Ca(2+) signals initiate at immobile IP(3) 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.