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Store-operated Ca 2+ entry signals are mediated by plasma membrane Orai channels activated through intermembrane coupling with Ca 2+ -sensing STIM proteins in the endoplasmic reticulum (ER). The nature of this elaborate Orai-gating mechanism has remained enigmatic. Based on the Drosophila Orai structure, mammalian Orai1 channels are hexamers comprising three dimeric subunit pairs. We utilized concatenated Orai1 dimers to probe the function of key domains within the channel pore and gating regions. The Orai1-E106Q selectivity-filter mutant, widely considered a dominant pore blocker, was surprisingly nondominant within concatenated heterodimers with Orai1-WT. The Orai1-E106Q/WT heterodimer formed STIM1-activated nonselective cation channels with significantly enlarged apparent pore diameter. Other Glu-106 substitutions entirely blocked the function of heterodimers with Orai1-WT. The hydrophobic pore-lining mutation V102C, which constitutively opens channels, was suppressed by Orai1-WT in the heterodimer. In contrast, the naturally occurring R91W pore-lining mutation associated with human immunodeficiency was completely dominant-negative over Orai-WT in heterodimers. Heterodimers containing the inhibitory K85E mutation extending outward from the pore helix gave an interesting partial effect on both channel activation and STIM1 binding, indicating an important allosteric link between the cytosolic Orai1 domains. The Orai1 C-terminal STIM1-binding domain mutation L273D powerfully blocked STIM1-induced channel activation. The Orai1-L273D/WT heterodimer had drastically impaired STIM1-induced channel gating but, unexpectedly, retained full STIM1 binding. This reveals the critical role of Leu-273 in transducing the STIM1-binding signal into the allosteric conformational change that initiates channel gating. Overall, our results provide important new insights into the role of key functional domains that mediate STIM1-induced gating of the Orai1 channel., (© 2018 Cai et al.)

The transmembrane docking of endoplasmic reticulum (ER) Ca 2+ -sensing STIM proteins with plasma membrane (PM) Orai Ca 2+ channels is a critical but poorly understood step in Ca 2+ signal generation. STIM1 protein dimers unfold to expose a discrete STIM-Orai activating region (SOAR1) that tethers and activates Orai1 channels within discrete ER-PM junctions. We reveal that each monomer within the SOAR dimer interacts independently with single Orai1 subunits to mediate cross-linking between Orai1 channels. Superresolution imaging and mobility measured by fluorescence recovery after photobleaching reveal that SOAR dimer cross-linking leads to substantial Orai1 channel clustering, resulting in increased efficacy and cooperativity of Orai1 channel function. A concatenated SOAR1 heterodimer containing one monomer point mutated at its critical Orai1 binding residue (F394H), although fully activating Orai channels, is completely defective in cross-linking Orai1 channels. Importantly, the naturally occurring STIM2 variant, STIM2.1, has an eight-amino acid insert in its SOAR unit that renders it functionally identical to the F394H mutant in SOAR1. Contrary to earlier predictions, the SOAR1-SOAR2.1 heterodimer fully activates Orai1 channels but prevents cross-linking and clustering of channels. Interestingly, combined expression of full-length STIM1 with STIM2.1 in a 5:1 ratio causes suppression of sustained agonist-induced Ca 2+ oscillations and protects cells from Ca 2+ overload, resulting from high agonist-induced Ca 2+ release. Thus, STIM2.1 exerts a powerful regulatory effect on signal generation likely through preventing Orai1 channel cross-linking. Overall, STIM-mediated cross-linking of Orai1 channels is a hitherto unrecognized functional paradigm that likely provides an organizational microenvironment within ER-PM junctions with important functional impact on Ca 2+ signal generation., Competing Interests: The authors declare no conflict of interest.

The ubiquitously expressed Orai Ca 2+ channels are gated through a unique process of intermembrane coupling with the Ca 2+ -sensing STIM proteins. Despite the significance of Orai1-mediated Ca 2+ signals, how gating of Orai1 is triggered by STIM1 remains unknown. A widely held gating model invokes STIM1 binding directly to Orai1 pore-forming helix. Here we report that an Orai1 C-terminal STIM1-binding site, situated far from the N-terminal pore helix, alone provides the trigger that is necessary and sufficient for channel gating. We identify a critical 'nexus' within Orai1 connecting the peripheral C-terminal STIM1-binding site to the Orai1 core helices. Mutation of the nexus transforms Orai1 into a persistently open state exactly mimicking the action of STIM1. We suggest that the Orai1 nexus transduces the STIM1-binding signal through a conformational change in the inner core helices, and that STIM1 remotely gates the Orai1 channel without the necessity for direct STIM1 contact with the pore-forming helix.

The endoplasmic reticulum (ER) Ca(2+) sensor, STIM1, becomes activated when ER-stored Ca(2+) is depleted and translocates into ER-plasma membrane junctions where it tethers and activates Orai1 Ca(2+) 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.

The coupling of ER Ca(2+)-sensing STIM proteins and PM Orai Ca(2+) entry channels generates "store-operated" Ca(2+) signals crucial in controlling responses in many cell types. The dimeric derivative of 2-aminoethoxydiphenyl borinate (2-APB), DPB162-AE, blocks functional coupling between STIM1 and Orai1 with an IC50 (200 nM) 100-fold lower than 2-APB. Unlike 2-APB, DPB162-AE does not affect L-type or TRPC channels or Ca(2+) pumps at maximal STIM1-Orai1 blocking levels. DPB162-AE blocks STIM1-induced Orai1 or Orai2, but does not block Orai3 or STIM2-mediated effects. We narrowed the DPB162-AE site of action to the STIM-Orai activating region (SOAR) of STIM1. DPB162-AE does not prevent the SOAR-Orai1 interaction but potently blocks SOAR-mediated Orai1 channel activation, yet its action is not as an Orai1 channel pore blocker. Using the SOAR-F394H mutant which prevents both physical and functional coupling to Orai1, we reveal DPB162-AE rapidly restores SOAR-Orai binding but only slowly restores Orai1 channel-mediated Ca(2+) entry. With the same SOAR mutant, 2-APB induces rapid physical and functional coupling to Orai1, but channel activation is transient. We infer that the actions of both 2-APB and DPB162-AE are directed toward the STIM1-Orai1 coupling interface. Compared to 2-APB, DPB162-AE is a much more potent and specific STIM1/Orai1 functional uncoupler. DPB162-AE provides an important pharmacological tool and a useful mechanistic probe for the function and coupling between STIM1 and Orai1 channels., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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.

There have been numerous publications linking Ca(2+) signaling and cancer, however, a clear explanation for this link has remained elusive. We recently identified the oncogenes/tumor suppressors Wilms Tumor Suppressor 1 (WT1) and Early Growth Response 1 (EGR1) as regulators of the expression of STIM1, an essential regulator of Ca(2+) entry in non-excitable cells. The current review focuses on the literature defining both differential Ca(2+) signaling and WT1/EGR1 expression patterns in 6 specific cancer subtypes: Acute Myeloid Leukemia, Wilms Tumor, breast cancer, ovarian cancer, glioblastoma and prostate cancer. For each tumor-type, we have assessed how specific changes in WT1 and EGR1 expression might contribute to aberrant Ca(2+) homeostasis as well as the therapeutic potential of these observations.

Store-operated calcium entry (SOCE) is a key evolutionarily conserved process whereby decreases in endoplasmic reticulum Ca(2+) content lead to the influx of Ca(2+) across the plasma membrane. How this process is regulated in specific tumor cell types is poorly understood. In an effort to address this concern, we obtained and tested primary Wilms tumor cells, finding no detectable SOCE in this cell type. Analysis of the expression levels of STIM1 and ORAI1 (the molecular mediators of SOC) revealed poor STIM1 expression. Analysis of the STIM1 promoter using the TESS search system (University of Pennsylvania) revealed four putative response elements to the zinc-finger proteins WT1 (Wilms tumor suppressor 1) and EGR1 (early growth response 1). Either overexpression of WT1 or knockdown of EGR1 resulted in loss of STIM1 expression and a resultant decrease in SOCE. Furthermore, examination of Egr1 knock-out animals revealed loss of STIM1 expression in multiple tissues. Finally, using chromatin immunoprecipitation, we reveal direct binding of both WT1 and EGR1 to putative response elements located within 500 bp of the transcriptional start site of STIM1. Considering that WT1 and EGR1 are well described oncogenes and tumor suppressors, these observations may reveal new mechanisms responsible for distinct Ca(2+) signals in cancer cells.

STIM1 and STIM2 are dynamic transmembrane endoplasmic reticulum Ca(2+) sensors, coupling directly to activate plasma membrane Orai Ca(2+) entry channels. Despite extensive sequence homology, the STIM proteins are functionally distinct. We reveal that the short variable N-terminal random coil sequences of STIM1 and STIM2 confer profoundly different activation properties. Using Orai1-expressing HEK293 cells, chimeric replacement of the 43-amino-acid STIM1 N terminus with that of STIM2 attenuates Orai1-mediated Ca(2+) entry and drastically slows store-induced Orai1 channel activation. Conversely, the 55-amino-acid STIM2 terminus substituted within STIM1 strikingly enhances both Orai1-mediated Ca(2+) entry and constitutive coupling to activate Orai1 channels. Hence, STIM N termini are powerful coupling modifiers, functioning in STIM2 to "brake" the otherwise constitutive activation of Orai1 channels afforded by its high sensitivity to luminal Ca(2+).