Ca2+ Puffs Originate from Preestablished Stable Clusters of Inositol Trisphosphate Receptors

Intracellular calcium ion (Ca2+) signaling crucially depends on the clustered organization of inositol trisphosphate receptors (IP(3)Rs) in the endoplasmic reticulum (ER) membrane. These ligand-gated ion channels liberate Ca2+ to generate local signals known as Ca2+ puffs. We tested the hypothesis that IP3 itself elicits rapid clustering of IP(3)Rs by using flash photolysis of caged IP3 in conjunction with high-resolution Ca2+ imaging to monitor the activity and localization of individual IP(3)Rs within intact mammalian cells. Our results indicate that Ca2+ puffs arising with latencies as short as 100 to 200 ms after photorelease of IP3 already involve at least four IP3R channels, and that this number does not subsequently grow. Moreover, single active IP(3)Rs show limited mobility, and stochastic simulations suggest that aggregation of IP(3)Rs at puff sites by a diffusional trapping mechanism would require many seconds. We thus conclude that puff sites represent preestablished, stable clusters of IP(3)Rs and that functional IP(3)Rs are not readily diffusible within the ER membrane.