A CONTINUUM OF INSP(3)-MEDIATED ELEMENTARY CA2+ SIGNALING EVENTS IN XENOPUS OOCYTES

1. The elementary release events underlying inositol 1,4,5-trisphospha te (InsP(3))-mediated calcium signalling were investigated in Xenopus oocytes by means of high-resolution confocal linescan imaging together with flash photolysis of caged InsP(3). 2. Weak photolysis flashes ev oked localized, transient calcium signals that arose at specific sites following random latencies of up to several seconds. The duration, sp atial spread and amplitude of these elementary events varied widely. E vent durations (at half-maximal amplitude) were distributed exponentia lly between about 100 and 600 ms. Fluorescence magnitudes (F/F-0 of Or egon Green 488 BAPTA-1) showed a skewed distribution with a peak at ab out 1.5 and a tail extending as high as 3.5. 3. Individual release sit es exhibited both small events (blips) and large events (puffs). The s patiotemporal distribution of calcium signals during puffs was consist ent with calcium diffusion from a point source (( a few hundred nanome tres), rather than with propagation of a microscopic calcium wave. 4. Estimates of the calcium flux associated with individual events were m ade by integrating fluorescence profiles along the scan line in three dimensions to derive the 'signal mass' at each time point. The smalles t resolved events corresponded to liberation of < 2 x 10(-20) mol Ca2, and large events to about 2 x 10(-18) mol Ca2+. The rise of signal m ass was more prolonged than that of the fluorescence intensity, sugges ting that calcium liberation persists even while the fluorescence begi ns to decline. Rates of rise of signal mass corresponded to Ca2+ curre nts of 0.4-2.5 pA. 5. Measurements of signal mass from different event s showed a continuous, exponential distribution, arising through varia bility in magnitude and duration of calcium flux. 6. We conclude that localized calcium transients in the oocyte represent a continuum of ev ents involving widely varying amounts of calcium liberation, rather th an falling into separate populations of 'fundamental' and 'elementary' events (blips and puffs) involving, respectively, single and multiple InsP(3) receptor channels. This variability probably arises through s tochastic variation in both the number of channels recruited and the d uration of channel opening.