Modeling of the Modulation by Buffers of Ca Release through Clusters of IP3 Receptors

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.     

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.     

Mitochondrial Calcium Signaling Driven by the IP3 Receptor

Many agonists bring about their effects on cellular functions through a rise incytosolic [Ca²⁺]([Ca²⁺]_c) mediated by the second messenger inositol 1,4,5-trisphosphate (IP₃). Imaging studiesof single cells have demonstrated that [Ca²⁺]_c signals display cell specific spatiotemporalorganization that is established by coordinated activation of IP₃ receptor Ca²⁺ channels.Evidence emerges that cytosolic calcium signals elicited by activation of the IP₃ receptors areefficiently transmitted to the mitochondria. An important function of mitochondrial calciumsignals is to activate the Ca²⁺-sensitive mitochondrial dehydrogenases, and thereby to meetdemands for increased energy in stimulated cells. Activation of the permeability transitionpore (PTP) by mitochondrial calcium signals may also be involved in the control of cell death.Furthermore, mitochondrial Ca²⁺ transport appears to modulate the spatiotemporal organizationof [Ca²⁺]_c responses evoked by IP₃ and so mitochondria may be important in cytosolic calciumsignaling as well. This paper summarizes recent research to elucidate the mechanisms andsignificance of IP₃-dependent mitochondrial calcium signaling.     

Caspase-3-truncated type 1 inositol 1,4,5-trisphosphate receptor enhances intracellular Ca2+ leak and disturbs Ca2+ signalling

Background information. The IP₃R (inositol 1,4,5‐trisphosphate receptor) is a tetrameric channel that accounts for a large part of the intracellular Ca²⁺ release in virtually all cell types. We have previously demonstrated that caspase‐3‐mediated cleavage of IP₃R1 during cell death generates a C‐terminal fragment of 95 kDa comprising the complete channel domain. Expression of this truncated IP₃R increases the cellular sensitivity to apoptotic stimuli, and it was postulated to be a constitutively active channel. Results. In the present study, we demonstrate that expression of the caspase‐3‐cleaved C‐terminus of IP₃R1 increased the rate of thapsigargin‐mediated Ca²⁺ leak and decreased the rate of Ca²⁺ uptake into the ER (endoplasmic reticulum), although it was not sufficient by itself to deplete intracellular Ca²⁺ stores. We detected the truncated IP₃R1 in different cell types after a challenge with apoptotic stimuli, as well as in aged mouse oocytes. Injection of mRNA corresponding to the truncated IP₃R1 blocked sperm factor‐induced Ca²⁺ oscillations and induced an apoptotic phenotype. Conclusions. In the present study, we show that caspase‐3‐mediated truncation of IP₃R1 enhanced the Ca²⁺ leak from the ER. We suggest a model in which, in normal conditions, the increased Ca²⁺ leak is largely compensated by enhanced Ca²⁺‐uptake activity, whereas in situations where the cellular metabolism is compromised, as occurring in aging oocytes, the Ca²⁺ leak acts as a feed‐forward mechanism to divert the cell into apoptosis.     

Hydrogen peroxide depolarizes mitochondria and inhibits IP3-evoked Ca2+ release in the endothelium of intact arteries

Hydrogen peroxide (H₂O₂) is a mitochondrial-derived reactive oxygen species (ROS) that regulates vascular signalling transduction, vasocontraction and vasodilation. Although the physiological role of ROS in endothelial cells is acknowledged, the mechanisms underlying H₂O₂ regulation of signalling in native, fully-differentiated endothelial cells is unresolved. In the present study, the effects of H₂O₂ on Ca²⁺ signalling were investigated in the endothelium of intact rat mesenteric arteries. Spontaneous local Ca²⁺ signals and acetylcholine evoked Ca²⁺ increases were inhibited by H₂O₂. H₂O₂ inhibition of acetylcholine-evoked Ca²⁺ signals was reversed by catalase. H₂O₂ exerts its inhibition on the IP₃ receptor as Ca²⁺ release evoked by photolysis of caged IP₃ was supressed by H₂O₂. H₂O₂ suppression of IP₃-evoked Ca²⁺ signalling may be mediated by mitochondria. H₂O₂ depolarized mitochondria membrane potential. Acetylcholine-evoked Ca²⁺ release was inhibited by depolarisation of the mitochondrial membrane potential by the uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP) or complex 1 inhibitor, rotenone. We propose that the suppression of IP₃-evoked Ca²⁺ release by H₂O₂ arises from the decrease in mitochondrial membrane potential. These results suggest that mitochondria may protect themselves against Ca²⁺ overload during IP₃-linked Ca²⁺ signals by a H₂O₂ mediated negative feedback depolarization of the organelle and inhibition of IP₃-evoked Ca²⁺ release.     

Analysis of IP3 receptors in and out of cells

Background

Inositol 1,4,5-trisphosphate receptors (IP₃R) are expressed in almost all animal cells. Three mammalian genes encode closely related IP₃R subunits, which assemble into homo- or hetero-tetramers to form intracellular Ca^2 + channels.

Scope of the review

In this brief review, we first consider a variety of complementary methods that allow the links between IP₃ binding and channel gating to be defined. How does IP₃ binding to the IP₃-binding core in each IP₃R subunit cause opening of a cation-selective pore formed by residues towards the C-terminal? We then describe methods that allow IP₃, Ca^2 + signals and IP₃R mobility to be examined in intact cells. A final section briefly considers genetic analyses of IP₃R signalling.

Major conclusions

All IP₃R are regulated by both IP₃ and Ca^2 +. This allows them to initiate and regeneratively propagate intracellular Ca^2 + signals. The elementary Ca^2 + release events evoked by IP₃ in intact cells are mediated by very small numbers of active IP₃R and the Ca^2 +-mediated interactions between them. The spatial organization of these Ca^2 + signals and their stochastic dependence on so few IP₃Rs highlight the need for methods that allow the spatial organization of IP₃R signalling to be addressed with single-molecule resolution.

General significance

A variety of complementary methods provide insight into the structural basis of IP₃R activation and the contributions of IP₃-evoked Ca^2 + signals to cellular physiology. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.     

Agonist‐evoked Ca2+ wave progression requires Ca2+ and IP3

Smooth muscle responds to IP₃‐generating agonists by producing Ca²⁺ waves. Here, the mechanism of wave progression has been investigated in voltage‐clamped single smooth muscle cells using localized photolysis of caged IP₃ and the caged Ca²⁺ buffer diazo‐2. Waves, evoked by the IP₃‐generating agonist carbachol (CCh), initiated as a uniform rise in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_c) over a single though substantial length (～30 µm) of the cell. During regenerative propagation, the wave‐front was about 1/3 the length (～9 µm) of the initiation site. The wave‐front progressed at a relatively constant velocity although amplitude varied through the cell; differences in sensitivity to IP₃ may explain the amplitude changes. Ca²⁺ was required for IP₃‐mediated wave progression to occur. Increasing the Ca²⁺ buffer capacity in a small (2 µm) region immediately in front of a CCh‐evoked Ca²⁺ wave halted progression at the site. However, the wave front does not progress by Ca²⁺‐dependent positive feedback alone. In support, colliding [Ca²⁺]_c increases from locally released IP₃ did not annihilate but approximately doubled in amplitude. This result suggests that local IP₃‐evoked [Ca²⁺]_c increases diffused passively. Failure of local increases in IP₃ to evoke waves appears to arise from the restricted nature of the IP₃ increase. When IP₃ was elevated throughout the cell, a localized increase in Ca²⁺ now propagated as a wave. Together, these results suggest that waves initiate over a surprisingly large length of the cell and that both IP₃ and Ca²⁺ are required for active propagation of the wave front to occur. J. Cell. Physiol. 224: 334–344, 2010. © 2010 Wiley‐Liss, Inc.     

Superresolution Localization of Single Functional IP3R Channels Utilizing Ca Flux as a Readout

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.     

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.     

Microfilament and microtubule assembly is required for the propagation of inositol trisphosphate receptor‐induced Ca2+ waves in bovine aortic endothelial cells

Ca²⁺ is a highly versatile second messenger that plays a key role in the regulation of numerous cell processes. One‐way cells ensure the specificity and reliability of Ca²⁺ signals is by organizing them spatially in the form of waves that propagate throughout the cell or within a specific subcellular region. In non‐excitable cells, the inositol 1,4,5‐trisphosphate receptor (IP₃R) is responsible for the release of Ca²⁺ from the endoplasmic reticulum. The spatial aspect of the Ca²⁺ signal depends on the organization of various elements of the Ca²⁺ signaling toolkit and varies from tissue to tissue. Ca²⁺ is implicated in many of endothelium functions that thus depend on the versatility of Ca²⁺ signaling. In the present study, we showed that the disruption of caveolae microdomains in bovine aortic endothelial cells (BAEC) with methyl‐ß‐cyclodextrin was not sufficient to disorganize the propagation of Ca²⁺ waves when the cells were stimulated with ATP or bradykinin. However, disorganizing microfilaments with latrunculin B and microtubules with colchicine both prevented the formation of Ca²⁺ waves. These results suggest that the organization of the Ca²⁺ waves mediated by IP₃R channels does not depend on the integrity of caveolae in BAEC, but that microtubule and microfilament cytoskeleton assembly is crucial. J. Cell. Biochem. 106: 344–352, 2009. © 2008 Wiley‐Liss, Inc.     

An efficient reduced-lattice model of IP3R for probing Ca2+ dynamics

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.     

Coupling switch of P2Y-IP<Subscript>3</Subscript> receptors mediates differential Ca<Superscript>2+</Superscript> signaling in human embryonic stem cells and derived cardiovascular progenitor cells

Purinergic signaling mediated by P2 receptors (P2Rs) plays important roles in embryonic and stem cell development. However, how it mediates Ca²⁺ signals in human embryonic stem cells (hESCs) and derived cardiovascular progenitor cells (CVPCs) remains unclear. Here, we aimed to determine the role of P2Rs in mediating Ca²⁺ mobilizations of these cells. hESCs were induced to differentiate into CVPCs by our recently established methods. Gene expression of P2Rs and inositol 1,4,5-trisphosphate receptors (IP₃Rs) was analyzed by quantitative/RT-PCR. IP₃R3 knockdown (KD) or IP₃R2 knockout (KO) hESCs were established by shRNA- or TALEN-mediated gene manipulations, respectively. Confocal imaging revealed that Ca²⁺ responses in CVPCs to ATP and UTP were more sensitive and stronger than those in hESCs. Consistently, the gene expression levels of most P2YRs except P2Y₁ were increased in CVPCs. Suramin or PPADS blocked ATP-induced Ca²⁺ transients in hESCs but only partially inhibited those in CVPCs. Moreover, the P2Y₁ receptor-specific antagonist MRS2279 abolished most ATP-induced Ca²⁺ signals in hESCs but not in CVPCs. P2Y₁ receptor-specific agonist MRS2365 induced Ca²⁺ transients only in hESCs but not in CVPCs. Furthermore, IP₃R2KO but not IP₃R3KD decreased the proportion of hESCs responding to MRS2365. In contrast, both IP₃R2 and IP₃R3 contributed to UTP-induced Ca²⁺ responses while ATP-induced Ca²⁺ responses were more dependent on IP₃R2 in the CVPCs. In conclusion, a predominant role of P2Y₁ receptors in hESCs and a transition of P2Y-IP₃R coupling in derived CVPCs are responsible for the differential Ca²⁺ mobilization between these cells.     

Bcl-2 interaction with the inositol 1,4,5-trisphosphate receptor: role in Ca(2+) signaling and disease

The Bcl-2 protein, best known for its ability to inhibit apoptosis, interacts with the inositol 1,4,5-trisphosphate receptor (IP₃R) Ca²⁺ channel to regulate IP₃-mediated Ca²⁺ release from the endoplasmic reticulum. This review summarizes the current state of knowledge regarding the interaction of Bcl-2, and also its homologue Bcl-xl, with the IP₃R and how these interactions regulate Ca²⁺ signaling. The dual role of these interactions in promoting prosurvival Ca²⁺ signals, while at the same time inhibiting proapoptotic Ca²⁺ signals, is discussed. Moreover, this review will elucidate the recently recognized importance of the Bcl-2-IP₃R interaction in human disease.     

Inositol 1,4,5‐trisphosphate receptor 1 degradation in mouse eggs and impact on [Ca2+]i oscillations

The initiation of normal embryo development depends on the completion of all events of egg activation. In all species to date, egg activation requires an increase(s) in the intracellular concentration of calcium ([Ca²⁺]_i), which is almost entirely mediated by inositol 1,4,5‐trisphosphate receptor 1 (IP₃R1). In mammalian eggs, fertilization‐induced [Ca²⁺]_i responses exhibit a periodic pattern that are called [Ca²⁺]_i oscillations. These [Ca²⁺]_i oscillations are robust at the beginning of fertilization, which occurs at the second metaphase of meiosis, but wane as zygotes approach the pronuclear stage, time after which in the mouse oscillations cease altogether. Underlying this change in frequency are cellular and biochemical changes associated with egg activation, including degradation of IP₃R1, progression through the cell cycle, and reorganization of intracellular organelles. In this study, we investigated the system requirements for IP₃R1 degradation and examined the impact of the IP₃R1 levels on the pattern of [Ca²⁺]_i oscillations. Using microinjection of IP₃ and of its analogs and conditions that prevent the development of [Ca²⁺]_i oscillations, we show that IP₃R1 degradation requires uniform and persistently elevated levels of IP₃. We also established that progressive degradation of the IP₃R1 results in [Ca²⁺]_i oscillations with diminished periodicity while a near complete depletion of IP₃R1s precludes the initiation of [Ca²⁺]_i oscillations. These results provide insights into the mechanism involved in the generation of [Ca²⁺]_i oscillations in mouse eggs. J. Cell. Physiol. 222:238–247, 2010. © 2009 Wiley‐Liss, Inc.     

Analyzing and Quantifying the Gain-of-Function Enhancement of IP3 Receptor Gating by Familial Alzheimer’s Disease-Causing Mutants in Presenilins

Familial Alzheimer’s disease (FAD)-causing mutant presenilins (PS) interact with inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R) Ca²⁺ release channels resulting in enhanced IP₃R channel gating in an amyloid beta (Aβ) production-independent manner. This gain-of-function enhancement of IP₃R activity is considered to be the main reason behind the upregulation of intracellular Ca²⁺ signaling in the presence of optimal and suboptimal stimuli and spontaneous Ca²⁺ signals observed in cells expressing mutant PS. In this paper, we employed computational modeling of single IP₃R channel activity records obtained under optimal Ca²⁺ and multiple IP₃ concentrations to gain deeper insights into the enhancement of IP₃R function. We found that in addition to the high occupancy of the high-activity (H) mode and the low occupancy of the low-activity (L) mode, IP₃R in FAD-causing mutant PS-expressing cells exhibits significantly longer mean life-time for the H mode and shorter life-time for the L mode, leading to shorter mean close-time and hence high open probability of the channel in comparison to IP₃R in cells expressing wild-type PS. The model is then used to extrapolate the behavior of the channel to a wide range of IP₃ and Ca²⁺ concentrations and quantify the sensitivity of IP₃R to its two ligands. We show that the gain-of-function enhancement is sensitive to both IP₃ and Ca²⁺ and that very small amount of IP₃ is required to stimulate IP₃R channels in the presence of FAD-causing mutant PS to the same level of activity as channels in control cells stimulated by significantly higher IP₃ concentrations. We further demonstrate with simulations that the relatively longer time spent by IP₃R in the H mode leads to the observed higher frequency of local Ca²⁺ signals, which can account for the more frequent global Ca²⁺ signals observed, while the enhanced activity of the channel at extremely low ligand concentrations will lead to spontaneous Ca²⁺ signals in cells expressing FAD-causing mutant PS.     

Unique Regulatory Properties of Heterotetrameric Inositol 1,4,5-Trisphosphate Receptors Revealed by Studying Concatenated Receptor Constructs

The ability of inositol 1,4,5-trisphosphate receptors (IP₃R) to precisely initiate and generate a diverse variety of intracellular Ca²⁺ signals is in part mediated by the differential regulation of the three subtypes (R1, R2, and R3) by key functional modulators (IP₃, Ca²⁺, and ATP). However, the contribution of IP₃R heterotetramerization to Ca²⁺ signal diversity has largely been unexplored. In this report, we provide the first definitive biochemical evidence of endogenous heterotetramer formation. Additionally, we examine the contribution of individual subtypes within defined concatenated heterotetramers to the shaping of Ca²⁺ signals. Under conditions where key regulators of IP₃R function are optimal for Ca²⁺ release, we demonstrate that individual monomers within heteromeric IP₃Rs contributed equally toward generating a distinct ''blended'' sensitivity to IP₃ that is likely dictated by the unique IP₃ binding affinity of the heteromers. However, under suboptimal conditions where [ATP] were varied, we found that one subtype dictated the ATP regulatory properties of heteromers. We show that R2 monomers within a heterotetramer were both necessary and sufficient to dictate the ATP regulatory properties. Finally, the ATP-binding site B in R2 critical for ATP regulation was mutated and rendered non-functional to address questions relating to the stoichiometry of IP₃R regulation. Two intact R2 monomers were sufficient to maintain ATP regulation in R2 homotetramers. In summary, we demonstrate that heterotetrameric IP₃R do not necessarily behave as the sum of the constituent subunits, and these properties likely extend the versatility of IP₃-induced Ca²⁺ signaling in cells expressing multiple IP₃R isoforms.     

Stochastic reaction-diffusion modeling of calcium dynamics in 3D dendritic spines of Purkinje cells

Calcium (Ca²⁺) is a second messenger assumed to control changes in synaptic strength in the form of both long-term depression and long-term potentiation at Purkinje cell dendritic spine synapses via inositol trisphosphate (IP₃)-induced Ca²⁺ release. These Ca²⁺ transients happen in response to stimuli from parallel fibers (PFs) from granule cells and climbing fibers (CFs) from the inferior olivary nucleus. These events occur at low numbers of free Ca²⁺, requiring stochastic single-particle methods when modeling them. We use the stochastic particle simulation program MCell to simulate Ca²⁺ transients within a three-dimensional Purkinje cell dendritic spine. The model spine includes the endoplasmic reticulum, several Ca²⁺ transporters, and endogenous buffer molecules. Our simulations successfully reproduce properties of Ca²⁺ transients in different dynamical situations. We test two different models of the IP₃ receptor (IP₃R). The model with nonlinear concentration response of binding of activating Ca²⁺ reproduces experimental results better than the model with linear response because of the filtering of noise. Our results also suggest that Ca²⁺-dependent inhibition of the IP₃R needs to be slow to reproduce experimental results. Simulations suggest the experimentally observed optimal timing window of CF stimuli arises from the relative timing of CF influx of Ca²⁺ and IP₃ production sensitizing IP₃R for Ca²⁺-induced Ca²⁺ release. We also model ataxia, a loss of fine motor control assumed to be the result of malfunctioning information transmission at the granule to Purkinje cell synapse, resulting in a decrease or loss of Ca²⁺ transients. Finally, we propose possible ways of recovering Ca²⁺ transients under ataxia.     

Probes for manipulating and monitoring IP3

Inositol 1,4,5-trisphosphate (IP₃) is an important second messenger produced via G-protein-coupled receptor- or receptor tyrosine kinase-mediated pathways. IP₃ levels induce Ca²⁺ release from the endoplasmic reticulum (ER) via IP₃ receptor (IP₃R) located in the ER membrane. The resultant spatiotemporal pattern of Ca²⁺ signals regulates diverse cellular functions, including fertilization, gene expression, synaptic plasticity, and cell death. Therefore, monitoring and manipulating IP₃ levels is important to elucidate not only the functions of IP₃-mediated pathways but also the encoding mechanism of IP₃R as a converter of intracellular signals from IP₃ to Ca²⁺.     

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.