1. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, USA
2. These authors contributed equally to this work.
3. Present addresses: Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, USA (B.W.); Department of Molecular Pharmacology and Biological Chemistry, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA (M.P.).

Correspondence to: Richard S. Lewis¹ Correspondence and requests for materials should be addressed to R.S.L. (Email: rslewis@stanford.edu).

Abstract

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^{1, 2}. Recent studies³ have revealed that the depletion of Ca²⁺ 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^{4, 5, 6, 7, 8} where the CRAC channel subunit ORAI1 accumulates in the plasma membrane and CRAC channels open^{9, 10, 11, 12}. 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 [Ca²⁺]_ER-dependent event that drives store-operated Ca²⁺ 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 M with a Hill coefficient of 4. Because STIM1 binds only a single Ca²⁺ ion⁵, 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 Ca²⁺-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.

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