Fig. 1: Working model for synthetic oligomerization-dependent CRAC activators.

A One component CRAC activators based on spontaneous STIM1ct oligomerization. To engineer constitutive activators of endogenous Orai1-channels, the ER-luminal part of native STIM1 was replaced by an N-terminal oligomeric protein domain, producing cytoplasmic STIM1ct proteins whose membrane translocation capability exclusively depends on its spontaneously formed oligomerization state. While fusion of monomers yields resting STIM1ct states incapable of Orai1-activation, CRAC activation efficiency directly correlates with the oligomerization status of synthetic STIM1ct. CRAC-mediated calcium influx can trigger activation of endogenous calmodulin/calcineurin-dependent nuclear factor of activated T-cells (NFAT) signaling and transcription from NFAT-specific promoters or Ca²⁺-dependent exocytosis in secretory cell types. B Oligomeric state of different fluorescent STIM1ct fusion proteins. Target proteins in each lysate were detected by native PAGE. Numbers on the left axis represent molecular weights (MW) of denatured target proteins. C Subcellular localization of different STIM1ct fusion proteins. Fluorescent images were acquired 48 h after transfection by confocal microscopy (scale bar: 50 µm). Representative images for 3 independent experiments are shown. D Stimulation of Ca²⁺-dependent gene expression by different fluorescent STIM1ct fusion proteins. E Ca²⁺-dependent gene expression mediated by chimeric STIM1-oligomers. Data are presented as the mean ± SD, n = 3 independent experiments. Error bars = SD. Two-tailed unpaired Student t-tests were used to evaluate the statistical significance of differences between two groups. P-values more than 0.05 were considered statistically not significant. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Source data are provided as a Source Data file.