N. Callamaras et I. Parker, Radial localization of inositol 1,4,5-trisphosphate-sensitive Ca2+ releasesites in Xenopus oocytes resolved by axial confocal linescan imaging, J GEN PHYSL, 113(2), 1999, pp. 199-213
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.