Ca2+ signals initiate at immobile IP3 receptors adjacent to ER-plasma membrane junctions

IP3 receptors (IP(3)Rs) release Ca2+ from the ER when they bind IP3 and Ca2+. The spatial organization of IP(3)Rs determines both the propagation of Ca2+ signals between IP(3)Rs and the selective regulation of cellular responses. Here we use gene editing to fluorescently tag endogenous IP(3)Rs, and super-resolution microscopy to determine the geography of IP(3)Rs and Ca2+ signals within living cells. We show that native IP(3)Rs cluster within ER membranes. Most IP3R clusters are mobile, moved by diffusion and microtubule motors. Ca2+ signals are generated by a small population of immobile IP(3)Rs. These IP(3)Rs are licensed to respond, but they do not readily mix with mobile IP(3)Rs. The licensed IP(3)Rs reside alongside ER-plasma membrane junctions where STIM1, which regulates store-operated Ca2+ entry, accumulates after depletion of Ca2+ stores. IP(3)Rs tethered close to ER-plasma membrane junctions are licensed to respond and optimally placed to be activated by endogenous IP3 and to regulate Ca2+ entry.