Local cytosolic Ca 2+ elevations are required for stromal interaction molecule 1 (STIM1) de-oligomerization and termination of store-operated Ca 2+ entry

Abstract

The Ca 2+ depletion of the endoplasmic reticulum (ER) activates the ubiquitous store-operated Ca 2+ entry (SOCE) pathway that sustains long-term Ca 2+ signals critical for cellular functions. ER Ca 2+ depletion initiates the oligomerization of stromal interaction molecules (STIM) that control SOCE activation, but whether ER Ca 2+ refilling controls STIM de-oligomerization and SOCE termination is not known. Here, we correlate the changes in free luminal ER Ca 2+ concentrations ([Ca 2+] ER) and in STIM1 oligomerization, using fluorescence resonance energy transfer (FRET) between CFP-STIM1 and YFP-STIM1. We observed that STIM1 de-oligomerized at much lower [Ca 2+] ERlevels during store refilling than it oligomerized during store depletion. We then refilled ER stores without adding exogenous Ca 2+ using a membrane-permeable Ca 2+ chelator to provide a large reservoir of buffered Ca 2+. This procedure rapidly restored pre-stimulatory [Ca 2+] ER levels but did not trigger STIM1 de-oligomerization, the FRET signals remaining elevated as long as the external [Ca 2+] remained low. STIM1 dissociation evoked by Ca 2+ readmission was prevented by SOC channel inhibition and was associated with cytosolic Ca 2+ elevations restricted to STIM1 puncta, indicating that Ca 2+ acts on a cytosolic target close to STIM1 clusters. These data indicate that the refilling of ER Ca 2+ stores is not sufficient to induce STIM1 de-oligomerization and that localized Ca 2+ elevations in the vicinity of assembled SOCE complexes are required for the termination of SOCE. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.