Comparison of Ca2+ puffs evoked by extracellular agonists and photoreleased IP3

The inositol trisphosphate (IP₃) signaling pathway evokes local Ca²⁺ signals (Ca²⁺ puffs) that arise from the concerted openings of clustered IP₃ receptor/channels in the ER membrane. Physiological activation is triggered by binding of agonists to G-protein-coupled receptors (GPCRs) on the cell surface, leading to cleavage of phosphatidyl inositol bisphosphate and release of IP₃ into the cytosol. Photorelease of IP₃ from a caged precursor provides a convenient and widely employed means to study the final stage of IP₃-mediated Ca²⁺ liberation, bypassing upstream signaling events to enable more precise control of the timing and relative concentration of cytosolic IP₃. Here, we address whether Ca²⁺ puffs evoked by photoreleased IP₃ fully replicate those arising from physiological agonist stimulation. We imaged puffs in individual SH-SY5Y neuroblastoma cells that were sequentially stimulated by picospritzing extracellular agonist (carbachol, CCH or bradykinin, BK) followed by photorelease of a poorly-metabolized IP₃ analog, i-IP₃. The centroid localizations of fluorescence signals during puffs evoked in the same cells by agonists and photorelease substantially overlapped (within ∼1 μm), suggesting that IP₃ from both sources accesses the same, or closely co-localized clusters of IP₃Rs. Moreover, the time course and spatial spread of puffs evoked by agonists and photorelease matched closely. Because photolysis generates IP₃ uniformly throughout the cytoplasm, our results imply that IP₃ generated in SH-SY5Y cells by activation of receptors to CCH and BK also exerts broadly distributed actions, rather than specifically activating a subpopulation of IP₃Rs that are scaffolded in close proximity to cell surface receptors to form a signaling nanodomain.     

Enhanced ER Ca2+ store filling by overexpression of SERCA2b promotes IP3-evoked puffs

Liberation of Ca²⁺ from the endoplasmic reticulum (ER) through inositol trisphosphate receptors (IP₃R) is modulated by the ER Ca²⁺ content, and overexpression of SERCA2b to accelerate Ca²⁺ sequestration into the ER has been shown to potentiate the frequency and amplitude of IP₃-evoked Ca²⁺ waves in Xenopus oocytes. Here, we examined the effects of SERCA overexpression on the elementary IP₃-evoked puffs to elucidate whether ER [Ca²⁺] may modulate IP₃R function via luminal regulatory sites in addition to simply determining the size of the available store and electrochemical driving force for Ca²⁺ release. SERCA2b and Ca²⁺ permeable nicotinic plasmalemmal channels were expressed in oocytes, and hyperpolarizing pulses were delivered to induce Ca²⁺ influx and thereby load ER stores. Puffs evoked by photoreleased IP₃ were significantly potentiated in terms of numbers of responding sites, frequency and amplitude following transient Ca²⁺ influx in SERCA-overexpressing cells, whereas little change was evident with SERCA overexpression alone or following Ca²⁺ influx in control cells not overexpressing SERCA. Intriguingly, we observed the appearance of a new population of puffs that arose after long latencies and had prolonged durations supporting the notion of luminal regulation of IP₃R gating kinetics.     

The sarcoplasmic reticulum Ca2+ store arrangement in vascular smooth muscle

In vascular smooth muscle cells, Ca²⁺ release via IP₃ receptors (IP₃R) and ryanodine receptors (RyR) on the sarcoplasmic reticulum (SR) Ca²⁺ store contributes significantly to the regulation of cellular events such as gene regulation, growth and contraction. Ca²⁺ release from various regions of a structurally compartmentalized SR, it is proposed, may selectively activate different cellular functions. Multiple SR compartments with various receptor arrangements are proposed also to exist at different stages of smooth muscle development and in proliferative vascular diseases such as atherosclerosis. The conclusions on SR organization have been derived largely from the outcome of functional studies. This study addresses whether the SR Ca²⁺ store is a single continuous interconnected network or multiple separate Ca²⁺ pools in single vascular myocytes. To do this, the consequences of depletion of the SR in small restricted regions on the Ca²⁺ available throughout the store was examined using localized photolysis of caged-IP₃ and focal application of ryanodine in guinea-pig voltage-clamped single portal vein myocytes. From one small site on the cell, the entire SR could be depleted via either RyR or IP₃R. The entire SR could also be refilled from one small site on the cell. The results suggest a single luminally continuous SR exists. However, the opening of IP₃R and RyR was regulated by the Ca²⁺ concentration within the SR (luminal [Ca²⁺]). As the luminal [Ca²⁺] declines, the opening of the receptors decline and stop, and there may appear to be stores with either only RyR or only IP₃R. The SR Ca²⁺ store is a single luminally continuous entity which contains both IP₃R and RyR and within which Ca²⁺ is accessed freely by each receptor. While the SR is a single continuous entity, regulation of IP₃R and RyR by luminal [Ca²⁺] explains the appearance of multiple stores in some functional studies.     

Endoplasmic reticulum Ca release through ryanodine and IP3 receptors contributes to neuronal excitotoxicity

Overactivation of ionotropic glutamate receptors induces a Ca²⁺ overload into the cytoplasm that leads neurons to excitotoxic death, a process that has been linked to several neurodegenerative disorders. While the role of mitochondria and its involvement in excitotoxicity have been widely studied, the contribution of endoplasmic reticulum (ER), another crucial intracellular store in maintaining Ca²⁺ homeostasis, is not fully understood. In this study, we analyzed the contribution of ER-Ca²⁺ release through ryanodine (RyR) and IP₃ (IP₃R) receptors to a neuronal in vitro model of excitotoxicity. NMDA induced a dose-dependent neuronal death, which was significantly decreased by ER-Ca²⁺ release inhibitors in cortical neurons as well as in organotypic slices. Furthermore, ryanodine and 2APB, RyR and IP₃R inhibitors respectively, attenuated NMDA-triggered intracellular Ca²⁺ increase and oxidative stress, whereas 2APB reduced mitochondrial membrane depolarization and caspase-3 cleavage. Consistent with ER-Ca²⁺ homeostasis disruption, we observed that NMDA-induced ER stress, characterized here by eIF2α phosphorylation and over-expression of GRP chaperones which were regulated by ER-Ca²⁺ release inhibitors. These results demonstrate that Ca²⁺ release from ER contributes to neuronal death by both promoting mitochondrial dysfunction and inducing specific stress and apoptosis pathways during excitotoxicity.     

IP3 receptors and Ca2+ entry

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.     

Intracellular Ca2+ release through ryanodine receptors contributes to AMPA receptor-mediated mitochondrial dysfunction and ER stress in oligodendrocytes

Overactivation of ionotropic glutamate receptors in oligodendrocytes induces cytosolic Ca²⁺ overload and excitotoxic death, a process that contributes to demyelination and multiple sclerosis. Excitotoxic insults cause well-characterized mitochondrial alterations and endoplasmic reticulum (ER) dysfunction, which is not fully understood. In this study, we analyzed the contribution of ER-Ca²⁺ release through ryanodine receptors (RyRs) and inositol triphosphate receptors (IP₃Rs) to excitotoxicity in oligodendrocytes in vitro. First, we observed that oligodendrocytes express all previously characterized RyRs and IP₃Rs. Blockade of Ca²⁺-induced Ca²⁺ release by TMB-8 following α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor-mediated insults attenuated both oligodendrocyte death and cytosolic Ca²⁺ overload. In turn, RyR inhibition by ryanodine reduced as well the Ca²⁺ overload whereas IP₃R inhibition was ineffective. Furthermore, AMPA-triggered mitochondrial membrane depolarization, oxidative stress and activation of caspase-3, which in all instances was diminished by RyR inhibition. In addition, we observed that AMPA induced an ER stress response as revealed by α subunit of the eukaryotic initiation factor 2α phosphorylation, overexpression of GRP chaperones and RyR-dependent cleavage of caspase-12. Finally, attenuating ER stress with salubrinal protected oligodendrocytes from AMPA excitotoxicity. Together, these results show that Ca²⁺ release through RyRs contributes to cytosolic Ca²⁺ overload, mitochondrial dysfunction, ER stress and cell death following AMPA receptor-mediated excitotoxicity in oligodendrocytes.     

Termination of calcium puffs and coupled closings of inositol trisphosphate receptor channels

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.     

Rapid Recycling of Ca2+ between IP3-Sensitive Stores and Lysosomes

Inositol 1,4,5-trisphosphate (IP₃) evokes release of Ca²⁺ from the endoplasmic reticulum (ER), but the resulting Ca²⁺ signals are shaped by interactions with additional intracellular organelles. Bafilomycin A₁, which prevents lysosomal Ca²⁺ uptake by inhibiting H⁺ pumping into lysosomes, increased the amplitude of the initial Ca²⁺ signals evoked by carbachol in human embryonic kidney (HEK) cells. Carbachol alone and carbachol in combination with parathyroid hormone (PTH) evoke Ca²⁺ release from distinct IP₃-sensitive Ca²⁺ stores in HEK cells stably expressing human type 1 PTH receptors. Bafilomycin A₁ similarly exaggerated the Ca²⁺ signals evoked by carbachol or carbachol with PTH, indicating that Ca²⁺ released from distinct IP₃-sensitive Ca²⁺ stores is sequestered by lysosomes. The Ca²⁺ signals resulting from store-operated Ca²⁺ entry, whether evoked by thapsigargin or carbachol, were unaffected by bafilomycin A₁. Using Gd³⁺ (1 mM) to inhibit both Ca²⁺ entry and Ca²⁺ extrusion, HEK cells were repetitively stimulated with carbachol to assess the effectiveness of Ca²⁺ recycling to the ER after IP₃-evoked Ca²⁺ release. Blocking lysosomal Ca²⁺ uptake with bafilomycin A₁ increased the amplitude of each carbachol-evoked Ca²⁺ signal without affecting the rate of Ca²⁺ recycling to the ER. This suggests that Ca²⁺ accumulated by lysosomes is rapidly returned to the ER. We conclude that lysosomes rapidly, reversibly and selectively accumulate the Ca²⁺ released by IP₃ receptors residing within distinct Ca²⁺ stores, but not the Ca²⁺ entering cells via receptor-regulated, store-operated Ca²⁺ entry pathways.     

cADPR stimulates SERCA activity in Xenopus oocytes

The intracellular second messenger cyclic ADP-ribose (cADPR) induces Ca²⁺ release through the activation of ryanodine receptors (RyRs). Moreover, it has been suggested that cADPR may serve an additional role to modulate sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) pump activity, but studies have been complicated by concurrent actions on RyR. Here, we explore the actions of cADPR in Xenopus oocytes, which lack RyRs. We examined the effects of cADPR on the sequestration of cytosolic Ca²⁺ following Ca²⁺ transients evoked by photoreleased inositol 1,4,5-trisphosphate (InsP₃), and by Ca²⁺ influx through expressed nicotinic acetylcholine receptors (nAChR) in the oocytes membrane. In both cases the decay of the Ca²⁺ transients was accelerated by intracellular injection of a non-metabolizable analogue of cADPR, 3-Deaza-cADPR, and photorelease of cADPR from a caged precursor demonstrated that this action is rapid (a few s). The acceleration was abolished by pre-treatment with thapsigargin to block SERCA activity, and was inhibited by two specific antagonists of cADPR, 8-NH₂-cADPR and 8-br-cADPR. We conclude that cADPR serves to modulate Ca²⁺ sequestration by enhancing SERCA pump activity, in addition to its well-established action on RyRs to liberate Ca²⁺.     

Dynamic Ca2+ imaging with a simplified lattice light-sheet microscope: A sideways view of subcellular Ca2+ puffs

We describe the construction of a simplified, inexpensive lattice light-sheet microscope, and illustrate its use for imaging subcellular Ca²⁺ puffs evoked by photoreleased i-IP₃ in cultured SH-SY5Y neuroblastoma cells loaded with the Ca²⁺ probe Cal520. The microscope provides sub-micron spatial resolution and enables recording of local Ca²⁺ transients in single-slice mode with a signal-to-noise ratio and temporal resolution (2 ms) at least as good as confocal or total internal reflection microscopy. Signals arising from openings of individual IP₃R channels are clearly resolved, as are stepwise changes in fluorescence reflecting openings and closings of individual channels during puffs. Moreover, by stepping the specimen through the light-sheet, the entire volume of a cell can be scanned within a few hundred ms. The ability to directly visualize a sideways (axial) section through cells directly reveals that IP₃-evoked Ca²⁺ puffs originate at sites in very close (≤a few hundred nm) to the plasma membrane, suggesting they play a specific role in signaling to the membrane.     

Interleukin-13 enhanced Ca2+ oscillations in airway smooth muscle cells

Physiological mechanisms associated with interleukin-13 (IL-13), a key cytokine in asthma, in intracellular Ca²⁺ signaling in airway smooth muscle cells (ASMCs) remain unclear. The aim of this study was to assess effects of IL-13 on Ca²⁺ oscillations in response to leukotriene D4 (LTD4) in human cultured ASMCs.LTD4-induced Ca²⁺ oscillations in ASMCs pretreated with IL-13 were imaged by confocal microscopy. mRNA expressions of cysteinyl leukotriene 1 receptors (CysLT1R), CD38, involved with the ryanodine receptors (RyR) system, and transient receptor potential canonical (TRPC), involved with store-operated Ca²⁺ entry (SOCE), were determined by real-time PCR. In IL-13-pretreated ASMCs, frequency of LTD4-induced Ca²⁺ oscillations and number of oscillating cells were significantly increased compared with untreated ASMCs. Both xestospongin C, a specific inhibitor of inositol 1,4,5-triphosphate receptors (IP₃R), and ryanodine or ruthenium red, inhibitors of RyR, partially blocked LTD4-induced Ca²⁺ oscillations. Ca²⁺ oscillations were almost completely inhibited by 50 μM of 2-aminoethoxydiphenyl borate (2-APB), which dominantly blocks SOCE but not IP₃R at this concentration. Pretreatment with IL-13 increased the mRNA expressions of CysLT1R and CD38, but not of TRPC1 and TRPC3.We conclude that IL-13 enhances frequency of LTD4-induced Ca²⁺ oscillations in human ASMCs, which may be cooperatively modulated by IP₃R, RyR systems and possibly by SOCE.     

A comparison of fluorescent Ca2+ indicators for imaging local Ca2+ signals in cultured cells

Localized subcellular changes in Ca²⁺ serve as important cellular signaling elements, regulating processes as diverse as neuronal excitability and gene expression. Studies of cellular Ca²⁺ signaling have been greatly facilitated by the availability of fluorescent Ca²⁺ indicators. The respective merits of different indicators to monitor bulk changes in cellular Ca²⁺ levels have been widely evaluated, but a comprehensive comparison for their use in detecting and analyzing local, subcellular Ca²⁺ signals is lacking. Here, we evaluated several fluorescent Ca²⁺ indicators in the context of local Ca²⁺ signals (puffs) evoked by inositol 1,4,5-trisphosphate (IP₃) in cultured human neuroblastoma SH-SY5Y cells, using high-speed video-microscopy. Altogether, nine synthetic Ca²⁺ dyes (Fluo-4, Fluo-8, Fluo-8 high affinity, Fluo-8 low affinity, Oregon Green BAPTA-1, Cal-520, Rhod-4, Asante Calcium Red, and X-Rhod-1) and three genetically-encoded Ca²⁺-indicators (GCaMP6-slow, -medium and -fast variants) were tested; criteria include the magnitude, kinetics, signal-to-noise ratio and detection efficiency of local Ca²⁺ puffs. Among these, we conclude that Cal-520 is the optimal indicator for detecting and faithfully tracking local events; that Rhod-4 is the red-emitting indicator of choice; and that none of the GCaMP6 variants are well suited for imaging subcellular Ca²⁺ signals.     

Modeling Ca2+ feedback on a single inositol 1,4,5-trisphosphate receptor and its modulation by Ca2+ buffers

The inositol 1,4,5-trisphosphate receptor/channel (IP₃R) is a major regulator of intracellular Ca²⁺ signaling, and liberates Ca²⁺ ions from the endoplasmic reticulum in response to binding at cytosolic sites for both IP₃ and Ca²⁺. Although the steady-state gating properties of the IP₃R have been extensively studied and modeled under conditions of fixed [IP₃] and [Ca²⁺], little is known about how Ca²⁺ flux through a channel may modulate the gating of that same channel by feedback onto activating and inhibitory Ca²⁺ binding sites. We thus simulated the dynamics of Ca²⁺ self-feedback on monomeric and tetrameric IP₃R models. A major conclusion is that self-activation depends crucially on stationary cytosolic Ca²⁺ buffers that slow the collapse of the local [Ca²⁺] microdomain after closure. This promotes burst-like reopenings by the rebinding of Ca²⁺ to the activating site; whereas inhibitory actions are substantially independent of stationary buffers but are strongly dependent on the location of the inhibitory Ca²⁺ binding site on the IP₃R in relation to the channel pore.     

Inositol (1,4,5)-Trisphosphate Receptor Microarchitecture Shapes Ca Puff Kinetics

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.     

The EF-hand Ca2+ Binding Domain Is Not Required for Cytosolic Ca2+ Activation of the Cardiac Ryanodine Receptor

Activation of the cardiac ryanodine receptor (RyR2) by elevating cytosolic Ca²⁺ is a central step in the process of Ca²⁺-induced Ca²⁺ release, but the molecular basis of RyR2 activation by cytosolic Ca²⁺ is poorly defined. It has been proposed recently that the putative Ca²⁺ binding domain encompassing a pair of EF-hand motifs (EF1 and EF2) in the skeletal muscle ryanodine receptor (RyR1) functions as a Ca²⁺ sensor that regulates the gating of RyR1. Although the role of the EF-hand domain in RyR1 function has been studied extensively, little is known about the functional significance of the corresponding EF-hand domain in RyR2. Here we investigate the effect of mutations in the EF-hand motifs on the Ca²⁺ activation of RyR2. We found that mutations in the EF-hand motifs or deletion of the entire EF-hand domain did not affect the Ca²⁺-dependent activation of [³H]ryanodine binding or the cytosolic Ca²⁺ activation of RyR2. On the other hand, deletion of the EF-hand domain markedly suppressed the luminal Ca²⁺ activation of RyR2 and spontaneous Ca²⁺ release in HEK293 cells during store Ca²⁺ overload or store overload-induced Ca²⁺ release (SOICR). Furthermore, mutations in the EF2 motif, but not EF1 motif, of RyR2 raised the threshold for SOICR termination, whereas deletion of the EF-hand domain of RyR2 increased both the activation and termination thresholds for SOICR. These results indicate that, although the EF-hand domain is not required for RyR2 activation by cytosolic Ca²⁺, it plays an important role in luminal Ca²⁺ activation and SOICR.     

STIM1 Positively Regulates the Ca2+ Release Activity of the Inositol 1,4,5-Trisphosphate Receptor in Bovine Aortic Endothelial Cells

The endothelium is actively involved in many functions of the cardiovascular system, such as the modulation of arterial pressure and the maintenance of blood flow. These functions require a great versatility of the intracellular Ca²⁺ signaling that resides in the fact that different signals can be encoded by varying the frequency and the amplitude of the Ca²⁺ response. Cells use both extracellular and intracellular Ca²⁺ pools to modulate the intracellular Ca²⁺ concentration. In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP₃R), located on the endoplasmic reticulum (ER), is responsible for the release of Ca²⁺ from the intracellular store. The proteins STIM1 and STIM2 are also located on the ER and they are involved in the activation of a store-operated Ca²⁺ entry (SOCE). Due to their Ca²⁺ sensor property and their close proximity with IP₃Rs on the ER, STIMs could modulate the activity of IP₃R. In this study, we showed that STIM1 and STIM2 are expressed in bovine aortic endothelial cells and they both interact with IP₃R. While STIM2 appears to play a minor role, STIM1 plays an important role in the regulation of agonist-induced Ca²⁺ mobilization in BAECs by a positive effect on both the SOCE and the IP₃R-dependent Ca²⁺ release.     

Bidirectional Ca2+ signaling occurs between the endoplasmic reticulum and acidic organelles

The endoplasmic reticulum (ER) and acidic organelles (endo-lysosomes) act as separate Ca²⁺ stores that release Ca²⁺ in response to the second messengers IP₃ and cADPR (ER) or NAADP (acidic organelles). Typically, trigger Ca²⁺ released from acidic organelles by NAADP subsequently recruits IP₃ or ryanodine receptors on the ER, an anterograde signal important for amplification and Ca²⁺ oscillations/waves. We therefore investigated whether the ER can signal back to acidic organelles, using organelle pH as a reporter of NAADP action. We show that Ca²⁺ released from the ER can activate the NAADP pathway in two ways: first, by stimulating Ca²⁺-dependent NAADP synthesis; second, by activating NAADP-regulated channels. Moreover, the differential effects of EGTA and BAPTA (slow and fast Ca²⁺ chelators, respectively) suggest that the acidic organelles are preferentially activated by local microdomains of high Ca²⁺ at junctions between the ER and acidic organelles. Bidirectional organelle communication may have wider implications for endo-lysosomal function as well as the generation of Ca²⁺ oscillations and waves.     

IP3 receptors in cell survival and apoptosis: Ca2+ release and beyond

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.     

TRPC3 Regulates Agonist-stimulated Ca2+ Mobilization by Mediating the Interaction between Type I Inositol 1,4,5-Trisphosphate Receptor, RACK1, and Orai1

There is a body of evidence suggesting that Ca²⁺ handling proteins assemble into signaling complexes required for a fine regulation of Ca²⁺ signals, events that regulate a variety of critical cellular processes. Canonical transient receptor potential (TRPC) and Orai proteins have both been proposed to form Ca²⁺-permeable channels mediating Ca²⁺ entry upon agonist stimulation. A number of studies have demonstrated that inositol 1,4,5-trisphosphate receptors (IP₃Rs) interact with plasma membrane TRPC channels; however, at present there is no evidence supporting the interaction between Orai proteins and IP₃Rs. Here we report that treatment with thapsigargin or cellular agonists results in association of Orai1 with types I and II IP₃Rs. In addition, we have found that TRPC3, RACK1 (receptor for activated protein kinase C-1), and STIM1 (stromal interaction molecule 1) interact with Orai1 upon stimulation with agonists. TRPC3 expression silencing prevented both the interaction of Orai1 with TRPC3 and, more interestingly, the association of Orai1 with the type I IP₃R, but not with the type II IP₃R, thus suggesting that TRPC3 selectively mediates interaction between Orai1 and type I IP₃R. In addition, TRPC3 expression silencing attenuated ATP- and CCh-stimulated interaction between RACK1 and the type I IP₃R, as well as Ca²⁺ release and entry. In conclusion, our results indicate that agonist stimulation results in the formation of an Orai1-STIM1-TRPC3-RACK1-type I IP₃R complex, where TRPC3 plays a central role. This Ca²⁺ signaling complex might be important for both agonist-induced Ca²⁺ release and entry.