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)P₂ for STIM1–SARAF interaction. Targeting STIM1 to PI(4,5)P₂-rich and -poor microdomains reveals that SARAF-dependent SCDI is observed only when STIM1–Orai1 are within the PI(4,5)P₂-rich microdomain. Notably, store depletion results in transient localization of STIM1–Orai1 in the PI(4,5)P₂-poor microdomain, which then translocates to the PI(4,5)P₂-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)P₂ involving translocation between PI(4,5)P₂ microdomains.