The Number and Spatial Distribution of IP 3 Receptors Underlying Calcium Puffs in Xenopus Oocytes

Abstract

Calcium puffs are local Ca 2+ release events that arise from a cluster of inositol 1,4,5-trisphosphate receptor channels (IP 3Rs) and serve as a basic “building block” from which global Ca 2+ waves are generated. Important questions remain as to the number of IP 3Rs that open during a puff, their spatial distribution within a cluster, and how much Ca 2+ current flows through each channel. The recent discovery of “trigger” events—small Ca 2+ signals that immediately precede puffs and are interpreted to arise through opening of single IP 3R channels—now provides a useful yardstick by which to calibrate the Ca 2+ flux underlying puffs. Here, we describe a deterministic numerical model to simulate puffs and trigger events. Based on confocal linescan imaging in Xenopus oocytes, we simulated Ca 2+ release in two sequential stages; representing the trigger by the opening of a single IP 3R in the center of a cluster for 12 ms, followed by the concerted opening of some number of IP 3Rs for 19 ms, representing the rising phase of the puff. The diffusion of Ca 2+ and Ca 2+-bound indicator dye were modeled in a three-dimensional cytosolic volume in the presence of immobile and mobile Ca 2+ buffers, and were used to predict the observed fluorescence signal after blurring by the microscope point-spread function. Optimal correspondence with experimental measurements of puff spatial width and puff/trigger amplitude ratio was obtained assuming that puffs arise from the synchronous opening of 25–35 IP 3Rs, each carrying a Ca 2+ current of ∼0.4 pA, with the channels distributed through a cluster 300–800 nm in diameter.