Oligomerization of STIM1 couples ER calcium depletion to CRAC channel activation

Ca²⁺-release-activated Ca²⁺ (CRAC) channels generate sustained Ca²⁺ signals that are essential for a range of cell functions, including antigen-stimulated T lymphocyte activation and proliferation. Recent studies have revealed that the depletion of Ca ²⁺ from the endoplasmic reticulum (ER) triggers the oligomerization of stromal interaction molecule 1 (STIM1), the ER Ca²⁺ sensor, and its redistribution to ER-plasma membrane (ER-PM) junctions where the CRAC channel subunit ORAI1 accumulates in the plasma membrane and CRAC channels open. However, how the loss of ER Ca²⁺ sets into motion these coordinated molecular rearrangements remains unclear. Here we define the relationships among [Ca²⁺]_ER, STIM1 redistribution and CRAC channel activation and identify STIM1 oligomerization as the critical [Ca ²⁺]_ER-dependent event that drives store-operated Ca ²⁺ entry. In human Jurkat leukaemic T cells expressing an ER-targeted Ca²⁺ indicator, CRAC channel activation and STIM1 redistribution follow the same function of [Ca²⁺]_ER, reaching half-maximum at ∼200 μM with a Hill coefficient of ∼4. Because STIM1 binds only a single Ca²⁺ ion, the high apparent cooperativity suggests that STIM1 must first oligomerize to enable its accumulation at ER-PM junctions. To assess directly the causal role of STIM1 oligomerization in store-operated Ca²⁺ entry, we replaced the luminal Ca ²⁺-sensing domain of STIM1 with the 12-kDa FK506- and rapamycin-binding protein (FKBP12, also known as FKBP1A) or the FKBP-rapamycin binding (FRB) domain of the mammalian target of rapamycin (mTOR, also known as FRAP1). A rapamycin analogue oligomerizes the fusion proteins and causes them to accumulate at ER-PM junctions and activate CRAC channels without depleting Ca²⁺ from the ER. Thus, STIM1 oligomerization is the critical transduction event through which Ca²⁺ store depletion controls store-operated Ca²⁺ entry, acting as a switch that triggers the self-organization and activation of STIM1-ORAI1 clusters at ER-PM junctions.