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

IP₃ receptors (IP₃Rs) release Ca²⁺ from the ER when they bind IP₃ and Ca²⁺. The spatial organization of IP₃Rs determines both the propagation of Ca²⁺ signals between IP₃Rs and the selective regulation of cellular responses. Here we use gene editing to fluorescently tag endogenous IP₃Rs, and super-resolution microscopy to determine the geography of IP₃Rs and Ca²⁺ signals within living cells. We show that native IP₃Rs 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 IP₃Rs. These IP₃Rs are licensed to respond, but they do not readily mix with mobile IP₃Rs. The licensed IP₃Rs reside alongside ER-plasma membrane junctions where STIM1, which regulates store-operated Ca²⁺ entry, accumulates after depletion of Ca²⁺ stores. IP₃Rs 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.