Single-Molecule Tracking of Inositol Trisphosphate Receptors Reveals Different Motilities and Distributions

Puffs are local Ca(2+) signals that arise by Ca(2+) liberation from the endoplasmic reticulum through the concerted opening of tightly clustered inositol trisphosphate receptors/channels (IP3Rs). The locations of puff sites observed by Ca(2+) imaging remain static over several minutes, whereas fluorescence recovery after photobleaching (FRAP) experiments employing overexpression of fluorescently tagged IP3Rs have shown that the majority of IP3Rs are freely motile. To address this discrepancy, we applied single-molecule imaging to locate and track type 1 IP3Rs tagged with a photoswitchable fluorescent protein and expressed in COS-7 cells. We found that ∼ 70% of the IP3R1 molecules were freely motile, undergoing random walk motility with an apparent diffusion coefficient of ∼ 0.095 μm s(-1), whereas the remaining molecules were essentially immotile. A fraction of the immotile IP3Rs were organized in clusters, with dimensions (a few hundred nanometers across) comparable to those previously estimated for the IP3R clusters underlying functional puff sites. No short-term (seconds) changes in overall motility or in clustering of immotile IP3Rs were apparent following activation of IP3/Ca(2+) signaling. We conclude that stable clusters of small numbers of immotile IP3Rs may underlie local Ca(2+) release sites, whereas the more numerous motile IP3Rs appear to be functionally silent.