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+)](ER) levels 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.
Highlights
STIM1 oligomerization upon endoplasmic reticulum (ER) Ca2ϩ depletion activates store-operated Ca2ϩ entry (SOCE) channels, but whether this mechanism is reversible is unknown
These data indicate that the refilling of ER Ca2؉ stores is not sufficient to induce STIM1 de-oligomerization and that localized Ca2؉ elevations in the vicinity of assembled SOCE complexes are required for the termination of SOCE
We show that during the activating phase of SOCE, [Ca2ϩ]ER levels are inversely correlated with the degree the mCherry fluorescence Distance along the 39-m cluster
Summary
STIM1 oligomerization upon endoplasmic reticulum (ER) Ca2ϩ depletion activates store-operated Ca2ϩ entry (SOCE) channels, but whether this mechanism is reversible is unknown. The high free Ca2ϩ concentration within the ER lumen ([Ca2ϩ]ER) of 300 – 600 M [22] stabilizes the protein in an inactive state via intra-molecular noglobulin protein; [Ca2ϩ]Cyt, cytosolic [Ca2ϩ]; [Ca2ϩ]ER, endoplasmic reticulum [Ca2ϩ]; CAD, CRAC activation domain; CRAC, Ca2ϩ-release activated Ca2ϩ current; ER, endoplasmic reticulum; PM, plasma membrane; SAM, sterile ␣ motif; SERCA, sarcoendoplasmic reticulum Ca2ϩ-ATPase; STIM, stromal interaction molecule; TG, thapsigargin; TIRF, total internal reflection fluorescence. In addition to ER Ca2ϩ refilling, a localized cytosolic Ca2ϩ elevation is required for the dissociation of STIM1 complexes and their retrieval from the plasma membrane
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