‘Trigger’ Events Precede Calcium Puffs in Xenopus Oocytes

Abstract

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.