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Figure 10. Cartoon illustrating the distribution of Journal of Physiology Physiology in Press

The liberation of calcium ions sequestered in the endoplasmic reticulum through inositol 1,4,5-trisphosphate receptors/channels (IP(3)Rs) 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(3)Rs 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(3)Rs within the cluster by a regenerative process of Ca(2+)-induced Ca(2+) release. Puff amplitudes (fluorescence ratio change) are on average approximately 6 times greater than that of the triggers, suggesting that at least six IP(3)Rs may simultaneously be open during a puff. Trigger events have average durations of approximately 12 ms, as compared to 19 ms for the mean rise time of puffs, and their spatial extent is approximately 3 times smaller than puffs (respective widths at half peak amplitude 0.6 and 1.6 micro m). All these parameters were relatively independent of IP(3) concentration, although the proportion of puffs showing resolved triggers was greatest (approximately 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(3)Rs within clusters.

SummaryWe 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., 2005 [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 18ms and that the underlying Ca 2+ currents range between 0.12 and 0.95pA. 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, 2006 [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.

2+ puffs following photolysis flashes that were subthreshold in controls, and the spatiotemporal properties of these localized events were differentially modulated by PV and CR. In comparison to results we previously obtained with exogenous Ca 2+ buffers, PV closely mimicked the actions of the slow buffer EGTA, whereas CR showed important differences from the fast buffer BAPTA. Most notably, puffs were never observed after loading BAPTA, and this exogenous buffer did not show the marked sensitization of IP 3 action evident with CR. The ability of Ca 2+ buffers and CaBPs with differing kinetics to fine-tune both global and local intracellular Ca 2+ signals is likely to have significant physiological implications.