Radial localization of inositol 1,4,5-trisphosphate-sensitive Ca2+ releasesites in Xenopus oocytes resolved by axial confocal linescan imaging

The radial localization and properties of elementary calcium release events ("puffs") were studied in Xenopus oocytes using a confocal microscope equi pped with a piezoelectric focussing unit to allow rapid (>100 Hz) imaging o f calcium signals along a radial line into the cell with a spatial resoluti on of <0.7 mu m. Weak photorelease of caged inositol 1,4,5-trisphosphate (I nsP(3)) evoked puffs arising predominantly within a 6-mu m thick band locat ed within a few micrometers of the cell surface. Approximately 25% of puffs had a restricted radial spread, consistent with calcium release from a sin gle site. Most puffs, however, exhibited a greater radial spread (3.25 mu m ), likely involving recruitment of radially neighboring release sites. Calc ium waves evoked by just suprathreshold stimuli exhibited radial calcium di stributions consistent with inward diffusion of calcium liberated at puff s ites, whereas stronger flashes evoked strong, short-latency signals at dept hs inward from puff sites, indicating deep InsP(3)-sensitive stores activat ed at higher concentrations of InsP(3). Immunolocalization of InsP(3) recep tors showed punctate staining throughout a region corresponding to the loca lization of puffs and subplasmalemmal endoplasmic reticulum. The radial org anization of puff sites a few micrometers inward from the plasma membrane m ay have important consequences for activation of calcium-dependent ion chan nels and "capacitative" calcium influx. However, on the macroscopic (hundre ds of micrometers) scale of global calcium waves, release can be considered to occur primarily within a thin, essentially two-dimensional subplasmalem mal shell.