Superresolution localization of single functional IP3R channels utilizing Ca2+ flux as a readout

The subcellular localization of membrane Ca2+ channels is crucial for their functioning, but is difficult to study because channels may be distributed more closely than the resolution of conventional microscopy is able to detect. We describe a technique, stochastic channel Ca2+ nanoscale resolution (SCCaNR), employing Ca2+-sensitive fluorescent dyes to localize stochastic openings and closings of single Ca2+-permeable channels within <50 nm, and apply it to examine the clustered arrangement of inositol trisphosphate receptor (IP3R) channels underlying local Ca2+ puffs. Fluorescence signals (blips) arising from single functional IP3Rs are almost immotile (diffusion coefficient<0.003 microm2 s(-1)), as are puff sites over prolonged periods, suggesting that the architecture of this signaling system is stable and not subject to rapid, dynamic rearrangement. However, rapid stepwise changes in centroid position of fluorescence are evident within the durations of individual puffs. These apparent movements likely result from asynchronous gating of IP3Rs distributed within clusters that have an overall diameter of approximately 400 nm, indicating that the nanoscale architecture of IP3R clusters is important in shaping local Ca2+ signals. We anticipate that SCCaNR will complement superresolution techniques such as PALM and STORM for studies of Ca2+ channels as it obviates the need for photoswitchable labels and provides functional as well as spatial information.