Translocation between PI(4,5)P2-poor and PI(4,5)P2-rich microdomains during store depletion determines STIM1 conformation and Orai1 gating

The Orai1-STIM1 current undergoes slow Ca2+-dependent inactivation (SCDI) mediated by the binding of SARAF to STIM1. Here we report the use of SCDI by SARAF as a probe of the conformation and microdomain localization of the Orai1-STIM1 complex. We find that the interaction of STIM1 with Orai1 carboxyl terminus (C terminus) and the STIM1 K-domain are required for the interaction of SARAF with STIM1 and SCDI. STIM1-Orai1 must be in a PM/ER microdomain tethered by E-Syt1, stabilized by septin4 and enriched in PI(4,5)P-2 for STIM1-SARAF interaction. Targeting STIM1 to PI(4,5)P-2-rich and -poor microdomains reveals that SARAF-dependent SCDI is observed only when STIM1-Orai1 are within the PI(4,5)P-2-rich microdomain. Notably, store depletion results in transient localization of STIM1-Orai1 in the PI(4,5)P-2-poor microdomain, which then translocates to the PI(4,5)P-2-rich domain. These findings reveal the role of PM/ER tethers in the regulation of Orai1 function and a mode of regulation by PI(4,5)P-2 involving translocation between PI(4,5)P-2 microdomains.