Xizhuo Wang

Distinct Orai-coupling domains in STIM1 and STIM2 define the Orai-activating site

STIM1 and STIM2 are widely expressed endoplasmic reticulum (ER) Ca(2+) sensor proteins able to translocate within the ER membrane to physically couple with and gate plasma membrane Orai Ca(2+) channels. Although they are structurally similar, we reveal critical differences in the function of the short STIM-Orai-activating regions (SOAR) of STIM1 and STIM2. We narrow these differences in Orai1 gating to a strategically exposed phenylalanine residue (Phe-394) in SOAR1, which in SOAR2 is substituted by a leucine residue. Remarkably, in full-length STIM1, replacement of Phe-394 with the dimensionally similar but polar histidine head group prevents both Orai1 binding and gating, creating an Orai1 non-agonist. Thus, this residue is critical in tuning the efficacy of Orai activation. While STIM1 is a full Orai1-agonist, leucine-replacement of this crucial residue in STIM2 endows it with partial agonist properties, which may be critical for limiting Orai1 activation stemming from its enhanced sensitivity to store-depletion.

STIM1 dimers undergo unimolecular coupling to activate Orai1 channels

The endoplasmic reticulum (ER) Ca2+ sensor, STIM1, becomes activated when ER-stored Ca2+ is depleted and translocates into ER–plasma membrane junctions where it tethers and activates Orai1 Ca2+ entry channels. The dimeric STIM1 protein contains a small STIM-Orai-activating region (SOAR)—the minimal sequence sufficient to activate Orai1 channels. Since SOAR itself is a dimer, we constructed SOAR concatemer–dimers and introduced mutations at F394, which is critical for Orai1 coupling and activation. The F394H mutation in both SOAR monomers completely blocks dimer function, but F394H introduced in only one of the dimeric SOAR monomers has no effect on Orai1 binding or activation. This reveals an unexpected unimolecular coupling between STIM1 and Orai1 and argues against recent evidence suggesting dimeric interaction between STIM1 and two adjacent Orai1 channel subunits. The model predicts that STIM1 dimers may be involved in crosslinking between Orai1 channels with implications for the kinetics and localization of Orai1 channel opening.

Distinct Orai-Coupling Domains in Stim1 and Stim2 Define the Orai-Activating Site

The ER membrane-spanning STIM1 protein is a finely-tuned sensor of ER luminal Ca2+. Small changes in ER Ca2+ induce STIM1 to undergo an intricate self-triggering process, causing it to translocate into ER-PM junctions where it couples with and activates the highly Ca2+-selective family of Orai channels in the PM. The entering Ca2+ sustains Ca2+ oscillations, maintains Ca2+ homeostasis, and provides crucial long-term Ca2+ signals in many cell types which control gene expression and cellular growth. Similar in structure and also widely expressed among cells, the little-studied STIM2 protein is reported to differ subtly from STIM1 in its N-terminal domain, affecting luminal Ca2+-sensitivity and the rate of unfolding and self-activation. The STIM1 cytoplasmic C-terminus contains the STIM-Orai activating region (SOAR) which has been structurally resolved. While the corresponding SOAR sequence in STIM2 is highly conserved, we reveal it has a profoundly diminished interaction with and ability to gate Orai1 channels. We narrowed this distinction in Orai1 activation to a small sequence in SOAR, within which substitution of a single phenylalanine in STIM1 with leucine in STIM2 confers a severe decrease in Orai1 channel-gating efficacy. This residue is strategically positioned at the structural apex of the SOAR domain. Modification of this single residue within the intact STIM1 protein reveals its pivotal role in both interaction with and gating of the Orai1 channel. The results not only pinpoint a crucial locus of STIM-Orai coupling but also reveal a physiologically profound distinction between STIM1 and STIM2.