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

Puffs are local Ca²⁺ signals that arise by Ca²⁺ liberation from the endoplasmic reticulum through the concerted opening of tightly clustered inositol trisphosphate receptors/channels (IP₃Rs). The locations of puff sites observed by Ca²⁺ 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/Ca²⁺ signaling. We conclude that stable clusters of small numbers of immotile IP₃Rs may underlie local Ca²⁺ release sites, whereas the more numerous motile IP₃Rs appear to be functionally silent.