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

IP 3 receptors (IP 3 Rs) release Ca 2+ from the ER when they bind IP 3 and Ca 2+ . The spatial organization of IP 3 Rs determines both the propagation of Ca 2+ 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 Ca 2+ signals within living cells. We show that native IP 3 Rs cluster within ER membranes. Most IP 3 R clusters are mobile, moved by diffusion and microtubule motors. Ca 2+ 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 Ca 2+ entry, accumulates after depletion of Ca 2+ stores. IP 3 Rs tethered close to ER-plasma membrane junctions are licensed to respond and optimally placed to be activated by endogenous IP 3 and to regulate Ca 2+ entry.