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1.
The subcellular localization of membrane Ca2+ channels is crucial for their functioning, but is difficult to study because channels may be distributed more closely than the resolution of conventional microscopy is able to detect. We describe a technique, stochastic channel Ca2+ nanoscale resolution (SCCaNR), employing Ca2+-sensitive fluorescent dyes to localize stochastic openings and closings of single Ca2+-permeable channels within <50 nm, and apply it to examine the clustered arrangement of inositol trisphosphate receptor (IP3R) channels underlying local Ca2+ puffs. Fluorescence signals (blips) arising from single functional IP3Rs are almost immotile (diffusion coefficient <0.003 μm2 s−1), as are puff sites over prolonged periods, suggesting that the architecture of this signaling system is stable and not subject to rapid, dynamic rearrangement. However, rapid stepwise changes in centroid position of fluorescence are evident within the durations of individual puffs. These apparent movements likely result from asynchronous gating of IP3Rs distributed within clusters that have an overall diameter of ∼400 nm, indicating that the nanoscale architecture of IP3R clusters is important in shaping local Ca2+ signals. We anticipate that SCCaNR will complement superresolution techniques such as PALM and STORM for studies of Ca2+ channels as it obviates the need for photoswitchable labels and provides functional as well as spatial information. 相似文献
2.
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
3.
Factors Determining the Recruitment of Inositol Trisphosphate Receptor Channels During Calcium Puffs
Puffs are localized, transient elevations in cytosolic Ca2+ that serve both as the building blocks of global cellular Ca2+ signals and as local signals in their own right. They arise from clustered inositol 1,4,5-trisphosphate receptor/channels (IP3Rs), whose openings are coordinated by Ca2+-induced Ca2+ release (CICR). We utilized total internal reflection fluorescence imaging of Ca2+ signals in neuroblastoma cells with single-channel resolution to elucidate the mechanisms determining the triggering, amplitudes, kinetics, and spatial spread of puffs. We find that any given channel in a cluster has a mean probability of ∼66% of opening following opening of an initial “trigger” channel, and the probability of puff triggering thus increases steeply with increasing number of channels in a cluster (cluster size). Mean puff amplitudes scale with cluster size, but individual amplitudes vary widely, even at sites of similar cluster size, displaying similar proportions of events involving any given number of the channels in the cluster. Stochastic variation in numbers of Ca2+-inhibited IP3Rs likely contributes to the variability of amplitudes of repeated puffs at a site but the amplitudes of successive puffs were uncorrelated, even though we observed statistical correlations between interpuff intervals and puff amplitudes. Initial puffs evoked following photorelease of IP3—which would not be subject to earlier Ca2+-inhibition—also showed wide variability, indicating that mechanisms such as stochastic variation in IP3 binding and channel recruitment by CICR further determine puff amplitudes. The mean termination time of puffs lengthened with increasing puff amplitude size, consistent with independent closings of channels after a given mean open time, but we found no correlation of termination time with cluster size independent of puff amplitude. The spatial extent of puffs increased with their amplitude, and puffs of similar size were of similar width, independent of cluster size. 相似文献
4.
Calcium puffs are localized Ca2+ signals mediated by Ca2+ release from the endoplasmic reticulum (ER) through clusters of inositol trisphosphate receptor (IP3R) channels. The recruitment of IP3R channels during puffs depends on Ca2+-induced Ca2+ release, a regenerative process that must be terminated to maintain control of cell signaling and prevent Ca2+ cytotoxicity. Here, we studied puff termination using total internal reflection microscopy to resolve the gating of individual IP3R channels during puffs in intact SH-SY5Y neuroblastoma cells. We find that the kinetics of IP3R channel closing differ from that expected for independent, stochastic gating, in that multiple channels tend to remain open together longer than predicted from their individual open lifetimes and then close in near-synchrony. This behavior cannot readily be explained by previously proposed termination mechanisms, including Ca2+-inhibition of IP3Rs and local depletion of Ca2+ in the ER lumen. Instead, we postulate that the gating of closely adjacent IP3Rs is coupled, possibly via allosteric interactions, suggesting an important mechanism to ensure robust puff termination in addition to Ca2+-inactivation. 相似文献
5.
Factors Determining the Recruitment of Inositol Trisphosphate Receptor Channels During Calcium Puffs
Puffs are localized, transient elevations in cytosolic Ca2+ that serve both as the building blocks of global cellular Ca2+ signals and as local signals in their own right. They arise from clustered inositol 1,4,5-trisphosphate receptor/channels (IP3Rs), whose openings are coordinated by Ca2+-induced Ca2+ release (CICR). We utilized total internal reflection fluorescence imaging of Ca2+ signals in neuroblastoma cells with single-channel resolution to elucidate the mechanisms determining the triggering, amplitudes, kinetics, and spatial spread of puffs. We find that any given channel in a cluster has a mean probability of ∼66% of opening following opening of an initial “trigger” channel, and the probability of puff triggering thus increases steeply with increasing number of channels in a cluster (cluster size). Mean puff amplitudes scale with cluster size, but individual amplitudes vary widely, even at sites of similar cluster size, displaying similar proportions of events involving any given number of the channels in the cluster. Stochastic variation in numbers of Ca2+-inhibited IP3Rs likely contributes to the variability of amplitudes of repeated puffs at a site but the amplitudes of successive puffs were uncorrelated, even though we observed statistical correlations between interpuff intervals and puff amplitudes. Initial puffs evoked following photorelease of IP3—which would not be subject to earlier Ca2+-inhibition—also showed wide variability, indicating that mechanisms such as stochastic variation in IP3 binding and channel recruitment by CICR further determine puff amplitudes. The mean termination time of puffs lengthened with increasing puff amplitude size, consistent with independent closings of channels after a given mean open time, but we found no correlation of termination time with cluster size independent of puff amplitude. The spatial extent of puffs increased with their amplitude, and puffs of similar size were of similar width, independent of cluster size. 相似文献
6.
《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
7.
Calcium puffs are local transient Ca2+ releases from internal Ca2+ stores such as the endoplasmic reticulum or the sarcoplasmic reticulum. Such release occurs through a cluster of inositol 1,4,5-trisphosphate receptors (IP3Rs). Based on the IP3R model (which is determined by fitting to stationary single-channel data) and nonstationary single-channel data, we construct a new IP3R model that includes time-dependent rates of mode switches. A point-source model of Ca2+ puffs is then constructed based on the new IP3R model and is solved by a hybrid Gillespie method with adaptive timing. Model results show that a relatively slow recovery of an IP3R from Ca2+ inhibition is necessary to reproduce most of the experimental outcomes, especially the nonexponential interpuff interval distributions. The number of receptors in a cluster could be severely underestimated when the recovery is sufficiently slow. Furthermore, we find that, as the number of IP3Rs increases, the average duration of puffs initially increases but then becomes saturated, whereas the average decay time keeps increasing linearly. This gives rise to the observed asymmetric puff shape. 相似文献
8.
The liberation of calcium ions sequestered in the endoplasmic reticulum through inositol 1,4,5-trisphosphate receptors/channels (IP3Rs) results in a spatiotemporal hierarchy of calcium signaling events that range from single-channel openings to local Ca2+ puffs believed to arise from several to tens of clustered IP3Rs to global calcium waves. Using high-resolution confocal linescan imaging and a sensitive Ca2+ indicator dye (fluo-4-dextran), we show that puffs are often preceded by small, transient Ca2+ elevations that we christen “trigger events”. The magnitude of triggers is consistent with their arising from the opening of a single IP3 receptor/channel, and we propose that they initiate puffs by recruiting neighboring IP3Rs within the cluster by a regenerative process of Ca2+-induced Ca2+ release. Puff amplitudes (fluorescence ratio change) are on average ~6 times greater than that of the triggers, suggesting that at least six IP3Rs may simultaneously be open during a puff. Trigger events have average durations of ~12 ms, as compared to 19 ms for the mean rise time of puffs, and their spatial extent is ~3 times smaller than puffs (respective widths at half peak amplitude 0.6 and 1.6 μm). All these parameters were relatively independent of IP3 concentration, although the proportion of puffs showing resolved triggers was greatest (~80%) at low [IP3]. Because Ca2+ puffs constitute the building blocks from which cellular IP3-mediated Ca2+ signals are constructed, the events that initiate them are likely to be of fundamental importance for cell signaling. Moreover, the trigger events provide a useful yardstick by which to derive information regarding the number and spatial arrangement of IP3Rs within clusters. 相似文献
9.
Intracellular Ca2+ release is a versatile second messenger system. It is modeled here by reaction-diffusion equations for the free Ca2+ and Ca2+ buffers, with spatially discrete clusters of stochastic IP3 receptor channels (IP3Rs) controlling the release of Ca2+ from the endoplasmic reticulum. IP3Rs are activated by a small rise of the cytosolic Ca2+ concentration and inhibited by large concentrations. Buffering of cytosolic Ca2+ shapes global Ca2+ transients. Here we use a model to investigate the effect of buffers with slow and fast reaction rates on single release spikes. We find that, depending on their diffusion coefficient, fast buffers can either decouple clusters or delay inhibition. Slow buffers have little effect on Ca2+ release, but affect the time course of the signals from the fluorescent Ca2+ indicator mainly by competing for Ca2+. At low [IP3], fast buffers suppress fluorescence signals, slow buffers increase the contrast between bulk signals and signals at open clusters, and large concentrations of buffers, either fast or slow, decouple clusters. 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.
Kevin Thurley Ian F. Smith Stephen C. Tovey Colin W. Taylor Ian Parker Martin Falcke 《Biophysical journal》2011,101(11):2638
The behavior of biological systems is determined by the properties of their component molecules, but the interactions are usually too complex to understand fully how molecular behavior generates cellular behavior. Ca2+ signaling by inositol trisphosphate receptors (IP3R) offers an opportunity to understand this relationship because the cellular behavior is defined largely by Ca2+-mediated interactions between IP3R. Ca2+ released by a cluster of IP3R (giving a local Ca2+ puff) diffuses and ignites the behavior of neighboring clusters (to give repetitive global Ca2+ spikes). We use total internal reflection fluorescence microscopy of two mammalian cell lines to define the temporal relationships between Ca2+ puffs (interpuff intervals, IPI) and Ca2+ spikes (interspike intervals) evoked by flash photolysis of caged IP3. We find that IPI are much shorter than interspike intervals, that puff activity is stochastic with a recovery time that is much shorter than the refractory period of the cell, and that IPI are not periodic. We conclude that Ca2+ spikes do not arise from oscillatory dynamics of IP3R clusters, but that repetitive Ca2+ spiking with its longer timescales is an emergent property of the dynamics of the whole cluster array. 相似文献
12.
Guiling Zhao Zachary P. Neeb M. Dennis Leo Judith Pachuau Adebowale Adebiyi Kunfu Ouyang Ju Chen Jonathan H. Jaggar 《The Journal of general physiology》2010,136(3):283-291
Plasma membrane large-conductance Ca2+-activated K+ (BKCa) channels and sarcoplasmic reticulum inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are expressed in a wide variety of cell types, including arterial smooth muscle cells. Here, we studied BKCa channel regulation by IP3 and IP3Rs in rat and mouse cerebral artery smooth muscle cells. IP3 activated BKCa channels both in intact cells and in excised inside-out membrane patches. IP3 caused concentration-dependent BKCa channel activation with an apparent dissociation constant (Kd) of ∼4 µM at physiological voltage (−40 mV) and intracellular Ca2+ concentration ([Ca2+]i; 10 µM). IP3 also caused a leftward-shift in BKCa channel apparent Ca2+ sensitivity and reduced the Kd for free [Ca2+]i from ∼20 to 12 µM, but did not alter the slope or maximal Po. BAPTA, a fast Ca2+ buffer, or an elevation in extracellular Ca2+ concentration did not alter IP3-induced BKCa channel activation. Heparin, an IP3R inhibitor, and a monoclonal type 1 IP3R (IP3R1) antibody blocked IP3-induced BKCa channel activation. Adenophostin A, an IP3R agonist, also activated BKCa channels. IP3 activated BKCa channels in inside-out patches from wild-type (IP3R1+/+) mouse arterial smooth muscle cells, but had no effect on BKCa channels of IP3R1-deficient (IP3R1−/−) mice. Immunofluorescence resonance energy transfer microscopy indicated that IP3R1 is located in close spatial proximity to BKCa α subunits. The IP3R1 monoclonal antibody coimmunoprecipitated IP3R1 and BKCa channel α and β1 subunits from cerebral arteries. In summary, data indicate that IP3R1 activation elevates BKCa channel apparent Ca2+ sensitivity through local molecular coupling in arterial smooth muscle cells. 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.
《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
15.
《生物化学与生物物理学报:生物膜》2023,1865(7):184195
Numerous cellular processes are regulated by Ca2+ signals, and the endoplasmic reticulum (ER) membrane's inositol triphosphate receptor (IP3R) is critical for modulating intracellular Ca2+ dynamics. The IP3Rs are seen to be clustered in a variety of cell types. The combination of IP3Rs clustering and IP3Rs-mediated Ca2+-induced Ca2+ release results in the hierarchical organization of the Ca2+ signals, which challenges the numerical simulation given the multiple spatial and temporal scales that must be covered. The previous methods rather ignore the spatial feature of IP3Rs or fail to coordinate the conflicts between the real biological relevance and the computational cost. In this work, a general and efficient reduced-lattice model is presented for the simulation of IP3Rs-mediated multiscale Ca2+ dynamics. The model highlights biological details that make up the majority of the calcium events, including IP3Rs clustering and calcium domains, and it reduces the complexity by approximating the minor details. The model's extensibility provides fresh insights into the function of IP3Rs in producing global Ca2+ events and supports the research under more physiological circumstances. Our work contributes to a novel toolkit for modeling multiscale Ca2+ dynamics and advances knowledge of Ca2+ signals. 相似文献
16.
We recently reported key physiologic roles for Ca2+-activated transient receptor potential melastatin 4 (TRPM4) channels in detrusor smooth muscle (DSM). However, the Ca2+-signaling mechanisms governing TRPM4 channel activity in human DSM cells are unexplored. As the TRPM4 channels are activated by Ca2+, inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release from the sarcoplasmic reticulum represents a potential Ca2+ source for TRPM4 channel activation. We used clinically-characterized human DSM tissues to investigate the molecular and functional interactions of the IP3Rs and TRPM4 channels. With in situ proximity ligation assay (PLA) and perforated patch-clamp electrophysiology, we tested the hypothesis that TRPM4 channels are tightly associated with the IP3Rs and are activated by IP3R-mediated Ca2+ release in human DSM. With in situ PLA, we demonstrated co-localization of the TRPM4 channels and IP3Rs in human DSM cells. As the TRPM4 channels and IP3Rs must be located within close apposition to functionally interact, these findings support the concept of a potential Ca2+-mediated TRPM4-IP3R regulatory mechanism. To investigate IP3R regulation of TRPM4 channel activity, we sought to determine the consequences of IP3R pharmacological inhibition on TRPM4 channel-mediated transient inward cation currents (TICCs). In freshly-isolated human DSM cells, blocking the IP3Rs with the selective IP3R inhibitor xestospongin-C significantly decreased TICCs. The data suggest that IP3Rs have a key role in mediating the Ca2+-dependent activation of TRPM4 channels in human DSM. The study provides novel insight into the molecular and cellular mechanisms regulating TRPM4 channels by revealing that TRPM4 channels and IP3Rs are spatially and functionally coupled in human DSM. 相似文献
17.
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
18.
Apical Vesicles Bearing Inositol 1,4,5-trisphosphate Receptors in the Ca2+Initiation Site of Ductal Epithelium of Submandibular Gland
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Miki Yamamoto-Hino Atsushi Miyawaki Akihisa Segawa Eijiro Adachi Shohei Yamashina Toyoshi Fujimoto Tomoyasu Sugiyama Teiichi Furuichi Mamoru Hasegawa Katsuhiko Mikoshiba 《The Journal of cell biology》1998,141(1):135-142
In polarized epithelial cells, agonists trigger Ca2+ waves and oscillations. These patterns may be caused by the compartmentalization of inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pools into specific regions. We have investigated the relationship between the distribution of IP3 receptors (IP3Rs) and the spatiotemporal pattern of Ca2+ signaling in the duct cells of the rat submandibular gland (SMG). Using immunofluorescence, although labeling was somewhat heterogeneous, the IP3Rs were colocalized to the apical pole of the duct cells. Immunoelectron microscopy identified small apical vesicles bearing IP3R2 in some types of duct cells. Real-time confocal imaging of intact ducts demonstrated that, after carbachol stimulation, an initial Ca2+ spike occurred in the apical region. Subsequently, repetitive Ca2+ spikes spread from the apical to the middle cytoplasm. These apical Ca2+ initiation sites were found only in some “pioneer cells,” rather than in all duct cells. We performed both Ca2+ imaging and immunofluorescence on the same ducts and detected the strongest immunosignals of IP3R2 in the Ca2+ initiation sites of the pioneer cells. The subcellular localization and expression level of IP3Rs correlated strongly with the spatiotemporal nature of the intracellular Ca2+ signal and distinct Ca2+ responses among the rat SMG duct cells. 相似文献
19.
Joseph SK Hajnóczky G 《Apoptosis : an international journal on programmed cell death》2007,12(5):951-968
Inositol 1,4,5-trisphosphate receptors (IP3Rs) serve to discharge Ca2+ from ER stores in response to agonist stimulation. The present review summarizes the role of these receptors in models of
Ca2+-dependent apoptosis. In particular we focus on the regulation of IP3Rs by caspase-3 cleavage, cytochrome c, anti-apoptotic proteins and Akt kinase. We also address the evidence that some of the effects of IP3Rs in apoptosis may be independent of their ion-channel function. The role of IP3Rs in delivering Ca2+ to the mitochondria is discussed from the perspective of the factors determining inter-organellar dynamics and the spatial
proximity of mitochondria and ER membranes. 相似文献
20.
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