‘Trigger’ Events Precede Calcium Puffs in Xenopus Oocytes

AbstractThe 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 ∼12ms, as compared to 19ms 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 1.6μm). 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