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

Abstract

The radial localization and properties of elementary calcium release events ('puffs') were studied in Xenopus oocytes using a confocal microscope equipped with a piezoelectric focussing unit to allow rapid (> 100 Hz) imaging of calcium signals along a radial line into the cell with a spatial resolution of <0.7 μm. Weak photo-release of caged inositol 1,4,5- trisphosphate (InsP3) evoked puffs arising predominantly within a 6-μ thick band located within a few micrometers of the cell surface. Approximately 25% of puffs had a restricted radial spread, consistent with calcium release from a single site. Most puffs, however, exhibited a greater radial spread (3.25 μm), likely involving recruitment of radially neighboring release sites. Calcium waves evoked by just suprathreshold stimuli exhibited radial calcium distributions consistent with inward diffusion of calcium liberated at puff sites, whereas stronger flashes evoked strong, short-latency signals at depths inward from puff sites, indicating deep InsP3-sensitive stores activated at higher concentrations of InsP3. Immunolocalization of InsP3 receptors showed punctate staining throughout a region corresponding to the localization of puffs and subplasmalemmal endoplasmic reticulum. The radial organization of puff sites a few micrometers inward from the plasma membrane may have important consequences for activation of calcium-dependent ion channels and 'capacitative' calcium influx. However, on the macroscopic (hundreds of micrometers) scale of global calcium waves, release can be considered to occur primarily within a thin, essentially two-dimensional subplasmalemmal shell.