, we observed reduced protein translation levels. Here, we discuss the possible molecular mechanism for this observation. We hypothesize that the cellular energy sensor, AMPK connects IP3R mediated Ca2+ release into the mitochondria, to protein translation, via the TOR pathway. 相似文献
8.
Diptiman D. Bose 《Biochemical and biophysical research communications》2009,379(2):594-133
Regulation of bi-directional communication between intracellular Ca2+ pools and surface Ca2+ channels remains incompletely characterized. We report Ca2+ release mediated by inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR) pathways is diminished under actin cytoskeleton disruption in NG115-401L (401L) neuronal cells, yet despite truncated Ca2+ release, Ca2+ influx was not significantly altered in these experiments. However, disruption of cortical actin networks completely abolished IP3R induced Ca2+ release, whereas RyR-mediated Ca2+ release was preserved, albeit attenuated. Moreover, cortical actin disruption completely abolished IP3R and RyR linked Ca2+ influx even though Ca2+ pool sensitivities were different. These findings suggest discrete Ca2+ store/Ca2+ channel coupling mechanisms in the IP3R and RyR pathways as revealed by the differential sensitivity to actin perturbation. 相似文献
9.
《Channels (Austin, Tex.)》2013,7(4):226-232
Inositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular Ca2+ channels. Their regulation by both IP3 and Ca2+ allows interactions between IP3Rs to generate a hierarchy of intracellular Ca2+ release events. These can progress from openings of single IP3R, through near-synchronous opening of a few IP3Rs within a cluster to much larger signals that give rise to regenerative Ca2+ waves that can invade the entire cell. We have used patch-clamp recording from excised nuclear membranes of DT40 cells expressing only IP3R3 and shown that low concentrations of IP3 rapidly and reversibly cause IP3Rs to assemble into small clusters. In addition to bringing IP3Rs close enough to allow Ca2+ released by one IP3R to regulate the activity of its neighbors, clustering also retunes the regulation of IP3Rs by IP3 and Ca2+. At resting cytosolic [Ca2+], lone IP3R are more sensitive to IP3 and the mean channel open time (~10ms) is twice as long as for clustered IP3R. When the cytosolic free [Ca2+] is increased to 1µM, to mimic the conditions that might prevail when an IP3R within a cluster opens, clustered IP3R are no longer inhibited and their gating becomes coupled. IP3, by dynamically regulating IP3R clustering, both positions IP3R for optimal interactions between them and it serves to exaggerate the effects of Ca2+ within a cluster. During the course of these studies, we have observed that nuclear IP3R stably express one of two single channel K + conductances (γK ~120 or 200pS). Here we demonstrate that for both states of the IP3R, the effects of IP3 on clustering are indistinguishable. These observations reinforce our conclusion that IP3 dynamically regulates assembly of IP3Rs into clusters that underlie the hierarchical recruitment of elementary Ca2+ release events. 相似文献
10.
Joon-Chul Kim Krishna P. Subedi Joung Real Ahn 《Progress in biophysics and molecular biology》2010,103(1):59-70
In atrial myocytes lacking t-tubules, action potential triggers junctional Ca2+ releases in the cell periphery, which propagates into the cell interior. The present article describes growing evidence on atrial local Ca2+ signaling and on the functions of inositol 1,4,5-trisphosphate receptors (IP3Rs) in atrial myocytes, and show our new findings on the role of IP3R subtype in the regulation of spontaneous focal Ca2+ releases in the compartmentalized areas of atrial myocytes. The Ca2+ sparks, representing focal Ca2+ releases from the sarcoplasmic reticulum (SR) through the ryanodine receptor (RyR) clusters, occur most frequently at the peripheral junctions in isolated resting atrial cells. The Ca2+ sparks that were darker and longer lasting than peripheral and non-junctional (central) sparks, were found at peri-nuclear sites in rat atrial myocytes. Peri-nuclear sparks occurred more frequently than central sparks. Atrial cells express larger amounts of IP3Rs compared with ventricular cells and possess significant levels of type 1 IP3R (IP3R1) and type 2 IP3R (IP3R2). Over the last decade the roles of atrial IP3R on the enhancement of Ca2+-induced Ca2+ release and arrhythmic Ca2+ releases under hormonal stimulations have been well documented. Using protein knock-down method and confocal Ca2+ imaging in conjunction with immunocytochemistry in the adult atrial cell line HL-1, we could demonstrate a role of IP3R1 in the maintenance of peri-nuclear and non-junctional Ca2+ sparks via stimulating a posttranslational organization of RyR clusters. 相似文献
11.
Inositol (1,4,5)-trisphosphate receptors (IP3Rs) release intracellular Ca2+ as localized Ca2+ signals (Ca2+ puffs) that represent the activity of small numbers of clustered IP3Rs spaced throughout the endoplasmic reticulum. Although much emphasis has been placed on estimating the number of active Ca2+ release channels supporting Ca2+ puffs, less attention has been placed on understanding the role of cluster microarchitecture. This is important as recent data underscores the dynamic nature of IP3R transitions between heterogeneous cellular architectures and the differential behavior of IP3Rs socialized into clusters. Here, we applied a high-resolution model incorporating stochastically gating IP3Rs within a three-dimensional cytoplasmic space to demonstrate: 1), Ca2+ puffs are supported by a broad range of clustered IP3R microarchitectures; 2), cluster ultrastructure shapes Ca2+ puff characteristics; and 3), loosely corralled IP3R clusters (>200 nm interchannel separation) fail to coordinate Ca2+ puffs, owing to inefficient triggering and impaired coupling due to reduced Ca2+-induced Ca2+ release microwave velocity (<10 nm/s) throughout the channel array. Dynamic microarchitectural considerations may therefore influence Ca2+ puff occurrence/properties in intact cells, contrasting with a more minimal role for channel number over the same simulated conditions in shaping local Ca2+ dynamics. 相似文献
12.
Joon-Chul Kim Min-Jeong Son Krishna P. Subedi Do Han Kim Sun-Hee Woo 《Molecules and cells》2010,29(4):387-395
HL-1 cells are the adult cardiac cell lines available that continuously divide while maintaining an atrial phenotype. Here
we examined the expression and localization of inositol 1,4,5-trisphosphate receptor (IP3R) subtypes, and investigated how pattern of IP3-induced subcellular local Ca2+ signaling is encoded by multiple IP3R subtypes in HL-1 cells. The type 1 IP3R (IP3R1) was expressed in the perinucleus with a diffuse pattern and the type 2 IP3R (IP3R2) was expressed in the cytosol with a punctate distribution. Extracellular ATP (1 mM) elicited transient intracellular Ca2+ releases accompanied by a Ca2+ oscillation, which was eliminated by the blocker of IP3Rs, 2-APB, and attenuated by ryanodine. Direct introduction of IP3 into the permeabilized cells induced Ca2+ transients with Ca2+ oscillations at ⩾ 20 μM of IP3, which was removed by the inhibition of IP3Rs using 2-APB and heparin. IP3-induced local Ca2+ transients contained two distinct time courses: a rapid oscillation and a monophasic Ca2+ transient. The magnitude of Ca2+ oscillation was significantly larger in the cytosol than in the nucleus, while the monophasic Ca2+ transient was more pronounced in the nucleus. These results provide evidence for the molecular and functional expression
of IP3R1 and IP3R2 in HL-1 cells, and suggest that such distinct local Ca2+ signaling may be correlated with the punctate distribution of IP3R2s in the cytosol and the diffuse localization of IP3R1 in the peri-nucleus. 相似文献
13.
Száva Bánsághi Tünde Golenár Muniswamy Madesh Gy?rgy Csordás Satish RamachandraRao Kumar Sharma David I. Yule Suresh K. Joseph Gy?rgy Hajnóczky 《The Journal of biological chemistry》2014,289(12):8170-8181
Reactive oxygen species (ROS) stimulate cytoplasmic [Ca2+] ([Ca2+]c) signaling, but the exact role of the IP3 receptors (IP3R) in this process remains unclear. IP3Rs serve as a potential target of ROS produced by both ER and mitochondrial enzymes, which might locally expose IP3Rs at the ER-mitochondrial associations. Also, IP3Rs contain multiple reactive thiols, common molecular targets of ROS. Therefore, we have examined the effect of superoxide anion (O2⨪) on IP3R-mediated Ca2+ signaling. In human HepG2, rat RBL-2H3, and chicken DT40 cells, we observed [Ca2+]c spikes and frequency-modulated oscillations evoked by a O2⨪ donor, xanthine (X) + xanthine oxidase (XO), dose-dependently. The [Ca2+]c signal was mediated by ER Ca2+ mobilization. X+XO added to permeabilized cells promoted the [Ca2+]c rise evoked by submaximal doses of IP3, indicating that O2⨪ directly sensitizes IP3R-mediated Ca2+ release. In response to X+XO, DT40 cells lacking two of three IP3R isoforms (DKO) expressing either type 1 (DKO1) or type 2 IP3Rs (DKO2) showed a [Ca2+]c signal, whereas DKO expressing type 3 IP3R (DKO3) did not. By contrast, IgM that stimulates IP3 formation, elicited a [Ca2+]c signal in every DKO. X+XO also facilitated the Ca2+ release evoked by submaximal IP3 in permeabilized DKO1 and DKO2 but was ineffective in DKO3 or in DT40 lacking every IP3R (TKO). However, X+XO could also facilitate the effect of suboptimal IP3 in TKO transfected with rat IP3R3. Although in silico studies failed to identify a thiol missing in the chicken IP3R3, an X+XO-induced redox change was documented only in the rat IP3R3. Thus, ROS seem to specifically sensitize IP3Rs through a thiol group(s) within the IP3R, which is probably inaccessible in the chicken IP3R3. 相似文献
14.
The inositol 1,4,5-trisphosphate (IP3)-mediated intracellular Ca2+ releases in secretory cells play vital roles in controlling not only the intracellular Ca2+ concentrations but also the Ca2+-dependent exocytotic processes. Of intracellular organelles that release Ca2+ in response to IP3, secretory granules stand out as the most prominent organelle and are responsible for the majority of IP3-dependent Ca2+ releases in the cytoplasm of chromaffin cells. Bovine chromaffin granules were the first granules that demonstrated the IP3-mediated Ca2+ release as well as the presence of the IP3 receptor (IP3R) in granule membranes. Secretory granules contain all three (type 1, 2, and 3) IP3R isoforms, and 58–69% of total cellular IP3R isoforms are expressed in bovine chromaffin granules. Moreover, secretory granules contain large amounts (2–4 mM) of chromogranins
and secretogranins; chromogranins A and B, and secretogranin II being the major species. Chromogranins A and B, and secretogranin
II are high-capacity, low-affinity Ca2+ binding proteins, binding 30–93 mol of Ca2+/mol of protein with dissociation constants of 1.5–4.0 mM. Due to this high Ca2+ storage properties of chromogranins secretory granules contain ~40 mM Ca2+. Furthermore, chromogranins A and B directly interact with the IP3Rs and modulate the IP3R/Ca2+ channels, i.e., increasing the open probability and the mean open time of the channels 8- to 16-fold and 9- to 42-fold, respectively.
Coupled chromogranins change the IP3R/Ca2+ channels to a more ordered, release-ready state, whereby making the IP3R/Ca2+ channels significantly more sensitive to IP3. 相似文献
15.
Veerle Vanderheyden Takuya Wakai Geert Bultynck Humbert De Smedt Jan B. Parys Rafael A. Fissore 《Cell calcium》2009,46(1):56-64
Egg activation and further embryo development require a sperm-induced intracellular Ca2+ signal at the time of fertilization. Prior to fertilization, the egg's Ca2+ machinery is therefore optimized. To this end, during oocyte maturation, the sensitivity, i.e. the Ca2+ releasing ability, of the inositol 1,4,5-trisphosphate receptor type 1 (IP3R1), which is responsible for most of this Ca2+ release, markedly increases. In this study, the recently discovered specific Polo-like kinase (Plk) inhibitor BI2536 was used to investigate the role of Plk1 in this process. BI2536 inactivates Plk1 in oocytes at the early stages of maturation and significantly decreases IP3R1 phosphorylation at an MPM-2 epitope at this stage. Moreover, this decrease in Plk1-dependent MPM-2 phosphorylation significantly lowers IP3R1 sensitivity. Finally, using in vitro phosphorylation techniques we identified T2656 as a major Plk1 site on IP3R1. We therefore propose that the initial increase in IP3R1 sensitivity during oocyte maturation is underpinned by IP3R1 phosphorylation at an MPM-2 epitope(s). 相似文献
16.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2019,1866(7):1092-1100
Inositol 1,4,5-trisphosphate receptors (IP3R) are the most widely expressed intracellular Ca2+ release channels. Their activation by IP3 and Ca2+ allows Ca2+ to pass rapidly from the ER lumen to the cytosol. The resulting increase in cytosolic [Ca2+] may directly regulate cytosolic effectors or fuel Ca2+ uptake by other organelles, while the decrease in ER luminal [Ca2+] stimulates store-operated Ca2+ entry (SOCE). We are close to understanding the structural basis of both IP3R activation, and the interactions between the ER Ca2+-sensor, STIM, and the plasma membrane Ca2+ channel, Orai, that lead to SOCE. IP3Rs are the usual means through which extracellular stimuli, through ER Ca2+ release, stimulate SOCE. Here, we review evidence that the IP3Rs most likely to respond to IP3 are optimally placed to allow regulation of SOCE. We also consider evidence that IP3Rs may regulate SOCE downstream of their ability to deplete ER Ca2+ stores. Finally, we review evidence that IP3Rs in the plasma membrane can also directly mediate Ca2+ entry in some cells. 相似文献
17.
Martijn Kerkhofs Bruno Seitaj Hristina Ivanova Giovanni Monaco Geert Bultynck Jan B. Parys 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2018,1865(11):1707-1717
Ca2+ signaling governs a diverse range of cellular processes and, as such, is subject to tight regulation. A main component of the complex intracellular Ca2+-signaling network is the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R), a tetrameric channel that mediates Ca2+ release from the endoplasmic reticulum (ER) in response to IP3. IP3R function is controlled by a myriad of factors, such as Ca2+, ATP, kinases and phosphatases and a plethora of accessory and regulatory proteins. Further complexity in IP3R-mediated Ca2+ signaling is the result of the existence of three main isoforms (IP3R1, IP3R2 and IP3R3) that display distinct functional characteristics and properties. Despite their abundant and overlapping expression profiles, IP3R1 is highly expressed in neurons, IP3R2 in cardiomyocytes and hepatocytes and IP3R3 in rapidly proliferating cells as e.g. epithelial cells. As a consequence, dysfunction and/or dysregulation of IP3R isoforms will have distinct pathophysiological outcomes, ranging from neurological disorders for IP3R1 to dysfunctional exocrine tissues and autoimmune diseases for IP3R2 and -3. Over the past years, several IP3R mutations have surfaced in the sequence analysis of patient-derived samples. Here, we aimed to provide an integrative overview of the clinically most relevant mutations for each IP3R isoform and the subsequent molecular mechanisms underlying the etiology of the disease. 相似文献
18.
Ito J Yoon SY Lee B Vanderheyden V Vermassen E Wojcikiewicz R Alfandari D De Smedt H Parys JB Fissore RA 《Developmental biology》2008,320(2):402-413
To initiate embryo development, the sperm induces in the egg release of intracellular calcium ([Ca2+]i). During oocyte maturation, the inositol 1,4,5-trisphosphate receptor (IP3R1), the channel implicated, undergoes modifications that enhance its function. We found that IP3R1 becomes phosphorylated during maturation at an MPM-2 epitope and that this persists until the fertilization-associated [Ca2+]i responses cease. We also reported that maturation without ERK activity diminishes IP3R1 MPM-2 reactivity and [Ca2+]i responses. Here, we show that IP3R1 is a novel target for Polo-like kinase1 (Plk1), a conserved M-phase kinase, which phosphorylates it at an MPM-2 epitope. Plk1 and IP3R1 interact in an M-phase preferential manner, and they exhibit close co-localization in the spindle/spindle poles area. This co-localization is reduced in the absence of ERK activity, as the ERK pathway regulates spindle organization and IP3R1 cortical re-distribution. We propose that IP3R1 phosphorylation by Plk1, and possibly by other M-phase kinases, underlies the delivery of spatially and temporally regulated [Ca2+]i signals during meiosis/mitosis and cytokinesis. 相似文献
19.
Nicolas Rosa Hristina Ivanova Larry E. Wagner nd Justin Kale Rita La Rovere Kirsten Welkenhuyzen Nikolaos Louros Spyridoula Karamanou Victoria Shabardina Irma Lemmens Elien Vandermarliere Kozo Hamada Hideaki Ando Frederic Rousseau Joost Schymkowitz Jan Tavernier Katsuhiko Mikoshiba Anastassios Economou David W. Andrews Jan B. Parys David I. Yule Geert Bultynck 《Cell death and differentiation》2022,29(4):788
Anti-apoptotic Bcl-2-family members not only act at mitochondria but also at the endoplasmic reticulum, where they impact Ca2+ dynamics by controlling IP3 receptor (IP3R) function. Current models propose distinct roles for Bcl-2 vs. Bcl-xL, with Bcl-2 inhibiting IP3Rs and preventing pro-apoptotic Ca2+ release and Bcl-xL sensitizing IP3Rs to low [IP3] and promoting pro-survival Ca2+ oscillations. We here demonstrate that Bcl-xL too inhibits IP3R-mediated Ca2+ release by interacting with the same IP3R regions as Bcl-2. Via in silico superposition, we previously found that the residue K87 of Bcl-xL spatially resembled K17 of Bcl-2, a residue critical for Bcl-2’s IP3R-inhibitory properties. Mutagenesis of K87 in Bcl-xL impaired its binding to IP3R and abrogated Bcl-xL’s inhibitory effect on IP3Rs. Single-channel recordings demonstrate that purified Bcl-xL, but not Bcl-xLK87D, suppressed IP3R single-channel openings stimulated by sub-maximal and threshold [IP3]. Moreover, we demonstrate that Bcl-xL-mediated inhibition of IP3Rs contributes to its anti-apoptotic properties against Ca2+-driven apoptosis. Staurosporine (STS) elicits long-lasting Ca2+ elevations in wild-type but not in IP3R-knockout HeLa cells, sensitizing the former to STS treatment. Overexpression of Bcl-xL in wild-type HeLa cells suppressed STS-induced Ca2+ signals and cell death, while Bcl-xLK87D was much less effective in doing so. In the absence of IP3Rs, Bcl-xL and Bcl-xLK87D were equally effective in suppressing STS-induced cell death. Finally, we demonstrate that endogenous Bcl-xL also suppress IP3R activity in MDA-MB-231 breast cancer cells, whereby Bcl-xL knockdown augmented IP3R-mediated Ca2+ release and increased the sensitivity towards STS, without altering the ER Ca2+ content. Hence, this study challenges the current paradigm of divergent functions for Bcl-2 and Bcl-xL in Ca2+-signaling modulation and reveals that, similarly to Bcl-2, Bcl-xL inhibits IP3R-mediated Ca2+ release and IP3R-driven cell death. Our work further underpins that IP3R inhibition is an integral part of Bcl-xL’s anti-apoptotic function.Subject terms: Cancer, Cell biology, Molecular biology 相似文献
20.
Yoo SH 《Cell calcium》2011,50(2):175-183
The majority of secretory cell calcium is stored in secretory granules that serve as the major IP3-dependent intracellular Ca2+ store. Even in unicellular phytoplankton secretory granules are responsible for the IP3-induced Ca2+ release that triggers exocytosis. The number of secretory granules in the cell is directly related not only to the magnitude of IP3-induced Ca2+ release, which accounts for the majority of the IP3-induced cytoplasmic Ca2+ release in neuroendocrine cells, but also to the IP3 sensitivity of the cytoplasmic IP3 receptor (IP3R)/Ca2+ channels. Moreover, secretory granules contain the highest IP3R concentrations and the largest amounts of IP3Rs in any subcellular organelles in neuroendocrine cells. Secretory granules from phytoplankton to mammals contain large amounts of polyanionic molecules, chromogranins being the major molecules in mammals, in addition to acidic intragranular pH and high Ca2+ concentrations. The polyanionic molecules undergo pH- and Ca2+-dependent conformational changes that serve as a molecular basis for condensation-decondensation phase transitions of the intragranular matrix. Likewise, chromogranins undergo pH- and Ca2+-dependent conformational changes with increased exposure of the structure and increased interactions with Ca2+ and other granule components at acidic pH. The unique physico-chemical properties of polyanionic molecules appear to be at the center of biogenesis, and physiological functions of secretory granules in living organisms from primitive to advanced species. 相似文献