Abstract
Store-operated Ca(2+) entry, essential for the adaptive immunity, is initiated by the endoplasmic reticulum (ER) Ca(2+) sensor STIM1. Ca(2+) entry occurs through the plasma membrane resident Ca(2+) channel Orai1 that directly interacts with the C-terminal STIM1 domain, named SOAR/CAD. Depletion of the ER Ca(2+) store controls this STIM1/Orai1 interaction via transition to an extended STIM1 C-terminal conformation, exposure of the SOAR/CAD domain, and STIM1/Orai1 co-clustering. Here we developed a novel approach termed FRET-derived Interaction in a Restricted Environment (FIRE) in an attempt to dissect the interplay of coiled-coil (CC) interactions in controlling STIM1 quiescent as well as active conformation and cluster formation. We present evidence of a sequential activation mechanism in the STIM1 cytosolic domains where the interaction between CC1 and CC3 segment regulates both SOAR/CAD exposure and CC3-mediated higher-order oligomerization as well as cluster formation. These dual levels of STIM1 auto-inhibition provide efficient control over the coupling to and activation of Orai1 channels.
Highlights
stromal interaction molecule 1 (STIM1) and Orai1 are key players in the store-operated Ca2ϩ entry
To validate the activation competence of the Y-TMG-CAD construct, with respect to the missing CC1, we co-expressed it with CFP (C)-Orai1 to functionally address the specificity of the endoplasmic reticulum (ER)-plasma membrane (PM) spacer function, proposed for CC1, in whole-cell patch-clamp recordings
The current density reached was in a similar range as that of Y-TMG-OASF (Fig. 2A) indicating that the absence of CC1 as a specific ER-PM spacer in Y-TMG-CAD did not interfere with its competence of fully activating Orai1 currents
Summary
STIM1 and Orai are key players in the store-operated Ca2ϩ entry. Results: The activation state of STIM1 is precisely controlled by heteromeric interaction between coiled-coil domains. We present evidence of a sequential activation mechanism in the STIM1 cytosolic domains where the interaction between CC1 and CC3 segment regulates both SOAR/CAD exposure and CC3-mediated higher-order oligomerization as well as cluster formation. These dual levels of STIM1 auto-inhibition provide efficient control over the coupling to and activation of Orai channels. We hypothesized that the CC3 domain of STIM1 provides homomerization function to arrange six STIM1 molecules in a higher-order oligomeric cluster within the Orai channel complex It is not known, whether these conformational rearrangements take place in live cells, and which additional, unidentified interactions they involve to tightly control both CAD/SOAR exposure and clustering in the STIM1 activation mechanism. We present a sequential, C-terminal switching mechanism providing dual levels of STIM1 auto-inhibition by linking CAD/SOAR exposure with cluster formation required for Orai channel coupling
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