'Trigger' events precede calcium puffs in Xenopus oocytes

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.     

Frequency and Relative Prevalence of Calcium Blips and Puffs in a Model of Small IP3R Clusters

In this work, we model the local calcium release from clusters with a few inositol 1,4,5-trisphosphate receptor (IP₃R) channels, focusing on the stochastic process in which an open channel either triggers other channels to open (as a puff) or fails to cause any channel to open (as a blip). We show that there are linear relations for the interevent interval (including blips and puffs) and the first event latency against the inverse cluster size. However, nonlinearity is found for the interpuff interval and the first puff latency against the inverse cluster size. Furthermore, the simulations indicate that the blip fraction among all release events and the blip frequency are increasing with larger basal [Ca²⁺], with blips in turn giving a growing contribution to basal [Ca²⁺]. This result suggests that blips are not just lapses to trigger puffs, but they may also possess a biological function to contribute to the initiation of calcium waves by a preceding increase of basal [Ca²⁺] in cells that have small IP₃R clusters.     

Frequency and Relative Prevalence of Calcium Blips and Puffs in a Model of Small IP3R Clusters

In this work, we model the local calcium release from clusters with a few inositol 1,4,5-trisphosphate receptor (IP₃R) channels, focusing on the stochastic process in which an open channel either triggers other channels to open (as a puff) or fails to cause any channel to open (as a blip). We show that there are linear relations for the interevent interval (including blips and puffs) and the first event latency against the inverse cluster size. However, nonlinearity is found for the interpuff interval and the first puff latency against the inverse cluster size. Furthermore, the simulations indicate that the blip fraction among all release events and the blip frequency are increasing with larger basal [Ca²⁺], with blips in turn giving a growing contribution to basal [Ca²⁺]. This result suggests that blips are not just lapses to trigger puffs, but they may also possess a biological function to contribute to the initiation of calcium waves by a preceding increase of basal [Ca²⁺] in cells that have small IP₃R clusters.     

Fluorescence Fluctuations and Equivalence Classes of Ca 2+ Imaging Experiments

release into the cytosol through inositol 1,4,5-trisphosphate receptors (IP₃Rs) plays a relevant role in numerous physiological processes. IP₃R-mediated signals involve -induced -release (CICR) whereby release through one open IP₃R induces the opening of other channels. IP₃Rs are apparently organized in clusters. The signals can remain localized (i.e., puffs) if CICR is limited to one cluster or become waves that propagate between clusters. puffs are the building blocks of waves. Thus, there is great interest in determining puff properties, especially in view of the current controversy on the spatial distribution of activatable IP₃Rs. puffs have been observed in intact cells with optical techniques proving that they are intrinsically stochastic. Obtaining a correct picture of their dynamics then entails being able to detect the whole range of puff sizes. puffs are observed using visible single-wavelength dyes, slow exogenous buffers (e.g., EGTA) to disrupt inter-cluster CICR and UV-photolyzable caged IP₃. Single-wavelength dyes increase their fluorescence upon calcium binding producing images that are strongly dependent on their kinetic, transport and photophysical properties. Determining the artifacts that the imaging setting introduces is particularly relevant when trying to analyze the smallest signals. In this paper we introduce a method to estimate the expected signal-to-noise ratio of imaging experiments that use single-wavelength dyes. The method is based on the Number and Brightness technique. It involves the performance of a series of experiments and their subsequent analysis in terms of a fluorescence fluctuation model with which the model parameters are quantified. Using the model, the expected signal-to-noise ratio is then computed. Equivalence classes between different experimental conditions that produce images with similar signal-to-noise ratios can then be established. The method may also be used to estimate the smallest signals that can reliably be observed with each setting.     

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.     

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.     

Abortive and Propagating Intracellular Calcium Waves: Analysis from a Hybrid Model

The functional properties of inositol(1,4,5)-triphosphate (IP₃) receptors allow a variety of intracellular Ca²⁺ phenomena. In this way, global phenomena, such as propagating and abortive Ca²⁺ waves, as well as local events such as puffs, have been observed. Several experimental studies suggest that many features of global phenomena (e.g., frequency, amplitude, speed wave) depend on the interplay of biophysical processes such as diffusion, buffering, efflux and influx rates, which in turn depend on parameters such as buffer concentration, Ca²⁺ pump density, cytosolic IP₃ level, and intercluster distance. Besides, it is known that cells are able to modify some of these parameters in order to regulate the Ca²⁺ signaling. By using a hybrid model, we analyzed different features of the hierarchy of calcium events as a function of two relevant parameters for the calcium signaling, the intercluster distance and the pump strength or intensity. In the space spanned by these two parameters, we found two modes of calcium dynamics, one dominated by abortive calcium waves and the other by propagating waves. Smaller distances between the release sites promote propagating calcium waves, while the increase of the efflux rate makes the transition from propagating to abortive waves occur at lower values of intercluster distance. We determined the frontier between these two modes, in the parameter space defined by the intercluster distance and the pump strength. Furthermore, we found that the velocity of simulated calcium waves accomplishes Luther’s law, and that an effective rate constant for autocatalytic calcium production decays linearly with both the intercluster distance and the pump strength.     

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.     

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.     

A Stochastic Model of Calcium Puffs Based on Single-Channel Data

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.     

ATP- and Gap Junction–dependent Intercellular Calcium Signaling in Osteoblastic Cells

Many cells coordinate their activities by transmitting rises in intracellular calcium from cell to cell. In nonexcitable cells, there are currently two models for intercellular calcium wave propagation, both of which involve release of inositol trisphosphate (IP₃)- sensitive intracellular calcium stores. In one model, IP₃ traverses gap junctions and initiates the release of intracellular calcium stores in neighboring cells. Alternatively, calcium waves may be mediated not by gap junctional communication, but rather by autocrine activity of secreted ATP on P₂ purinergic receptors. We studied mechanically induced calcium waves in two rat osteosarcoma cell lines that differ in the gap junction proteins they express, in their ability to pass microinjected dye from cell to cell, and in their expression of P2Y₂ (P_2U) purinergic receptors. ROS 17/2.8 cells, which express the gap junction protein connexin43 (Cx43), are well dye coupled, and lack P_2U receptors, transmitted slow gap junction-dependent calcium waves that did not require release of intracellular calcium stores. UMR 106-01 cells predominantly express the gap junction protein connexin 45 (Cx45), are poorly dye coupled, and express P_2U receptors; they propagated fast calcium waves that required release of intracellular calcium stores and activation of P_2U purinergic receptors, but not gap junctional communication. ROS/P_2U transfectants and UMR/Cx43 transfectants expressed both types of calcium waves. Gap junction–independent, ATP-dependent intercellular calcium waves were also seen in hamster tracheal epithelia cells. These studies demonstrate that activation of P_2U purinergic receptors can propagate intercellular calcium, and describe a novel Cx43-dependent mechanism for calcium wave propagation that does not require release of intracellular calcium stores by IP₃. These studies suggest that gap junction communication mediated by either Cx43 or Cx45 does not allow passage of IP₃ well enough to elicit release of intracellular calcium stores in neighboring cells.     

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.     

Cytosolic and nuclear calcium signaling in atrial myocytes: IP3-mediated calcium release and the role of mitochondria

In rabbit atrial myocytes Ca signaling has unique features due to the lack of transverse (t) tubules, the spatial arrangement of mitochondria and the contribution of inositol-1,4,5-trisphosphate (IP₃) receptor-induced Ca release (IICR). During excitation-contraction coupling action potential-induced elevation of cytosolic [Ca] originates in the cell periphery from Ca released from the junctional sarcoplasmic reticulum (j-SR) and then propagates by Ca-induced Ca release from non-junctional (nj-) SR toward the cell center. The subsarcolemmal region between j-SR and the first array of nj-SR Ca release sites is devoid of mitochondria which results in a rapid propagation of activation through this domain, whereas the subsequent propagation through the nj-SR network occurs at a velocity typical for a propagating Ca wave. Inhibition of mitochondrial Ca uptake with the Ca uniporter blocker Ru360 accelerates propagation and increases the amplitude of Ca transients (CaTs) originating from nj-SR. Elevation of cytosolic IP₃ levels by rapid photolysis of caged IP₃ has profound effects on the magnitude of subcellular CaTs with increased Ca release from nj-SR and enhanced CaTs in the nuclear compartment. IP₃ uncaging restricted to the nucleus elicites ‘mini’-Ca waves that remain confined to this compartment. Elementary IICR events (Ca puffs) preferentially originate in the nucleus in close physical association with membrane structures of the nuclear envelope and the nucleoplasmic reticulum. The data suggest that in atrial myocytes the nucleus is an autonomous Ca signaling domain where Ca dynamics are primarily governed by IICR.     

Cytosolic and nuclear calcium signaling in atrial myocytes: IP3-mediated calcium release and the role of mitochondria

In rabbit atrial myocytes Ca signaling has unique features due to the lack of transverse (t) tubules, the spatial arrangement of mitochondria and the contribution of inositol-1,4,5-trisphosphate (IP₃) receptor-induced Ca release (IICR). During excitation-contraction coupling action potential-induced elevation of cytosolic [Ca] originates in the cell periphery from Ca released from the junctional sarcoplasmic reticulum (j-SR) and then propagates by Ca-induced Ca release from non-junctional (nj-) SR toward the cell center. The subsarcolemmal region between j-SR and the first array of nj-SR Ca release sites is devoid of mitochondria which results in a rapid propagation of activation through this domain, whereas the subsequent propagation through the nj-SR network occurs at a velocity typical for a propagating Ca wave. Inhibition of mitochondrial Ca uptake with the Ca uniporter blocker Ru360 accelerates propagation and increases the amplitude of Ca transients (CaTs) originating from nj-SR. Elevation of cytosolic IP₃ levels by rapid photolysis of caged IP₃ has profound effects on the magnitude of subcellular CaTs with increased Ca release from nj-SR and enhanced CaTs in the nuclear compartment. IP₃ uncaging restricted to the nucleus elicites ‘mini’-Ca waves that remain confined to this compartment. Elementary IICR events (Ca puffs) preferentially originate in the nucleus in close physical association with membrane structures of the nuclear envelope and the nucleoplasmic reticulum. The data suggest that in atrial myocytes the nucleus is an autonomous Ca signaling domain where Ca dynamics are primarily governed by IICR.     

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.     

Calcium Events in Smooth Muscles and Their Associated Cells

Calcium is essential for contraction of smooth muscle cells (SMC). The contractile proteins are activated by calcium released from the stores within the cell in response to calcium entry through voltage-dependent channels and/or activation of receptors, which often increase D-myoinositol 1,4,5-trisphosphate (IP₃) concentration in the cell through stimulation of phospholipase C (PLC). A global rise in the concentration of ionized calcium, [Ca²⁺] _i , which gives rise to contraction or shortening, is initiated at preferred locations in the cell, termed frequent discharge sites (FDS). In many SMC these sites often spontaneously discharge calcium packets; this is caused by bursts of openings of calcium channels (commonly ryanodine receptors, RyR, or IP₃ receptors) in the sarcoplasmic reticulum (SR). The rise in [Ca²⁺] _i may be detected by introducing calcium indicator dyes into the cell; the release of a calcium packet then gives rise to a rapid increase in fluorescence, or “spark.” A spark may activate a burst of openings of calcium-activated potassium or chloride channels in the cell membrane, so giving rise to spontaneous transient outward currents (STOC) or spontaneous transient inward currents (STIC), respectively. The term “spark” should probably be reserved for a calcium event resulting from the discharge of a single cluster, or domain, of RyR channels; when IP₃; concentrations are raised, adjacent domains may discharge closely in time, giving rise to larger calcium events, activation of more distant domains by a fire-diffuse-fire mechanism, and saltatory propagation of a calcium wave leading to a global rise in [Ca²⁺] _i and contraction of the cell. In many smooth muscle tissues, including some blood vessels, SMC are associated with interstitial cells (IC); well-known examples are the IC of Cajal in the gut muscles. In the media of small mesenteric arteries and portal vein, the IC share many properties with the SMC but, unlike the latter, have many thin processes and do not contract to agents, which contract the SMC. The role of these IC in blood vessels is unknown.     

Calcium-dependent inactivation and the dynamics of calcium puffs and sparks

Localized intracellular Ca²⁺ elevations known as puffs and sparks arise from the cooperative activity of inositol 1,4,5-trisphosphate receptor Ca²⁺ channels (IP₃Rs) and ryanodine receptor Ca²⁺ channels (RyRs) clustered at Ca²⁺ release sites on the surface of the endoplasmic reticulum or sarcoplasmic reticulum. When Markov chain models of these intracellular Ca²⁺-regulated Ca²⁺ channels are coupled via a mathematical representation of a Ca²⁺ microdomain, simulated Ca²⁺ release sites may exhibit the phenomenon of “stochastic Ca²⁺ excitability” reminiscent of Ca²⁺ puffs and sparks where channels open and close in a concerted fashion. To clarify the role of Ca²⁺ inactivation of IP₃Rs and RyRs in the dynamics of puffs and sparks, we formulate and analyze Markov chain models of Ca²⁺ release sites composed of 10–40 three-state intracellular Ca²⁺ channels that are inactivated as well as activated by Ca²⁺. We study how the statistics of simulated puffs and sparks depend on the kinetics and dissociation constant of Ca²⁺ inactivation and find that puffs and sparks are often less sensitive to variations in the number of channels at release sites and strength of coupling via local [Ca²⁺] when the average fraction of inactivated channels is significant. Interestingly, we observe that the single channel kinetics of Ca²⁺ inactivation influences the thermodynamic entropy production rate of Markov chain models of puffs and sparks. While excessively fast Ca²⁺ inactivation can preclude puffs and sparks, moderately fast Ca²⁺ inactivation often leads to time-irreversible puffs and sparks whose termination is facilitated by the recruitment of inactivated channels throughout the duration of the puff/spark event. On the other hand, Ca²⁺ inactivation may be an important negative feedback mechanism even when its time constant is much greater than the duration of puffs and sparks. In fact, slow Ca²⁺ inactivation can lead to release sites with a substantial fraction of inactivated channels that exhibit puffs and sparks that are nearly time-reversible and terminate without additional recruitment of inactivated channels.     

Initiation and propagation of a neuronal intracellular calcium wave

The ability to image calcium movement within individual neurons inspires questions of functionality including whether calcium entry into the nucleus is related to genetic regulation for phenomena such as long term potentiation. Calcium waves have been initiated in hippocampal pyramidal cells with glutmatergic signals both in the presence and absence of back propagating action potentials (BPAPs). The dendritic sites of initiation of these calcium waves within about 100 μm of the soma are thought to be localized near oblique junctions. Stimulation of synapses on oblique dendrites leads to production of inositol 1,4,5-trisphosphate (IP₃) which diffuses to the apical dendrite igniting awaiting IP₃ receptors (IP₃Rs) and initiating and propagating catalytic calcium release from the endoplasmic reticulum. We construct a reduced mathematical system which accounts for calcium wave initiation and propagation due to elevated IP₃. Inhomogeneity in IP₃ distribution is responsible for calcium wave initiation versus subthreshold or spatially uniform suprathreshold activation. However, the likelihood that a calcium wave is initiated does not necessarily increase with more calcium entering from BPAPs. For low transient synaptic stimuli, timing between IP₃ generation and BPAPs is critical for calcium wave initiation. We also show that inhomogeneity in IP₃R density can account for calcium wave directionality. Simulating somatic muscarinic receptor production of IP₃, we can account for the critical difference between calcium wave entry into the soma and failure to do so.     

Inositol 1,4,5-Trisphosphate Receptor Subtype-Specific Regulation of Calcium Oscillations

Oscillatory fluctuations in the cytosolic concentration of free calcium ions (Ca²⁺) are considered a ubiquitous mechanism for controlling multiple cellular processes. Inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R) are intracellular Ca²⁺ release channels that mediate Ca²⁺ release from endoplasmic reticulum (ER) Ca²⁺ stores. The three IP₃R subtypes described so far exhibit differential structural, biophysical, and biochemical properties. Subtype specific regulation of IP₃R by the endogenous modulators IP₃, Ca²⁺, protein kinases and associated proteins have been thoroughly examined. In this article we will review the contribution of each IP₃R subtype in shaping cytosolic Ca²⁺ oscillations.     

Glucocorticoids downregulate Fyn and inhibit IP3-mediated calcium signaling to promote autophagy in T lymphocytes

T cell receptor activation induces inositol 1,4,5 trisphosphate (IP₃)-mediated calcium signaling that is essential for cell metabolism and survival. Moreover, inhibitors of IP₃ or pharmacological agents that disrupt calcium homeostasis readily induce autophagy. Using a glucocorticoid-sensitive CD4/CD8 positive T cell line, we found that dexamethasone prevented both IP₃-mediated and spontaneous calcium signals within a timeframe that correlated with the induction of autophagy. We determined that this loss in IP₃-mediated calcium signaling was dependent upon the downregulation of the Src kinase Fyn at the mRNA and protein level. Because it has previously been shown that Fyn positively regulates IP₃-mediated calcium release by phosphorylating Type I IP₃ receptors (IP₃R1), we investigated the effect of glucocorticoids on IP₃R1 phosphorylation at Tyr353. Accordingly, glucocorticoid-mediated downregulation of Fyn prevented IP₃R1 phosphorylation at Tyr353. Moreover, selective knockdown of Fyn or treatment with a Src inhibitor also attenuated IP₃-mediated calcium release and induced autophagy. Collectively, these data indicate that glucocorticoids promote autophagy by inhibiting IP₃-dependent calcium signals. These findings carry important therapeutic implications given the widespread use of dexamethasone as both a chemotherapeutic and immunosuppressive agent.