A model-based method for estimating Ca2+ release fluxes from linescan images in Xenopus oocytes.

ABSTRACT

We propose a model-based method of interpreting linescan images observed in Xenopus oocytes with the use of Oregon Green-1 as a fluorescent dye. We use a detailed modeling formalism based on numerical simulations that incorporate physical barriers for local diffusion, and, by assuming a Gaussian distribution of release durations, we derive the distributions of release Ca(2+) amounts and currents, fluorescence amplitudes, and puff widths. We analyze a wide set of available data collected from 857 and 281 events observed in the animal and the vegetal hemispheres of the oocyte, respectively. A relatively small fraction of events appear to involve coupling of two or three adjacent clusters of Ca(2+) releasing channels. In the animal hemisphere, the distribution of release currents with a mean of 1.4 pA presents a maximum at 1.0 pA and a rather long tail extending up to 5 pA. The overall distribution of liberated Ca(2+) amounts exhibits a dominant peak at 120 fC, a smaller peak at 375 fC, and an average of 166 fC. Ca(2+) amounts and release fluxes in the vegetal hemisphere appear to be 3.6 and 1.6 times smaller than in the animal hemisphere, respectively. Predicted diameters of elemental release sites are approximately 1.0 microm in the animal and approximately 0.5 microm in the vegetal hemisphere, but the side-to-side separation between adjacent sites appears to be identical (approximately 0.4 microm). By fitting the model to individual puffs we can estimate the quantity of liberated calcium, the release current, the orientation of the scan line, and the dimension of the corresponding release site.