‘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(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.

Mitochondrial Ca2+ Uptake Increases Ca2+ Release from Inositol 1,4,5-Trisphosphate Receptor Clusters in Smooth Muscle Cells

“…Above 500 M EGTA significantly attenuated the amplitude of Ca 2ϩ puffs and, in some cells, they could not be evoked. EGTA (300 M) at a concentration similar to that used in experiments examining Ca 2ϩ puffs in Xenopus oocytes was used in the present study to enable Ca 2ϩ puffs to be evoked and measured (27,36). EGTA (300 M) neither affects the magnitude nor slows the kinetics of Ca 2ϩ puffs (27,37).…”

`Quantal' Ca2+ release at the cytoplasmic aspect of the Ins(1,4,5)P3R channel in smooth muscle

“…amplitude) of several Ins(1,4,5)P 3 -mediated Ca 2+ signals, such as puffs, waves and spikes. For example, the transition of a signal from a 'blip' to a 'puff' or from a 'puff' to a wave might each require local feedback in a CICR-like process; in the former case to recruit within individual clustered Ins(1,4,5)P 3 Rs (Bootman et al, 1997a;Rose et al, 2006) and, in the latter, to recruit among clusters (Bootman et al, 1997a;MacMillan et al, 2005;McCarron et al, 2004).…”

Imaging the quantal substructure of single IP 3 R channel activity during Ca 2+ puffs in intact mammalian cells

“…Local, ''elementary'' Ca 2ϩ transients (i.e., Ca 2ϩ puffs) are generated by the concerted openings of multiple IP 3 Rs within a cluster, and serve autonomous signaling functions as well as constituting the building blocks from which global cellular Ca 2ϩ waves are constructed (2)(3)(4)(5)(6). In turn, puffs are composed from ''fundamental'' signals (i.e., Ca 2ϩ blips), representing Ca 2ϩ flux through individual IP 3 Rs (7)(8)(9)(10). Information concerning the spatial localization and numbers of IP 3 Rs involved in a puff, and the mechanisms and kinetics by which their activity is coordinated, is essential to understand how these channels act to initiate and terminate local Ca 2ϩ liberation (11).…”