Fig. 6: Use of Chemo-CRAB to suppress Ca²⁺ signaling downstream of GPCRs and RTKs.

a Schematic of two independent strategies to activate CRAC channels and their inhibition by Chemo-CRAB. (i) Opto-CRAC: a LOV2-STIM1(336-486) module is activated by blue light, exposing STIM1(336-486) to gate ORAI channels and drive Ca²⁺ influx. (ii) Caf-CRAC: a caffeine-responsive COSMO-STIM1(233-685) module dimerizes upon caffeine addition, activating STIM1 cytosolic domain to trigger Ca²⁺ influx through ORAI channels. In the presence of 1 µM rapamycin, Chemo-CRAB inhibits both Opto-CRAC- and Caf-CRAC-mediated Ca²⁺ entry. Created in BioRender. Liu, X. (2026) https://BioRender.com/enohm3h. b Monitoring of light-induced Ca²⁺ influx in HeLa cells stably expressing GCaMP6s and transiently co-expressing Opto-CRAC and Chemo-CRAB. Cells were recorded in the dark for baseline, exposed to blue light to activate Opto-CRAC for Ca²⁺ influx, and subsequently treated with rapamycin to activate Chemo-CRAB and suppress Ca²⁺ signals. Arrows mark timing of light exposure and rapamycin addition. Data are shown as mean ± sem. n = 15 cells from three independent biological replicates. c Quantification of GCaMP6s signals in cells from panel (b) at baseline, after blue light stimulation, and following rapamycin treatment. Data are shown as mean ± sem. n = 15 cells from three independent biological replicates. **** P < 0.0001 (Two tailed Welch’s t-test). d Monitoring of caffeine-induced Ca²⁺ influx in HeLa cells stably expressing GCaMP6s and transiently co-expressing Caf-CRAC and Chemo-CRAB. Baseline was recorded prior to 1 µM caffeine addition, after which caffeine was applied to activate Caf-CRAC and induce Ca²⁺ influx, followed by 1 µM rapamycin treatment to activate Chemo-CRAB and suppress Ca²⁺ signal. Arrows indicate timing of caffeine and rapamycin addition. Data are presented as mean ± sem; n = 29 cells from three independent biological replicate. e Quantification of GCaMP6s signals in cells from panel (d) at baseline before caffeine addition, peak after caffeine stimulation, and following rapamycin treatment. Data are shown as mean ± sem. n = 29 cells from three independent biological replicates. **** P < 0.0001 (Two tailed Welch’s t-test). f Schematic of GPCR- and RTK-mediated signaling pathways that converge on store depletion to initiate CRAC channel opening. Ligand-bound GPCRs couple to Gq and stimulate phospholipase C beta (PLC-β), whereas RTKs engage PLC-γ. PLC-β/γ hydrolyzes PIP₂ into IP₃ and DAG, with IP₃ inducing Ca²⁺ release from ER stores via IP₃R. Store depletion subsequently drives CRAC channel activation, leading to Ca²⁺ influx and NFAT_1-460 nuclear translocation. Rapamycin switches on Chemo-CRAB, thereby inhibiting CRAC channel activity downstream of GPCR or RTK signaling. Created in BioRender. Liu, X. (2026) https://BioRender.com/enohm3h. g Quantification of the nucleus-to-cytosol ratios of NFAT_1-460-GFP intensities (F_nuc/F_cyto) in HeLa cells co-expressing NFAT_1-460-GFP and SAMBA-TrkA together with Chemo-CRAB, with or without rapamycin, followed by stimulation with salicylic acid to activate RTK signaling. Data are shown as mean ± sem. n = 30 cells from three independent biological replicates. ****P < 0.0001 (Two tailed Welch’s t-test). h Quantification of the nucleus-to-cytosol ratios of NFAT_1-460-GFP intensities (F_nuc/F_cyto) in HeLa or HEK293 cells co-expressing NFAT_1-460-GFP and the indicated GPCRs together with Chemo-CRAB, with or without rapamycin treatment. Cells were stimulated with the corresponding GPCR agonists: 10 µM for hM3Dq, 100 µM histamine for histamine receptor 1 (H1R), 100 µM carbachol for muscarinic receptor 3 (M3R), 10 µM angiotensin II for angiotensin II receptor 1 (AT1R), and 4 mM Ca²⁺ for calcium-sensing receptor (CaSR). Data are shown as mean ± sem. n = 30 cells from three independent biological replicates. ****P < 0.0001 (Two tailed Welch’s t-test). Source data are provided as a Source Data file.