Functional communication between IP₃R and STIM2 at subthreshold stimuli is a critical checkpoint for initiation of...

Stromal interaction molecules, STIM1 and STIM2, sense decreases in the endoplasmic reticulum (ER) [Ca²⁺] ([Ca²⁺]_ER) and cluster in ER-plasma membrane (ER-PM) junctions where they recruit and activate Orai1. While STIM1 responds when [Ca²⁺]_ER is relatively low, STIM2 displays constitutive clustering in the junctions and is suggested to regulate basal Ca²⁺ entry. The cellular cues that determine STIM2 clustering under basal conditions is not known. By using gene editing to fluorescently tag endogenous STIM2, we report that endogenous STIM2 is constitutively localized in mobile and immobile clusters. The latter associate with ER-PM junctions and recruit Orai1 under basal conditions. Agonist stimulation increases immobile STIM2 clusters, which coordinate recruitment of Orai1 and STIM1 to the junctions. Extended synaptotagmin (E-Syt)2/3 are required for forming the ER-PM junctions, but are not sufficient for STIM2 clustering. Importantly, inositol 1,4,5-triphosphate receptor (IP₃R) function and local [Ca²⁺]_ER are the main drivers of immobile STIM2 clusters. Enhancing, or decreasing, IP₃R function at ambient [IP₃] causes corresponding increase, or attenuation, of immobile STIM2 clusters. We show that immobile STIM2 clusters denote decreases in local [Ca²⁺]_ER mediated by IP₃R that is sensed by the STIM2 N terminus. Finally, under basal conditions, ambient PIP₂-PLC activity of the cell determines IP₃R function, immobilization of STIM2, and basal Ca²⁺ entry while agonist stimulation augments these processes. Together, our findings reveal that immobilization of STIM2 clusters within ER-PM junctions, a first response to ER-Ca²⁺ store depletion, is facilitated by the juxtaposition of IP₃R and marks a checkpoint for initiation of Ca²⁺ entry.