Dynamic regulation of IP3 receptor clustering and activity by IP3

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 (γ_K ~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.     

Development of a functional assay for Ca2+ release activity of IP3R and expression of an IP3R gene fragment in the baculovirus-insect cell system

Receptor-stimulated phosphoinositide (PI) hydrolysis is an important and ubiquitous mechanism of intracellular signaling. Inositol 1,4,5-trisphosphate (IP₃), generated by phosphoinositide (PI) hydrolysis, binds to and gates an intracellular Ca²⁺ channel, the IP₃ receptor (IP₃R), which is therefore a central component of this signaling cascade. Here we describe the development of a baculovirus (BV)/Sf(S. frugiperda) cell system that can be used to look at IP₃R function. Agonist-evoked changes in intracellular Ca²⁺ levels [Ca²⁺]_i were measured (using Fura2) in Sf cells expressing the gene encoding the muscarinic acetylcholine receptor (vmlAchR). Furthermore, we have constructed a recombinant BV (vlP₃R), with the core of the IP₃R ligand-binding domain from the Drosophila IP₃R, under the polyhedrin promoter. The recombinant protein from such a virus was expected to act as a large ligand sink for IP3, generated by stimulation of vmlAchR. Cells coinfected with recombinant BV carrying the potential dominant-negative vIP₃R construct and vmlAchR have been used to assay the modulation of IP₃R-mediated Ca²⁺ release, by the ligand sink.     

Phospholipase C-η1 is activated by intracellular Ca(2+) mobilization and enhances GPCRs/PLC/Ca(2+) signaling

Phospholipase C-η1 (PLC-η1) is the most recently identified PLC isotype and is primarily expressed in nerve tissue. However, its functional role is unclear. In the present study, we report for the first time that PLC-η1 acts as a signal amplifier in G protein-coupled receptor (GPCR)-mediated PLC and Ca²⁺ signaling. Short-hairpin RNA (shRNA)-mediated knockdown of endogenous PLC-η1 reduced lysophosphatidic acid (LPA)-, bradykinin (BK)-, and PACAP-induced PLC activity in mouse neuroblastoma Neuro2A (N2A) cells, indicating that PLC-η1 participates in GPCR-mediated PLC activation. Interestingly, ionomycin-induced PLC activity was significantly decreased by PLC-η1, but not PLC-η2, knockdown. In addition, we found that intracellular Ca²⁺ source is enough for PLC-η1 activation. Furthermore, the IP₃ receptor inhibitor, 2-APB, inhibited LPA-induced PLC activity in control N2A cells, whereas this effect was not observed in PLC-η1 knockdown N2A cells, suggesting a pivotal role of intracellular Ca²⁺ mobilization in PLC-η1 activation. Finally, we found that LPA-induced ERK1/2 phosphorylation and expression of the downstream target gene, krox-24, were significantly decreased by PLC-η1 knockdown, and these knockdown effects were abolished by 2-APB. Taken together, our results strongly suggest that PLC-η1 is activated via intracellular Ca²⁺ mobilization from the ER, and therefore amplifies GPCR-mediated signaling.     

Cyanide-stimulated inositol 1,4,5-trisphosphate formation: An intracellular neurotoxic signaling cascade

Cyanide-induced neurotoxicity is associated with altered cellular Ca²⁺ homeostasis resulting in sustained elevation of cytosolic Ca²⁺. In order to characterize the effect of cyanide on intracellular signaling mechanisms, the interaction of KCN with the inositol 1,4,5-triphosphate Ca²⁺ signaling system was determined in the PC12 cell line. KCN in the concentration range of 1.0–100 μM produced a rapid rise in intracellular IP₃ levels (peak level occurred within 60 sec); 10 μM KCN elevated intracellular levels of IP₃ to 148% of control levels. This response was mediated by phospholipase C (PLC) since U73122, a specific PLC inhibitor, blocked the response. Removal of Ca²⁺ from the incubation medium and chelation of intracellular Ca²⁺ with BAPTA partially attenuate the cyanide-stimulated IP₃ generation, showing that the response is partially Ca²⁺ dependent. Also, treatment of cells with nifedipine or LaCl₃, Ca²⁺ channel blockers, partially blocked the generation of IP₃. This study shows that cyanide in concentrations as low as 1 μM stimulates IP₃ generation that may be mediated by receptor and nonreceptor IP₃ production since they have differential dependence on Ca²⁺. It is proposed that this response is an early intracellular signaling action that can contribute to altered Ca²⁺ homeostasis characteristic of cyanide neurotoxicity. © 1997 John Wiley & Sons, Inc.     

The role of inositol 1,4,5-trisphosphate in mobilizing calcium from intracellular stores in the salivary glands of Amblyomma americanum (L.)

Isolated tick salivary glands, permeabilized with digitonin in the presence of the Ca²⁺ uptake inhibitors, sodium azide and vanadate, released Ca²⁺ in response to 20 μM inositol-1,4,5-trisphosphate (IP₃). Inositol-1-phosphate (IP₁) and inositol-1,4-bisphosphate (IP₂) appeared to stimulate an uptake of Ca²⁺ into whole glands. Inositol-1,4,5-trisphosphate caused release of Ca²⁺ from a 100,000 g microsome enriched pellet; however, IP₁ and IP₂ were ineffective in stimulating an uptake or efflux of Ca²⁺. The combined 900 and 11,500 g pellets showed no significant release of Ca²⁺ in response to addition of IP₃. Inositol-1,4,5-trisphosphate concentrations as low as 1 μM are capable of stimulating a significant release of Ca²⁺ from microsomes. Results suggest that intracellular Ca²⁺ is mobilized from microsomal intracellular stores in response to agonists which increase cytosolic IP₃ in tick salivary glands. Results also suggest a possible role for IP₁ and IP₂ or both in stimulating an uptake of Ca²⁺ into vanadate and azide-insensitive intracellular pools.     

Type 1 IP3 receptors activate BKCa channels via local molecular coupling in arterial smooth muscle cells

Plasma membrane large-conductance Ca²⁺-activated K⁺ (BK_Ca) 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 BK_Ca channel regulation by IP₃ and IP₃Rs in rat and mouse cerebral artery smooth muscle cells. IP₃ activated BK_Ca channels both in intact cells and in excised inside-out membrane patches. IP₃ caused concentration-dependent BK_Ca channel activation with an apparent dissociation constant (K_d) of ∼4 µM at physiological voltage (−40 mV) and intracellular Ca²⁺ concentration ([Ca²⁺]_i; 10 µM). IP₃ also caused a leftward-shift in BK_Ca channel apparent Ca²⁺ sensitivity and reduced the K_d for free [Ca²⁺]_i from ∼20 to 12 µM, but did not alter the slope or maximal P_o. BAPTA, a fast Ca²⁺ buffer, or an elevation in extracellular Ca²⁺ concentration did not alter IP₃-induced BK_Ca channel activation. Heparin, an IP₃R inhibitor, and a monoclonal type 1 IP₃R (IP₃R1) antibody blocked IP₃-induced BK_Ca channel activation. Adenophostin A, an IP₃R agonist, also activated BK_Ca channels. IP₃ activated BK_Ca channels in inside-out patches from wild-type (IP₃R1^+/+) mouse arterial smooth muscle cells, but had no effect on BK_Ca channels of IP₃R1-deficient (IP₃R1^−/−) mice. Immunofluorescence resonance energy transfer microscopy indicated that IP₃R1 is located in close spatial proximity to BK_Ca α subunits. The IP₃R1 monoclonal antibody coimmunoprecipitated IP₃R1 and BK_Ca channel α and β1 subunits from cerebral arteries. In summary, data indicate that IP₃R1 activation elevates BK_Ca channel apparent Ca²⁺ sensitivity through local molecular coupling in arterial smooth muscle cells.     

Inositol 1,4,5-Trisphosphate Receptor in Chromaffin Secretory Granules and Its Relation to Chromogranins

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.     

Regulation of inositol 1,4,5-trisphosphate receptor type 1 function during oocyte maturation by MPM-2 phosphorylation

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).     

Polycystin-2 Activation by Inositol 1,4,5-Trisphosphate-induced Ca2+ Release Requires Its Direct Association with the Inositol 1,4,5-Trisphosphate Receptor in a Signaling Microdomain

Autosomal dominant polycystic kidney disease is characterized by the loss-of-function of a signaling complex involving polycystin-1 and polycystin-2 (TRPP2, an ion channel of the TRP superfamily), resulting in a disturbance in intracellular Ca²⁺ signaling. Here, we identified the molecular determinants of the interaction between TRPP2 and the inositol 1,4,5-trisphosphate receptor (IP₃R), an intracellular Ca²⁺ channel in the endoplasmic reticulum. Glutathione S-transferase pulldown experiments combined with mutational analysis led to the identification of an acidic cluster in the C-terminal cytoplasmic tail of TRPP2 and a cluster of positively charged residues in the N-terminal ligand-binding domain of the IP₃R as directly responsible for the interaction. To investigate the functional relevance of TRPP2 in the endoplasmic reticulum, we re-introduced the protein in TRPP2^−/− mouse renal epithelial cells using an adenoviral expression system. The presence of TRPP2 resulted in an increased agonist-induced intracellular Ca²⁺ release in intact cells and IP₃-induced Ca²⁺ release in permeabilized cells. Using pathological mutants of TRPP2, R740X and D509V, and competing peptides, we demonstrated that TRPP2 amplified the Ca²⁺ signal by a local Ca²⁺-induced Ca²⁺-release mechanism, which only occurred in the presence of the TRPP2-IP₃R interaction, and not via altered IP₃R channel activity. Moreover, our results indicate that this interaction was instrumental in the formation of Ca²⁺ microdomains necessary for initiating Ca²⁺-induced Ca²⁺ release. The data strongly suggest that defects in this mechanism may account for the altered Ca²⁺ signaling associated with pathological TRPP2 mutations and therefore contribute to the development of autosomal dominant polycystic kidney disease.     

Differential Regulation of Multiple Steps in Inositol 1,4,5-Trisphosphate Signaling by Protein Kinase C Shapes Hormone-stimulated Ca2+ Oscillations

How Ca²⁺ oscillations are generated and fine-tuned to yield versatile downstream responses remains to be elucidated. In hepatocytes, G protein-coupled receptor-linked Ca²⁺ oscillations report signal strength via frequency, whereas Ca²⁺ spike amplitude and wave velocity remain constant. IP₃ uncaging also triggers oscillatory Ca²⁺ release, but, in contrast to hormones, Ca²⁺ spike amplitude, width, and wave velocity were dependent on [IP₃] and were not perturbed by phospholipase C (PLC) inhibition. These data indicate that oscillations elicited by IP₃ uncaging are driven by the biphasic regulation of the IP₃ receptor by Ca²⁺, and, unlike hormone-dependent responses, do not require PLC. Removal of extracellular Ca²⁺ did not perturb Ca²⁺ oscillations elicited by IP₃ uncaging, indicating that reloading of endoplasmic reticulum stores via plasma membrane Ca²⁺ influx does not entrain the signal. Activation and inhibition of PKC attenuated hormone-induced Ca²⁺ oscillations but had no effect on Ca²⁺ increases induced by uncaging IP₃. Importantly, PKC activation and inhibition differentially affected Ca²⁺ spike frequencies and kinetics. PKC activation amplifies negative feedback loops at the level of G protein-coupled receptor PLC activity and/or IP₃ metabolism to attenuate IP₃ levels and suppress the generation of Ca²⁺ oscillations. Inhibition of PKC relieves negative feedback regulation of IP₃ accumulation and, thereby, shifts Ca²⁺ oscillations toward sustained responses or dramatically prolonged spikes. PKC down-regulation attenuates phenylephrine-induced Ca²⁺ wave velocity, whereas responses to IP₃ uncaging are enhanced. The ability to assess Ca²⁺ responses in the absence of PLC activity indicates that IP₃ receptor modulation by PKC regulates Ca²⁺ release and wave velocity.     

Characterization of a flatworm inositol (1,4,5) trisphosphate receptor (IP3R) reveals a role in reproductive physiology

Inositol 1,4,5-trisphosphate receptors (IP₃Rs) are intracellular Ca²⁺ channels that elevate cytoplasmic Ca²⁺ in response to the second messenger IP₃. Here, we describe the identification and in vivo functional characterization of the planarian IP₃R, the first intracellular Ca²⁺ channel to be defined in flatworms. A single IP₃R gene in Dugesia japonica encoded a 2666 amino acid protein (Dj.IP₃R) that shared well conserved structural features with vertebrate IP₃R counterparts. Expression of an NH₂-terminal Dj.IP₃R region (amino acid residues 223–585) recovered high affinity ³H-IP₃ binding (0.9 ± 0.1 nM) which was abolished by a single point mutation of an arginine residue (R495L) important for IP₃ coordination. In situ hybridization revealed that Dj.IP₃R mRNA was most strongly expressed in the pharynx and optical nerve system as well as the reproductive system in sexualized planarians. Consistent with this observed tissue distribution, in vivo RNAi of Dj.IP₃R resulted in a decreased egg-laying behavior suggesting Dj.IP₃R plays an upstream role in planarian reproductive physiology.     

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.     

The actin cytoskeleton differentially regulates NG115-401L cell ryanodine receptor and inositol 1,4,5-trisphosphate receptor induced calcium signaling pathways

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.     

Structure and function of IP3 receptors

The Molecular, structural and functional characteristics of the intracellular Ca²⁺ release channel activated by inositol 1,4,5-trisphosphate (IP₃), also named IP₃ receptor (IP₃R), are described here. We also discuss the differences in primary structure, expression and modulation of the receptor subtypes and their physiological roles. The similarity and differences between the IP₃R and the other intracellular Ca²⁺ channel, the ryanodine receptor, are briefly presented.     

Regulation of endogenous and heterologous Ca release-activated Ca currents by pH

Deviations from physiological pH (∼pH 7.2) as well as altered Ca²⁺ signaling play important roles in immune disease and cancer. One of the most ubiquitous pathways for cellular Ca²⁺ influx is the store-operated Ca²⁺ entry (SOCE) or Ca²⁺ release-activated Ca²⁺ current (I_CRAC), which is activated upon depletion of intracellular Ca²⁺ stores. We here show that extracellular and intracellular changes in pH regulate both endogenous I_CRAC in Jurkat T lymphocytes and RBL2H3 cells, and heterologous I_CRAC in HEK293 cells expressing the molecular components STIM1/2 and Orai1/2/3 (CRACM1/2/3). We find that external acidification suppresses, and alkalization facilitates IP₃-induced I_CRAC. In the absence of IP₃, external alkalization did not elicit endogenous I_CRAC but was able to activate heterologous I_CRAC in HEK293 cells expressing Orai1/2/3 and STIM1 or STIM2. Similarly, internal acidification reduced IP₃-induced activation of endogenous and heterologous I_CRAC, while alkalization accelerated its activation kinetics without affecting overall current amplitudes. Mutation of two aspartate residues to uncharged alanine amino acids (D110/112A) in the first extracellular loop of Orai1 significantly attenuated both the inhibition of I_CRAC by external acidic pH as well as its facilitation by alkaline conditions. We conclude that intra- and extracellular pH differentially regulates I_CRAC. While intracellular pH might affect aggregation and/or binding of STIM to Orai, external pH seems to modulate I_CRAC through its channel pore, which in Orai1 is partially mediated by residues D110 and D112.     

Mammalian target of rapamycin (mTOR) phosphorylates inositol 1,4,5-trisphosphate receptor type 2 and increases its Ca release activity

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.     

Exact Stochastic Simulation of a Calcium Microdomain Reveals the Impact of Ca2+ Fluctuations on IP3R Gating

In this study, we numerically analyzed the nonlinear Ca²⁺-dependent gating dynamics of a single, nonconducting inositol 1,4,5-trisphosphate receptor (IP₃R) channel, using an exact and fully stochastic simulation algorithm that includes channel gating, Ca²⁺ buffering, and Ca²⁺ diffusion. The IP₃R is a ubiquitous intracellular Ca²⁺ release channel that plays an important role in the formation of complex spatiotemporal Ca²⁺ signals such as waves and oscillations. Dynamic subfemtoliter Ca²⁺ microdomains reveal low copy numbers of Ca²⁺ ions, buffer molecules, and IP₃Rs, and stochastic fluctuations arising from molecular interactions and diffusion do not average out. In contrast to models treating calcium dynamics deterministically, the stochastic approach accounts for this molecular noise. We varied Ca²⁺ diffusion coefficients and buffer reaction rates to tune the autocorrelation properties of Ca²⁺ noise and found a distinct relation between the autocorrelation time τ_ac, the mean channel open and close times, and the resulting IP₃R open probability P_O. We observed an increased P_O for shorter noise autocorrelation times, caused by increasing channel open times and decreasing close times. In a pure diffusion model the effects become apparent at elevated calcium concentrations, e.g., at [Ca²⁺] = 25 μM, τ_ac = 0.082 ms, the IP₃R open probability increased by ≈20% and mean open times increased by ≈4 ms, compared to a zero noise model. We identified the inactivating Ca²⁺ binding site of IP₃R subunits as the primarily noise-susceptible element of the De Young and Keizer model. Short Ca²⁺ noise autocorrelation times decrease the probability of Ca²⁺ association and consequently increase IP₃R activity. These results suggest a functional role of local calcium noise properties on calcium-regulated target molecules such as the ubiquitous IP₃R. This finding may stimulate novel experimental approaches analyzing the role of calcium noise properties on microdomain behavior.     

Bcl-xL acts as an inhibitor of IP3R channels,thereby antagonizing Ca2+-driven apoptosis

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-xL^K87D, 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-xL^K87D was much less effective in doing so. In the absence of IP₃Rs, Bcl-xL and Bcl-xL^K87D 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