Puff Amplitudes Research Articles

IP3Rs puff along: A SNAPpy dance with IP3 and Ca2+

Concerted openings of clustered inositol 1,4,5-trisphosphate receptors (IP3Rs) result in short, localized Ca2+ bursts, also called puffs, which are crucial regulators of Ca2+-dependent signaling processes. However, the processes regulating Ca2+ puff amplitude (average ∼0.5 ΔF/F0) and duration (at half-maximal; average ∼25-30ms) have yet to be elucidated. A recent study in JBC by Smith and Taylor determined that Ca2+ puff amplitude is independent of IP3R cluster density and that the termination of IP3R Ca2+ puff is regulated by IP3 dissociation, illuminating the steps of this regulatory dance.

Termination of Ca2+ puffs during IP3-evoked global Ca2+ signals

We previously described that cell-wide cytosolic Ca2+ transients evoked by inositol trisphosphate (IP3) are generated by two modes of Ca2+ liberation from the ER; ‘punctate’ release via an initial flurry of transient Ca2+ puffs from local clusters of IP3 receptors, succeeded by a spatially and temporally ‘diffuse’ Ca2+ liberation. Those findings were derived using statistical fluctuation analysis to monitor puff activity which is otherwise masked as global Ca2+ levels rise. Here, we devised imaging approaches to resolve individual puffs during global Ca2+ elevations to better investigate the mechanisms terminating the puff flurry. We find that puffs contribute about 40% (∼90 attomoles) of the total Ca2+ liberation, largely while the global Ca2+ signal rises halfway to its peak. The major factor terminating punctate Ca2+ release is an abrupt decline in puff frequency. Although the amplitudes of large puffs fall during the flurry, the amplitudes of more numerous small puffs remain steady, so overall puff amplitudes decline only modestly (∼30%). The Ca2+ flux through individual IP3 receptor/channels does not measurably decline during the flurry, or when puff activity is depressed by pharmacological lowering of Ca2+ levels in the ER lumen, indicating that the termination of punctate release is not a simple consequence of reduced driving force for Ca2+ liberation. We propose instead that the gating of IP3 receptors at puff sites is modulated such that their openings become suppressed as the bulk [Ca2+] in the ER lumen falls during global Ca2+ signals.

Suppressing effect of Ca^{2+} blips on puff amplitudes by inhibiting channels to prevent recovery.

As local signals, calcium puffs arise from the concerted opening of a few nearby inositol 1,4,5-trisphospate receptor channels to release Ca^{2+} ions from the endoplasmic reticulum. Although Ca^{2+} puffs have been well studied, little is known about the modulation of cytosolic basal Ca^{2+} concentration ([Ca^{2+}]_{Basal}) on puff dynamics. In this paper we consider a puff model to study how the statistical properties of puffs are modulated by [Ca^{2+}]_{Basal}. The puff frequency and lifetime trivially increase with the increasing [Ca^{2+}]_{Basal}, but an unexpected result is that the puff amplitude and the maximum open-channel number of the puff show decreasing relationship with the increasing [Ca^{2+}]_{Basal}. The underlying dynamics is related not only to the increasing puff frequency which gives a shorter recovery time, but also to the increasing frequency of blips with only one channel open. We indicate that Ca^{2+} blips cause the channels to be inhibited and prevent their recovery during interpuff intervals, resulting in the suppressing effect on puff amplitudes. With increasing [Ca^{2+}]_{Basal}, more blips occur to cause more channels to be inhibited, leaving fewer channels available for puff events. This study shows that the blips may play relevant functions in global Ca^{2+} waves through modulating puff dynamics.

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.

Cytosolic [Ca2+] regulation of InsP3-evoked puffs

InsP3-mediated puffs are fundamental building blocks of cellular Ca2+ signalling, and arise through the concerted opening of clustered InsP3Rs (InsP3 receptors) co-ordinated via Ca2+-induced Ca2+ release. Although the Ca2+ dependency of InsP3Rs has been extensively studied at the single channel level, little is known as to how changes in basal cytosolic [Ca2+] would alter the dynamics of InsP3-evoked Ca2+ signals in intact cells. To explore this question, we expressed Ca2+-permeable channels (nicotinic acetylcholine receptors) in the plasma membrane of voltage-clamped Xenopus oocytes to regulate cytosolic [Ca2+] by changing the electrochemical gradient for extracellular Ca2+ entry, and imaged Ca2+ liberation evoked by photolysis of caged InsP3. Elevation of basal cytosolic [Ca2+] strongly increased the amplitude and shortened the latency of global Ca2+ waves. In oocytes loaded with EGTA to localize Ca2+ signals, the number of sites at which puffs were observed and the frequency and latency of puffs were strongly dependent on cytosolic [Ca2+], whereas puff amplitudes were only weakly affected. The results of the present study indicate that basal cytosolic [Ca2+] strongly affects the triggering of puffs, but has less of an effect on puffs once they have been initiated.

Intracellular calcium signals display an avalanche-like behavior over multiple lengthscales

Many natural phenomena display “self-organized criticality” (SOC), (Bak et al., 1987). This refers to spatially extended systems for which patterns of activity characterized by different lengthscales can occur with a probability density that follows a power law with pattern size. Differently from power laws at phase transitions, systems displaying SOC do not need the tuning of an external parameter. Here we analyze intracellular calcium (Ca2+) signals, a key component of the signaling toolkit of almost any cell type. Ca2+ signals can either be spatially restricted (local) or propagate throughout the cell (global). Different models have suggested that the transition from local to global signals is similar to that of directed percolation. Directed percolation has been associated, in turn, to the appearance of SOC. In this paper we discuss these issues within the framework of simple models of Ca2+ signal propagation. We also analyze the size distribution of local signals (“puffs”) observed in immature Xenopus Laevis oocytes. The puff amplitude distribution obtained from observed local signals is not Gaussian with a noticeable fraction of large size events. The experimental distribution of puff areas in the spatio-temporal record of the image has a long tail that is approximately log-normal. The distribution can also be fitted with a power law relationship albeit with a smaller goodness of fit. The power law behavior is encountered within a simple model that includes some coupling among individual signals for a wide range of parameter values. An analysis of the model shows that a global elevation of the Ca2+ concentration plays a major role in determining whether the puff size distribution is long-tailed or not. This suggests that Ca2+-clearing from the cytosol is key to determine whether IP3-mediated Ca2+ signals can display a SOC-like behavior or not.

Quantifying calcium fluxes underlying calcium puffs in Xenopus laevis oocytes

We determine the calcium fluxes through inositol 1,4,5-trisphosphate receptor/channels underlying calcium puffs of Xenopus laevis oocytes using a simplified version of the algorithm of Ventura et al. [1]. An analysis of 130 puffs obtained with Fluo-4 indicates that Ca 2+ release comes from a region of width ∼450 nm, that the release duration is peaked around 18 ms and that the underlying Ca 2+ currents range between 0.12 and 0.95 pA. All these parameters are independent of IP 3 concentration. We explore what distributions of channels that open during a puff, N p , and what relations between current and number of open channels, I( N p ), are compatible with our findings and with the distribution of puff-to-trigger amplitude ratio reported in Rose et al. [2]. To this end, we use simple “mean field” models in which all channels open and close simultaneously. We find that the variability among clusters plays an important role in shaping the observed puff amplitude distribution and that a model for which I( N p ) ∼ N p for small N p and I ( N p ) ∼ N p 1 / α ( α > 1) for large N p , provides the best agreement. Simulations of more detailed models in which channels open and close stochastically show that this nonlinear behavior can be attributed to the limited time resolution of the observations and to the averaging procedure that is implicit in the mean-field models. These conclusions are also compatible with observations of ∼400 puffs obtained using the dye Oregon green.

A simple sequential-binding model for calcium puffs

Calcium puffs describe the transient release of Ca(2+) ions into the cytosol, through small clusters of 1,4,5-inositol triphosphate (IP(3)) receptors, present on internal stores such as the endoplasmic reticulum. Statistical properties of puffs, such as puff amplitudes and durations, have been well characterized experimentally. We model calcium puffs using a simple, sequential-binding model for the IP(3) receptor in conjunction with a computationally inexpensive point-source approximation. We follow two different protocols, a sequential protocol and a renewal protocol. In the sequential protocol, puffs are generated successively by the same cluster; in the renewal protocol, the system is reset after each puff. In both cases for a single set of parameters our results are in excellent agreement with experimental results for puff amplitudes and durations but indicate puff-to-puff correlations for the sequential protocol, consistent with recent experimental findings [H. J. Rose, S. Dargan, J. W. Shuai, and I. Parker, Biophys. J. 91, 4024 (2006)]. The model is then used to test the consistency of the hypothesized steep Ca(2+) gradients around single channels with the experimentally observed features of puff durations and amplitudes. A three-dimensional implementation of our point-source model suggests that a peak Ca(2+) concentration of the order of 10 muM at the cluster site (not channel) is consistent with the statistical features of observed calcium puffs.

Localization of puff sites adjacent to the plasma membrane: Functional and spatial characterization of Ca 2+ signaling in SH-SY5Y cells utilizing membrane-permeant caged IP 3

The Xenopus oocyte has been a favored model system in which to study spatio-temporal mechanisms of intracellular Ca2+ dynamics, in large part because this giant cell facilitates intracellular injections of Ca2+ indicator dyes, buffers and caged compounds. However, the recent commercial availability of membrane-permeant ester forms of caged IP3 (ci-IP3) and EGTA, now allows for facile loading of these compounds into smaller mammalian cells, permitting control of [IP3]i and cytosolic Ca2+ buffering. Here, we establish the human neuroblastoma SH-SY5Y cell line as an advantageous experimental system for imaging Ca2+ signaling, and characterize IP3-mediated Ca2+ signaling mechanisms in these cells. Flash photo-release of increasing amounts of i-IP3 evokes Ca2+ puffs that transition to waves, but intracellular loading of EGTA decouples release sites, allowing discrete puffs to be studied over a wide range of [IP3]. Puff activity persists for minutes following a single photo-release, pointing to a slow rate of i-IP3 turnover in these cells and suggesting that repetitive Ca2+ spikes with periods of 20-30s are not driven by oscillations in [IP3]. Puff amplitudes are independent of [IP3], whereas their frequencies increase with increasing photo-release. Puff sites in SH-SY5Y cells are not preferentially localized near the nucleus, but instead are concentrated close to the plasma membrane where they can be visualized by total internal reflection microscopy, offering the potential for unprecedented spatio-temporal resolution of Ca2+ puff kinetics.

Multi-dimensional resolution of elementary Ca 2+ signals by simultaneous multi-focal imaging

Elementary events such as puffs and sparks are cytosolic microdomains of Ca 2+ from which cellular Ca 2+ signals are constructed. Because of the tight localization and fast kinetics of elementary events, imaging studies have been hindered by instrumental limitations of confocal and deconvolution fluorescence microscopy which necessitate compromises between spatial and temporal resolution. Here, we describe a novel, yet simple ‘multi-focal’ fluorescence microscopy system that employs three high-speed cameras focused at different axial depths to enable 4-dimensional imaging with millisecond resolution. We demonstrate the utility of this system for studies of puffs in Xenopus oocytes by mapping the axial distribution of puff sites, by obtaining measurements of puff amplitudes undistorted by focus error, and by deriving deblurred images that reveal novel sub-micron jumps of Ca 2+ release sites.

‘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 (IP 3Rs) results in a spatiotemporal hierarchy of calcium signaling events that range from single-channel openings to local Ca 2+ puffs believed to arise from several to tens of clustered IP 3Rs to global calcium waves. Using high-resolution confocal linescan imaging and a sensitive Ca 2+ indicator dye (fluo-4-dextran), we show that puffs are often preceded by small, transient Ca 2+ elevations that we christen “trigger events”. The magnitude of triggers is consistent with their arising from the opening of a single IP 3 receptor/channel, and we propose that they initiate puffs by recruiting neighboring IP 3Rs within the cluster by a regenerative process of Ca 2+-induced Ca 2+ release. Puff amplitudes (fluorescence ratio change) are on average ∼6 times greater than that of the triggers, suggesting that at least six IP 3Rs 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 IP 3 concentration, although the proportion of puffs showing resolved triggers was greatest (∼80%) at low [IP 3]. Because Ca 2+ puffs constitute the building blocks from which cellular IP 3-mediated Ca 2+ 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 IP 3Rs within clusters.

Analysis of Puff Dynamics in Oocytes: Interdependence of Puff Amplitude and Interpuff Interval

Puffs are localized Ca 2+ signals that arise in oocytes in response to inositol 1,4,5-trisphosphate (IP 3). They are analogous to the sparks of myocytes and are believed to be the result of the liberation of Ca 2+ from the endoplasmic reticulum through the coordinated opening of IP 3 receptor/channels clustered at a functional release site. In this article, we analyze sequences of puffs that occur at the same site to help elucidate the mechanisms underlying puff dynamics. In particular, we show a dependence of the interpuff time on the amplitude of the preceding puff, and of the amplitude of the following puff on the preceding interval. These relationships can be accounted for by an inhibitory role of the Ca 2+ that is liberated during puffs. We construct a stochastic model for a cluster of IP 3 receptor/channels that quantitatively replicates the observed behavior, and we determine that the characteristic time for a channel to escape from the inhibitory state is of the order of seconds.

Stochastic Properties of Ca2+ Release of Inositol 1,4,5-Trisphosphate Receptor Clusters

Intracellular Ca2+ release is controlled by inositol 1,4,5-trisphosphate (IP3) receptors or ryanodine receptors. These receptors are typically distributed in clusters with several or tens of channels. The random opening and closing of these channels introduces stochasticity into the elementary calcium release mechanism. Stochastic release events have been experimentally observed in a variety of cell types and have been termed sparks and puffs. We put forward a stochastic version of the Li-Rinzel model (the deactivation binding process is described by a Markovian scheme) and a computationally more efficient Langevin approach to model the stochastic Ca2+ oscillation of single clusters. Statistical properties such as Ca2+ puff amplitudes, lifetimes, and interpuff intervals are studied with both models and compared with experimental observations. For clusters with tens of channels, a simply decaying amplitude distribution is typically observed at low IP3 concentration, while a single peak distribution appears at high IP3 concentration.

Ca2+ transients associated with openings of inositol trisphosphate-gated channels in Xenopus oocytes.

1. The mechanisms underlying inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ liberation were studied in Xenopus oocytes by using scanning and stationary-point confocal fluorescence microscopy to record Ca2+ signals evoked by photorelease of InsP3 from a caged precursor. 2. Fluorescence measurements from confocal images showed that increasing [InsP3] evoked three distinct modes of Ca2+ liberation: a diffuse 'pacemaker' signal, localized transient puffs, and propagating waves. Peak free Ca2+ concentrations during waves and puffs (respectively, 2-5 microM and 100-200 nM) varied only slightly with [InsP3], whereas the pacemaker amplitude varied over a wider range (at least 1-30 nM Ca2+). 3. The improved resolution provided by confocal point recording revealed discontinuous Ca2+ 'blips' during pacemaker release. These events were resolved only at particular locations and had time courses similar to the puffs (rise, approximately 50 ms; decay, a few hundred milliseconds) but with amplitudes one-fifth or less of puff amplitudes. 4. We conclude that blips may arise through opening of single InsP3-gated channels, whereas puffs reflect the concerted opening of several clustered channels due to local regenerative feedback by Ca2+.