Engineering of genetically encoded programmable calcium channel inhibitory binders

Liu, Xiaoxuan; Ali, Sher; Lan, Tien-Hung; Wang, Decheng; McKee, Brendan; Nonomura, Tatsuki; Liu, Siyao; Zhao, Feng; Zhu, Michael X.; Huang, Yun; Deng, Qing; Ma, Guolin; Zhou, Yubin

doi:10.1038/s41467-026-71769-2

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Published: 13 April 2026

Engineering of genetically encoded programmable calcium channel inhibitory binders

Nature Communications volume 17, Article number: 3472 (2026) Cite this article

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Abstract

Store-operated Ca²⁺ release-activated Ca²⁺ (CRAC) channels, composed of STIM and ORAI, are essential for immune and developmental processes, and their dysregulation underlies channelopathies such as Stormorken syndrome. Here, we report the engineering of genetically encoded CRAC channel inhibitory binders (CRABs) derived from the ORAI C-terminal tail. Guided by deep mutational scanning, we optimize a membrane-anchored CRAB variant that potently inhibits Ca²⁺ influx and NFAT signaling, and rescues thrombocytopenia-like phenotypes in a zebrafish model of Stormorken syndrome. To enable tunable inhibition, we further design oligomeric, optogenetic (Opto-CRAB), and chemogenetic (Chemo-CRAB) variants, providing graded and real-time control of CRAC activity. Chemo-CRAB further suppresses Ca²⁺ signaling downstream of RTKs, GPCRs, and CAR-T cell activation, establishing broad applicability across physiological and synthetic contexts. Together, these programmable peptide-based inhibitors provide a versatile platform to dissect SOCE dynamics and hold promise as a therapeutic strategy against autoimmune, inflammatory, and neoplastic disorders driven by CRAC channel hyperactivity.

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Introduction

Store-operated Ca²⁺ entry (SOCE) is one of the principal pathways by which mammalian cells replenish the endoplasmic reticulum (ER) Ca²⁺ store and couple external stimuli to downstream signaling¹. In its prototypical form, store-operated Ca²⁺ release-activated Ca²⁺ (CRAC) channels are activated when the ER-resident Ca²⁺ sensor stromal interaction molecule 1 (STIM1) detects Ca²⁺ store depletion in the ER lumen^2,3,4, undergoes substantial conformational changes, and exposes its STIM-ORAI activating region (SOAR, also known as the CRAC activation domain or CAD) and polybasic (PB) C-terminal tail^5,6,7. Activated STIM1 oligomerizes and accumulates at ER-plasma-membrane (PM) junctions, where it opens CRAC channels to allow Ca²⁺ influx through engaging the N- and C-terminal regions of PM-resident ORAI proteins^8,9. The resulting Ca²⁺ signals, encoded in their amplitude, frequency, and spatial patterning, control a wide range of physiological processes ranging from short-term cell motility and secretion to long-term metabolism, proliferation, and gene transcription^2,3,4.

SOCE plays a critical role in the immune system^2,3,4. Engagement of immunoreceptors, including antigen receptors on lymphocytes, Fc receptors on myeloid cells, and chemokine receptors on neutrophils, rapidly depletes ER Ca²⁺ stores and activates STIM1-ORAI coupling to trigger Ca²⁺ influx¹⁰. The ensuing Ca²⁺ oscillations activate downstream effectors such as calcineurin, calmodulin kinase II, and Erk1/2, and drive the nuclear entry of transcription factors, including NF-κB and nuclear factor of activated-T cells (NFAT). These pathways are intimately involved in T cell activation, dendritic cell maturation, macrophage inflammasome signaling, and antigen presentation^11,12,13,14. Dysregulation of SOCE has been implicated in a broad spectrum of human diseases^11,15. Loss-of-function mutations that abolish SOCE are linked to severe combined immunodeficiency (SCID), autoimmunity, and ectodermal dysplasia, whereas gain-of-function mutations drive constitutive or excessive Ca²⁺ influx and cause multisystem disorders such as York platelet syndrome, and Stormorken syndrome^11,15. Beyond these channelopathies, aberrant SOCE contributes to diverse pathological conditions, including cancer, cardiovascular and metabolic disorders, and neurodegeneration^16,17,18. In the immune context, hyperactive Ca²⁺/NFAT signaling promotes tonic signaling and T cell exhaustion, a barrier frequently encountered in CAR-T cell-based immunotherapy^14,19. Notably, dampening Ca²⁺ influx has been shown to alleviate exhaustion and enhance anti-leukemia efficacy of CAR-T cells by modulating both the SOCE-calcineurin-NFAT axis and glycolytic pathways²⁰. However, despite remarkable progress in the development of small-molecule CRAC channel inhibitors, none have reached the milestone of FDA approval^21,22,23. For example, the widely studied compound BTP2 inhibits not only CRAC but also TRPC3, TRPC5, and TRPM4 channels, and induces hepatic, renal, and metabolic side effects. These limitations highlight the need for alternative therapeutic strategies, particularly genetically encoded inhibitors that can be switched on by light or small molecules, to enable precise and context-dependent modulation of SOCE and its downstream signaling pathways.

We and others have recently reported the development of a series of genetically encoded CRAC channel actuators (GECAs) that enable photon- or chemical-inducible Ca²⁺ influx through CRAC channels, thus providing powerful tools for immunomodulation and mechanistic dissection of SOCE in health and disease^{6,13,14,24,25,26,27}. In contrast, molecular tools for direct and tunable inhibition of CRAC channels remain underdeveloped, despite the clear therapeutic and mechanistic value. Given the well-characterized STIM1-ORAI interaction interface^9,28, agents that selectively disrupt this coupling represent promising candidates with minimal off-target effects.

Here, we describe the engineering of a suite of genetically encoded peptide-based CRAC channel inhibitory binders (designated CRABs) derived from the ORAI C-terminal region, a defined STIM1-binding interface. Unlike small-molecule channel blockers, CRABs act as competitive, genetically encoded decoys that intercept STIM1 before it can engage ORAI, thereby shutting channels upstream of ion conduction. We establish a potent PM-anchored variant (PM-CRAB) optimized through deep mutational scanning, and further design oligomeric variants that provide graded inhibitory strength. To achieve reversible and user-defined control, we introduce optogenetic (Opto-CRAB) and chemogenetic (Chemo-CRAB) versions, enabling precise inhibition of SOCE. Importantly, we demonstrate that CRABs not only suppress Ca²⁺/NFAT signaling downstream of diverse G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) but also restore thrombocyte progenitor production in a zebrafish model of Stormorken syndrome. Together, CRABs constitute a programmable platform for targeted endogenous CRAC channel inhibition in vertebrates. By combining molecular specificity with tunable and orthogonal modes of control, CRABs fill a longstanding gap in the Ca²⁺ signaling toolkit and reveal therapeutic potential in both rare CRAC channelopathies and immune modulation for precision cancer immunotherapy.

Results

Design and optimization of CRABs

Previous biophysical and biochemical evidence suggests that STIM1 might gate the ORAI1 channel through direct interactions with its N- and C-termini^9,29,30. The C-terminal extension of ORAI1 facilitates STIM1 binding and assists in recruitment of ORAI1 at ER-PM junctions, while the N-terminal region contributes to the stability of the STIM1-ORAI1 association and is involved in channel activation^30,31. Building upon these insights, we envisioned that overexpression of STIM1-interacting peptides derived from ORAI could competitively disrupt the endogenous ORAI-STIM1 interaction, thereby inhibiting CRAC channel activation. To test this, we fused either N- or C-terminal fragments derived from three ORAI homologs (ORAI1, ORAI2, and ORAI3) to a PM-targeting motif, either at the N-terminal side (Lyn11 from Lyn kinase) or at the C-terminal side (CAAX motif from KRAS4B) of the fragment, and co-expressed these constructs with the cytosolic SOAR domain of STIM1 in HeLa cells (Fig. 1a, and Supplementary Fig. 1a). Among a dozen of candidates screened, the C-terminal fragment from ORAI3 fused to CAAX (mCh-O3CT-CAAX) induced marked PM-translocation of the cytosolic SOAR domain, while that fused to Lyn11 (Lyn11-mCh-O3CT) only had a moderate effect (Fig. 1b, c, and Supplementary Fig. 1b, c). The interaction between PM-CRAB to the STIM1 activating domain was further validated using a bioluminescence resonance energy transfer (BRET) assay. In this system, NanoLuc-fused CRAB variants (nLuc-CRAB or membrane-tethered nLuc-CRAB-CAAX) served as the bioluminescent donors, while YFP-SOAR or full-length STIM1-YFP served as the fluorescent acceptors. We observed that the PM-tethered construct (nLuc-CRAB-CAAX) yielded a higher BRET ratio compared to its cytosolic version (nLuc-CRAB) when YFP-SOAR or STIM1-YFP was used as the acceptor (Supplementary Fig. 1d). Importantly, BRET signals from nLuc-CRAB-CAAX were substantially higher than those obtained with the non-interacting membrane-localized control (nLuc-CAAX), indicating that the signal is not due to nonspecific membrane proximity. Together, these results provide independent evidence to support the interaction between CRAB and the SOAR domain within STIM1.

**Fig. 1: Engineering of a genetically encoded CRAC channel inhibitor binder (CRAB).**

To further enhance the binding strength of mCh-O3CT-CAAX toward the SOAR domain, we performed deep mutational scanning of the 14-mer O3CT peptide (residues 282-295) by generating a library of 280 variants. Several substitutions, including G290V, G290R, and Q289Y, exhibited strong colocalization with SOAR in the initial screen (Fig. 1c–e). Among these candidates, G290V was prioritized for further characterization based on its combined experimental performance and predicted structural stabilization. To gain structural insight into this enhancement, we modeled the ORAI3 CT peptide in complex with a SOAR dimer using AlphaFold. The resulting model (pTM = 0.80; ipTM = 0.77) positioned the O3CT helix within the CC2–CC3 binding groove of SOAR, consistent with prior structural and biochemical studies of STIM-ORAI coupling. In this predicted interface, residue 290 lies directly at the contact surface. Substitution of glycine with valine introduces a hydrophobic side chain that enhances packing within the CC2-CC3 groove, providing a structural rationale for the increased binding efficiency. To evaluate how this mutation might affect protein stability, we calculated the change in Gibbs free energy (ΔΔG) using the ddg_monomer protocol in the Rosetta modeling suite^32,33,34. A negative ΔΔG value indicates a stabilizing effect relative to the wild-type (WT) O3CT peptide. Among the top-performing variants, G290V exhibited the most favorable (substantially negative) ΔΔG, supporting enhanced local stability (Fig. 1f). These findings suggest that the native glycine introduces structural instability at this position, whereas substitution with a helix-stabilizing valine likely reinforces the local conformation and promotes stronger engagement with SOAR.

Next, we assessed the effects of three O3CT-based constructs on CRAC channel activity: cytosolic mCh-O3CT, membrane-anchored O3CT (mCh-O3CT-CAAX), and the G290V mutant (mCh-O3CT-G290V-CAAX). In line with their relative binding strengths to SOAR in the colocalization assay (Fig. 1b, c), the membrane-anchored G290V variant (thereafter termed as “PM-CRAB”) exhibited the most pronounced inhibition of thapsigargin (TG)-induced Ca²⁺ influx in HeLa cells, as measured by the stably expressed Ca²⁺ indicator GCaMP6s (Fig. 1g, and Supplementary Movie 1). By contrast, membrane-anchored wild-type O3CT showed less inhibition than the G290V variant, whereas the cytosolic O3CT fragment had minimal effect (Fig. 1h).

To further evaluate the functional consequence of CRAC channel inhibition, we monitored nuclear NFAT transcription factor that undergoes Ca²⁺-dependent dephosphorylation and subsequent nuclear entry upon sustained cytosolic Ca²⁺ elevation, in HeLa cells stably expressing NFAT_1-460-GFP²⁴. Consistent with the Ca²⁺ imaging data (Fig. 1g, h), TG-induced nuclear translocation of NFAT_1-460 was markedly suppressed in cells expressing mCh-O3CT-G290V-CAAX (Supplementary Movie 2), but largely unaffected in those expressing the cytosolic O3CT fragment (Fig. 1i, j). Taken together, these results demonstrate that the engineered membrane-anchored O3CT-G290V variant (mCh-O3CT-G290V-CAAX) functions as a potent PM-anchored CRAC channel inhibitor, capable of suppressing CRAC channel-mediated Ca²⁺ influx and downstream Ca²⁺-dependent signaling.

To further define the molecular specificity and potential off-target effects of PM-CRAB, we evaluated its efficacy across different ORAI and STIM isoforms. Using HEK293 triple ORAI1/2/3 knockout cells reconstituted with individual ORAI isoforms together with STIM1, whole-cell patch-clamp recordings demonstrated that PM-CRAB robustly suppressed CRAC currents mediated by ORAI1, ORAI2, and ORAI3 to a comparable extent (Supplementary Fig. 2a–c). These results indicate that the inhibitory mechanism is not isoform-selective at the level of ORAI but instead reflects interference with STIM-dependent channel activation. Furthermore, in HEK293 STIM1/STIM2 double-knockout cells, PM-CRAB effectively inhibited STIM2-mediated current (Supplementary Fig. 2d). These results are consistent with the conserved ORAI-interacting interface shared by STIM1 and STIM2 (particularly the corresponding SOAR regions), suggesting that CRAB functions as a general competitive antagonist of STIM-ORAI coupling.

CRAB rescues thrombocytopenia in a zebrafish model of Stormorken syndrome

A gain-of-function mutation in STIM1 (R304W), which induces constitutive Ca²⁺ influx through ORAI channels (Fig. 2a), is causally linked to Stormorken syndrome, a disorder characterized by thrombocytopenia, bleeding diathesis, muscle weakness, and asplenia^35,36. In HEK293T cells expressing this mutant, we observed spontaneous development of store-independent CRAC current (I_CRAC), which was effectively suppressed upon co-expression of PM-CRAB (Fig. 2b–d). Moreover, PM-CRAB attenuated the aberrant activation of the Ca²⁺/NFAT pathway induced by STIM1-R304W, as reflected by inhibition of NFAT_1-460 nuclear translocation (Fig. 2e).

**Fig. 2: PM-CRAB counteracts aberrant activity of the Stormorken syndrome (SS)-associated STIM1 mutant in mammalian cells and zebrafish embryos.**

Next, to assess the in vivo application and therapeutic potential of PM-CRAB, we used a well-established zebrafish model of Stormorken syndrome by microinjecting STIM1(R304W) mutant mRNA, with or without PM-CRAB, into the Tg(CD41:GFP) line^35,36,37. This line expresses GFP in mature thrombocytes and hematopoietic stem and progenitor cells, thereby allowing easy visualization and quantification of thrombocyte populations during embryonic development^35,36,37. As expected, embryos expressing STIM1(R304W) displayed prominent pathophysiological features, including a notable reduction in GFP fluorescence in the caudal veins and hematopoietic tissues (Fig. 2f, g), indicative of thrombocytopenia. This phenotype mimics the platelet deficiency observed in patients with Stormorken syndrome^35,36. Notably, co-expression of PM-CRAB with STIM1(R304W) effectively restored the production of thrombocyte progenitors, as evidenced by robust gain of GFP signals in embryos (from ~20% to over 75%; Fig. 2f, g). Collectively, these findings demonstrate that PM-CRAB can mitigate STIM1(R304W)-induced thrombocytopenia in a vertebrate model, supporting its potential as a therapeutic agent for CRAC channelopathies such as Stormorken syndrome.

Engineering CRAB oligomers to enable tunable CRAB channel inhibition

To assess how PM-CRAB affects the functional coupling between STIM1 and ORAI1 at ER-PM contact sites, we co-expressed PM-CRAB with full-length STIM1 in HeLa cells, with the cytosolic CRAB as a negative control. Unlike the cytosolic CRAB, which did not promote spontaneous STIM1 localization to ER-PM junctions (Supplementary Fig. 3a), PM-CRAB induced robust puncta formation even in the absence of TG (Supplementary Fig. 3b). These findings suggest that PM-CRAB engages resting STIM1 and overrides its autoinhibitory “folded-back” conformation. This likely occurs through PM-CRAB competing with the intramolecular CC1-SOAR interaction, ultimately exposing SOAR/CAD and the polybasic C-tail necessary for PM targeting. To further validate this model, we examined the STIM1 R426L mutant, which stabilizes the inactive conformation by strengthening the intramolecular CC1-SOAR interaction³⁸. PM-CRAB was still capable of recruiting STIM1 R426L to ER-PM junctions, although to a lesser extent than wild-type STIM1 (Supplementary Fig. 3c). This attenuation is consistent with a competitive mechanism in which PM-CRAB counteracts intramolecular CC1-SOAR interaction, partially overcoming stabilized autoinhibition to expose the SOAR domain. Importantly, this recruitment of STIM1 to ER-PM junctions does not translate into functional activation. PM-CRAB competitively occupies the SOAR domain and masks the ORAI-binding interface, preventing the repositioned STIM1 from engaging and gating ORAI channels. While Ca²⁺ influx or NFAT nuclear translocation were not detected in cells expressing PM-CRAB (Fig. 1g-j), the constitutive recruitment of STIM1 to ER-PM junctions could nonetheless interfere with the dynamics of ER-PM contact sites to perturb other cellular events^39,40,41,42. Moreover, as CRAC channels serve as the predominant Ca²⁺ entry route in non-excitable cells⁴³, persistent blockade by PM-CRAB may limit its utility in mechanistic studies and therapeutic settings. These findings prompted us to engineer additional CRAC channel inhibitors with tunable inhibitory strength, thus allowing for context-dependent modulation of Ca²⁺ signaling.

Since both cysteine prenylation and polybasic amino acid sequence are essential for PM targeting mediated by the CAAX motif^44,45, we initiated our design strategy by truncating the CVIM sequence within the CAAX motif and titrating the polybasic tail avidity through modulation of the oligomeric state of CRAB peptides. Specifically, we generated a monomeric CRAB (Mo-CRAB) by removing the CVIM sequence while retaining the poly-lysine residues in the CAAX motif from PM-CRAB (Fig. 3a). The dimeric (Di-CRAB) and tetrameric (TD-CRAB) variants were subsequently engineered by fusing the dimeric glutathione S-transferase (GST) and the tetrameric domain derived from p53 (residues 324-356), respectively^46,47, to the N-terminus of CRAB (Fig. 3a). As expected, Mo-CRAB exhibited negligible PM targeting, whereas Di-CRAB and TD-CRAB showed progressively enhanced PM targeting when co-expressed with SOAR (Fig. 3b-c). Next, we evaluated the inhibitory effects of these oligomeric CRAB variants on CRAC channel activity and downstream signaling. Consistent with their PM targeting and SOAR binding capabilities, Mo-CRAB only exhibited marginal inhibition of STIM1-induced CRAC channel activity, while Di-CRAB showed nearly 50% inhibition, as assessed by fluorescence signals from the Ca²⁺ indicator GCaMP6s (Fig. 3d, e). Similar inhibitory effects were also observed in Ca²⁺-dependent downstream signaling, as determined by the degree of nuclear translocation of NFAT_1-460 (Fig. 3f). Notably, TD-CRAB demonstrated robust inhibitory efficacy comparable to PM-CRAB by almost completely inhibiting store depletion-triggered Ca²⁺ influx and nuclear entry of NFAT (Fig. 3d–f).

**Fig. 3: Oligomeric CRABs differentially inhibit CRAC channel activity and downstream signaling.**

Next, we set out to determine whether TD-CRAB interacts with inactive STIM1 under resting conditions. We observed an ER network-like distribution of GFP-STIM1 in HeLa cells co-expressing mCherry-tagged TD-CRAB (mCh-TD-CRAB) in the absence of TG, while mCh-TD-CRAB was evenly distributed throughout the cytoplasm (Supplementary Fig. 3d). Following TG-induced Ca²⁺ store depletion, GFP-STIM1 formed discrete puncta and concurrently recruited TD-CRAB to ER-PM junctions (Supplementary Fig. 3d). These findings support the notion that TD-CRAB functions as a potent, genetically encoded CRAC channel inhibitor that preferentially targets activated STIM1 without disturbing the autoinhibition of resting STIM1.

Opto-CRAB allows optical inhibition of the CRAC channel

While TD-CRAB provides proof-of-principle for CRAC inhibition, its constitutive activity restricts broader biological applications. To address this limitation and achieve higher temporal precision, we sought to engineer an optically controllable CRAB variant that affords spatiotemporal regulation and reversible inhibition. The photolyase-homology domain (PHR) of cryptochrome 2 (CRY2; aa 1-498) from Arabidopsis thaliana has been widely utilized in the development of optogenetic tools due to its blue light-dependent homo-oligomerization properties^{25,48,49,50,51,52}. To leverage this feature, we designed a construct in which CRY2-PHR was fused to the N-terminus of Mo-CRAB, creating a light-controllable CRAB variant termed Opto-CRAB (Fig. 4a). We reasoned that light-induced oligomerization of CRY2 would confer Opto-CRAB with potent inhibitory effects on CRAC channels comparable to those of TD-CRAB.

**Fig. 4: Engineering of light-switchable CRAB for reversible control of the CRAC channel and its downstream activities.**

Indeed, we observed notable light-inducible co-localization of YFP-SOAR with mCh-Opto-CRAB in HeLa cells (Fig. 4b), with activation and deactivation half-lives of 0.5 and 3.2 min, respectively (Fig. 4c, and Supplementary Movie 3, Supplementary Movie 4). This deactivation reflects the dissociation of the Opto-CRAB/SOAR complex as the photosensitive module returns to its dark-state conformation. The resulting loss of multivalency reduces both the polybasic tail avidity for PM targeting and its binding strength for SOAR domain. In line with its PM targeting and SOAR binding properties, Opto-CRAB effectively inhibited store depletion-triggered Ca²⁺ influx under blue light illumination, as measured by the red Ca²⁺ indicator R-GECO1.2 (Fig. 4d). Extending the dark phase in the light-pulse cycle progressively diminished inhibition (Fig. 4e), suggesting a light-dependent, time-sensitive window for CRAC channel inhibition. Consistently, TG-induced NFAT_1-460 nuclear translocation was markedly suppressed by Opto-CRAB under photostimulation (Fig. 4f).

To accelerate the reversibility, we further engineered an Opto-CRAB variant (Opto-CRAB-2) by replacing the CRY2 module with the iLID-sspB optical dimerization system (Fig. 4g). This system exploits the oat light-oxygen-voltage 2 (LOV2) domain to enable rapid and reversible light-dependent interaction between bacterial ssrA and sspB^53,54. We fused sspB to CRAB and co-expressed iLID-CAAX in HeLa cells. Upon photostimulation, we detected notable translocation of sspB-CRAB toward the PM-anchored iLID (t_{1/2, ON} = 8.2 sec; t_{1/2, OFF} = 37.9 sec; Fig. 4h–i), accompanied by suppression of Ca²⁺ influx in a light-dependent manner (Fig. 4j). Together, these results support Opto-CRAB as a genetically encoded photo-switchable inhibitor for CRAC channel activity.

Chemo-CRAB enables chemogenetic intervention of CRAC channelopathy

Despite the unmatched spatiotemporal resolution and reversibility of optogenetic tools, the requirement for light delivery poses a challenge for translational applications^14,50. Chemogenetic strategies, by contrast, provide a practical alternative through simple drug administration. Among these, the rapamycin-inducible FRB (FKBP-rapamycin binding) and FKBP (FK506-binding protein) heterodimerization system is one of the most widely used chemically induced dimerization (CID) modules in synthetic biology^55,56,57. Building on this system, an FRB-FKBP fusion protein capable of self-assembly into a tetramer or higher-order oligomer upon addition of rapamycin has been reported^58,59. Leveraging this property, we fused the FRB-FKBP module to the N-terminus of Mo-CRAB, generating a rapamycin-controllable variant termed Chemo-CRAB. We reasoned that rapamycin-induced oligomerization of Chemo-CRAB would mimic TD-CRAB activity in inhibiting CRAC channel function (Fig. 5a).

**Fig. 5: Design of Chemo-CRAB to conditionally inhibit CRAC channel activity in mammalian cells and in a zebrafish model of Stormorken Syndrome.**

As designed, rapamycin treatment triggered PM targeting of both YFP-SOAR and mCherry-tagged Chemo-CRAB in HeLa cells with an activation half-life of 13.9 s (Fig. 5b, c, and Supplementary Movie 5), indicating induced heterodimerization between the two otherwise cytosolic components. Functionally, this translated into strong suppression of store depletion-evoked Ca²⁺ influx, as reflected by reduced GCaMP6s signals (Fig. 5d, e). Notably, Chemo-CRAB could effectively act on activated STIM1, as rapamycin addition immediately curtailed ongoing Ca²⁺ influx (Fig. 5f), thus establishing its utility as an inducible inhibitory binder of active CRAC channels.

Having established the inducible activity of Chemo-CRAB, we next examined whether it could directly engage disease-associated STIM1 mutants that drive pathological CRAC hyperactivity^35,36. Specifically, we monitored the real-time interaction between Chemo-CRAB and the constitutively active STIM1 R304W mutant in mammalian cells. Rapamycin addition induced strong colocalization of the two proteins (Fig. 5g, and Supplementary Movie 6), confirming that Chemo-CRAB can effectively target a gain-of-function STIM1 mutant. To further exploit the potential of Chemo-CRAB in alleviating pathological conditions driven by constitutive activation of CRAC channel, we again turned to the aforementioned zebrafish model of Stormorken syndrome, in which expression of the constitutively active STIM1 R304W mutant causes thrombocytopenia^35,36,37, as evidenced by a reduced GFP-labeled thrombocyte progenitors in the tail region of embryos (Fig. 5h, i). Remarkably, treatment with rapamycin restored GFP-positive thrombocyte progenitor levels to near-normal levels in zebrafish embryos co-expressing both STIM1 R304W and Chemo-CRAB.

To address the inherent immunosuppressive effects associated with rapamycin^60,61,62, we engineered an alternative Chemo-CRAB variant (Chemo-CRAB-2) by replacing the FRB-FKBP module with a tetrameric FKBP(F36V) cassette, which enables higher-order oligomerization in the presence of the B/B homodimerizer (also known as AP20187), a non-immunosuppressive rapalog (Supplementary Fig. 4a)⁶³. Upon addition of B/B homodimerizer, we observed robust PM targeting of both YFP-SOAR and mCherry-tagged Chemo-CRAB-2 in HeLa cells (Supplementary Fig. 4b), along with suppression of both TG-induced Ca²⁺ influx and NFAT_1-460 nuclear translocation (Supplementary Fig. 4c, d).

Together, these findings establish Chemo-CRAB as an effective chemogenetic tool for modulating CRAC channel activity and downstream NFAT signaling, and demonstrate its therapeutic potential for mitigating pathological conditions associated with CRAC channel hyperactivity, such as Stormorken syndrome.

Chemo-CRAB can conditionally perturb GPCR and RTK signaling

To assess the versatility of Chemo-CRAB in more sophisticated synthetic biology applications, we further tested its compatibility with other genetically encoded Ca²⁺ channel actuators, including Opto-CRAC and Caf-CRAC^24,64. Opto-CRAC is an optogenetic actuator composed of a LOV2-caged STIM1 fragment (LOV2-STIM1_336-486) that activates endogenous ORAI channels via blue light-induced uncaging²⁴. Caf-CRAC is a chemogenetic system based on a caffeine-operated synthetic module (COSMO), in which caffeine triggers dimerization of the cytoplasmic domain of STIM1 (COSMO-STIM1_233-685) to activate ORAI channels⁶⁴. Both actuators function by exposing STIM1 cytosolic fragments that engage ORAI channels to trigger Ca²⁺ flux from the extracellular space into the cytosol (Fig. 6a). We reasoned that Chemo-CRAB, once oligomerized by rapamycin, could antagonize these systems by competitively binding to the exposed STIM1 fragments. Indeed, co-expression of Chemo-CRAB with either Opto-CRAC (Fig. 6b, c) or Caf-CRAC (Fig. 6d, e) in HeLa cells resulted in robust suppression of light- or caffeine-induced Ca²⁺ entry following rapamycin treatment, validating the inhibitory activity of Chemo-CRAB against orthogonal STIM1-based actuators. As expected, Chemo-CRAB failed to suppress Ca²⁺ influx mediated by LOCa3, an engineered ORAI1-LOV2 hybrid channel that can be photo-activated independent on STIM1⁶⁵ (Supplementary Fig. 5a–c), confirming its working mechanism as a competitive inhibitory binder of STIM1-ORAI interactions. To further validate this mechanism, we evaluated Chemo-CRAB against three well-characterized, constitutively active ORAI gain-of-function (GOF) mutants: A137V, P245L, and V181K^35,66,67. Consistent with the results observed with LOCa3, Chemo-CRAB failed to attenuate NFAT activation driven by these STIM1-independent ORAI1 mutants (Supplementary Fig. 5d). These findings further support that the inhibitory activity of Chemo-CRAB depends on competitive interference at the STIM1-ORAI1 binding interface and does not result from direct or nonspecific inhibition of the ORAI channel pore.

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

Beyond directly antagonizing synthetic CRAC channel actuators, we evaluated the ability of Chemo-CRAB to inhibit Ca²⁺ signaling initiated by RTKs, using TrkA as an example. TrkA is the high-affinity receptor for nerve growth factor (NGF) and, upon ligand binding, undergoes autophosphorylation to activate downstream effectors, including phospholipase C γ (PLCγ), which catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) to generate inositol-1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG), thereby promoting the intracellular Ca²⁺ mobilization from internal stores and subsequent activation of Ca²⁺-dependent effectors, including NFAT (Fig. 6f)^68,69. To enable orthogonal control of this pathway, we recently engineered a synthetic TrkA construct by fusing its intracellular kinase domain (TrkA-ICD) to a salicylic acid-mediated binary association system (SAMBA)^70,71, which allows salicylic acid-inducible PM recruitment and activation of TrkA signaling. In HeLa cells co-expressing SAMBA-TrkA and NFAT_1-460, salicylic acid stimulation induced prominent nuclear translocation of NFAT_1-460. Notably, this Ca²⁺-dependent response was suppressed upon rapamycin-induced activation of Chemo-CRAB (Fig. 6g), indicating its ability to effectively intercept TrkA-driven Ca²⁺ signaling.

We next examined whether Chemo-CRAB could modulate Ca²⁺ signaling initiated by G_q-coupled GPCRs^72,73. As a model system, we used hM3Dq, a Designer Receptor Exclusively Activated by Designer Drug (DREADD) engineered from the human M3 muscarinic receptor⁷⁴. Upon activation by its orthogonal ligand clozapine-N-oxide (CNO), hM3Dq activates G_q signaling pathway, in which phospholipase C β (PLCβ)-catalyzed PIP₂ hydrolysis leads to IP₃ production and subsequent release of Ca²⁺ from intracellular stores, ultimately promoting transcriptional responses such as NFAT activation (Fig. 6f). As expected, CNO-induced hM3Dq activation led to robust nuclear translocation of NFAT_1-460, which was inhibited by Chemo-CRAB in the presence of rapamycin (Fig. 6h). To extend the applicability of Chemo-CRAB beyond synthetic systems, we examined its efficacy in modulating downstream Ca²⁺/NFAT signaling downstream of GPCR activation by native ligands, thereby testing the generalizability of this inhibitory strategy in physiologically relevant contexts (Fig. 6f). Across all four GPCRs examined (including histamine receptor 1 (H1R), muscarinic receptor 3 (M3R), angiotensin II receptor 1 (AT1R), and calcium-sensing receptor (CaSR), we consistently observed rapamycin-dependent suppression of downstream effectors, shown as reduced NFAT_1-460 nuclear entry (Fig. 6h). Together, these findings establish Chemo-CRAB as a versatile and modular tool capable of conditionally perturbing diverse receptor-initiated Ca²⁺ signaling pathways, spanning synthetic actuators, RTKs, and GPCRs.

Chemo-CRAB enables programmable control of immune responses

Since CRAC channel-mediated Ca²⁺ signaling is indispensable for immune response, its modulation represents a promising strategy for immunomodulatory therapies^10,11,19. To evaluate the inhibitory capacity of CRAB in cells derived from the immune system, we first examined CRAC channel activity in Jurkat, an immortalized human T-cell line derived from the peripheral blood of a 14-year-old boy with acute T-cell leukemia. Whole-cell patch clamp recordings showed that store depletion-induced CRAC current (I_CRAC) was abolished in Jurkat T cells expressing PM-CRAB, whereas wild-type Jurkat cells exhibited the inwardly rectifying current typical of CRAC channels (Fig. 7a–c). We then assessed downstream Ca²⁺-dependent transcriptional activity using an NFAT-driven luciferase (NFAT-Luc) reporter. In Jurkat-Lucia NFAT reporter cells, stimulation of Ca²⁺/NFAT signaling by ionomycin, together with the activation of Ras/Raf/MAPK/AP-1 by the pleiotropic PKC activator PMA, induced a robust increase in luciferase expression. In contrast, ectopic expression of PM-CRAB, but not the cytosolic control construct mCh-O3CT, markedly suppressed Ca²⁺/NFAT-dependent reporter activation, indicating potent inhibition of CRAC-mediated signaling in a disease-relevant cellular model (Fig. 7d, e).

**Fig. 7: Chemo-CRAB enables chemogenetic immunomodulation.**

To further evaluate the inhibitory potential of PM-CRAB in a more physiologically and clinically relevant context, we extended our study to primary human T cells. CD3⁺ T cells were engineered to co-express a CD19-targeted chimeric antigen receptor (CD19-CAR) together with PM-CRAB. Upon co-culture with CD19-positive Raji cells, CAR-T cells expressing PM-CRAB exhibited a notable reduction in the secretion of key effector cytokines, including IL-2, IFN-γ, and TNF-α (Fig. 7f–h), compared to control groups lacking the inhibitor. These findings demonstrate that PM-CRAB effectively attenuates CAR-T cell effector functions and offer a strategy to modulate Ca²⁺-dependent T cell activation.

We next asked whether Chemo-CRAB could modulate CRAC channel activity and downstream signaling in a chemically inducible manner. Indeed, robust luciferase expression was observed in Jurkat-Lucia NFAT reporter cells stably co-expressing Chemo-CRAB upon PMA/ionomycin stimulation, which was suppressed by rapamycin in a dose-dependent manner (Supplementary Fig. 6a). To further validate these findings in a more physiologically relevant context, we quantified the level of interleukin-2 (IL-2), a hallmark cytokine released by activated T cells. Consistent with the reporter assay, IL-2 secretion was significantly reduced in Jurkat cells expressing Chemo-CRAB in a rapamycin dose-dependent manner, whereas rapamycin treatment failed to inhibit IL-2 secretion in control cells lacking Chemo-CRAB expression (Supplementary Fig. 6b). Similar trend was observed using Chemo-CRAB-2 in the presence of the B/B homodimerizer (Supplementary Fig. 4e-f).

CAR-T cell therapy has demonstrated remarkable efficacy in hematological immunotherapy and immunoinflammatory disorders^75,76,77, where engagement of tumor-associated antigens (TAAs) triggers immunoreceptor tyrosine-based activation motif (ITAM) signaling, CRAC-mediated Ca²⁺ influx, and a series of effector functions^10,11,18. However, excessive Ca²⁺/NFAT signaling has been implicated in tonic signaling and T cell exhaustion^10,78, which remain major obstacles to sustaining durable responses in CAR-T cell therapy. To establish the proof of concept that CRAB can serve as a tool to mitigate overactivated Ca²⁺/NFAT signaling that partially accounts for T cell exhaustion and tonic signaling, we examined whether Chemo-CRAB could inhibit CRAC-mediated Ca²⁺ signaling in anti-CD19 CAR-T cells upon stimulation with Raji lymphoma cells (Supplementary Fig. 6c). In Jurkat-Lucia NFAT reporter cells stably co-expressing anti-CD19 CAR and Chemo-CRAB, co-culture with CD19⁺ Raji cells triggered robust luciferase activity and IL-2 secretion, both of which were markedly suppressed upon rapamycin treatment. By contrast, co-culture with CD19-negative K562 chronic myelogenous leukemia cells failed to induce NFAT-dependent transcription or IL-2 secretion, and rapamycin had no effect in cells lacking Chemo-CRAB (Supplementary Fig. 6d, e).

Together, these results demonstrate that CRAB effectively suppresses CRAC channel activity and downstream NFAT-dependent responses in T cells. In particular, the inducible Chemo-CRAB system enables chemogenetic control of Ca²⁺ signaling triggered by CD19-CAR activation, providing a promising strategy to fine-tune signaling strength and mitigate T cell exhaustion.

Discussion

SOCE is indispensable for transducing receptor stimulation to downstream transcriptional programs and effector functions, and it plays critical roles in immunity, development, and tissue homeostasis^2,3,4. Dysregulated SOCE is associated with a spectrum of diseases, ranging from immunodeficiency and autoimmunity to muscular disorders and cancer. Despite its importance, tools to manipulate CRAC channel activity with precise spatial, temporal, and cell-type specificity remain limited. Small-molecule inhibitors such as BTP2, 2-APB, Synta-66, and CM4620, are hampered by poor ion/channel selectivity, cytotoxicity, and/or incompletely understood mechanisms of action⁷⁹. In contrast, CRAB expression resulted in minimal cytotoxicity and negligible disturbance to cell functions. CCK-8 assays confirmed that CRAB expression does not compromise cell viability (Supplementary Fig. 7a), while Fluo-4 imaging demonstrated that resting Ca²⁺ levels remain unperturbed (Supplementary Fig. 7b). By comparison, genetic approaches such as ORAI knockout or dominant-negative mutants impose more irreversible perturbations on Ca²⁺ signaling pathways.

Recent progress has been made in engineering genetically encoded inhibitors for other ion channels, including RGK- and nanobody-based modulators for voltage-gated calcium channels^80,81,82 and de novo designed peptides that suppress pathogenic voltage-gated sodium channels⁸³. In contrast, development of molecular tools for direct and reversible inhibition of CRAC channels has lagged behind, despite their clear mechanistic and therapeutic value. In the current study, we introduce CRABs, a series of genetically encoded CRAC channel inhibitors that selectively inhibit CRAC channel activation by competitively intercepting STIM1-ORAI coupling. Through deep mutational scanning of an ORAI C-terminal peptide, we optimized a PM-anchored variant (PM-CRAB) that potently suppressed Ca²⁺ influx, and rescued thrombocytopenia in a zebrafish model of Stormorken syndrome. Building on this foundation, we generated oligomeric, optogenetic, and chemogenetic CRAB formats that provide tunable and reversible control of SOCE with desired spatiotemporal precision. Notably, Chemo-CRAB effectively suppressed Ca²⁺/NFAT signaling downstream of GPCRs, RTKs, and CAR-T cell activation, thereby establishing CRABs as versatile tools for dissecting calcium dynamics and promising candidates for therapeutic intervention of aberrant CRAC channel activity-driven pathologies.

SOCE and associated Ca²⁺ oscillations are typically initiated by activation of surface receptors coupled to PLC, which hydrolyzes PIP₂ to generate DAG and IP₃, thereby activating downstream effectors. A genetically encoded peptide inhibitor that disrupts Gα_q-PLCβ interactions has been shown to block GPCR -induced IP₃ production, Ca²⁺ influx, and DAG-dependent signaling, with a concomitant shift toward alternative effectors such as RhoGEF and GRKs⁸⁴. In this context, CRABs complement such upstream inhibitors by directly targeting the STIM1-ORAI coupling, offering a distinct and mechanistically precise strategy to modulate Ca²⁺ entry and its downstream consequences without perturbing upstream events or parallel signaling pathways.

The ability of CRABs to inhibit CD19-CAR-induced NFAT activation and cytokine release points to their translational potential for immune modulation. Efficacy loss remains a critical barrier in CAR T cell therapy, as chronic or excessive Ca²⁺/NFAT activation promotes premature exhaustion, increases the risk of off-target cytotoxicity, and enhance excessive cytokine production that can culminate into cytokine release syndrome, collectively compromising safety and therapeutic durability^19,20,85. Approaches such as rational optimization of CAR architecture, incorporation of tailored costimulatory domains, and pharmacological interventions of downstream pathways have shown promise but are often limited by the lack of specificity or temporal control^86,87,88. CRABs offer a complementary solution by directly targeting the distal STIM1-ORAI-mediated Ca²⁺ signaling, providing a mechanistically precise means to temper activation without disturbing upstream receptor and kinase function. Looking forward, the integration of CRAB modules into CAR constructs could serve as a built-in molecular switch to fine-tune Ca²⁺ signaling, prevent exhaustion, mitigate toxicity, thereby improving therapeutic persistence and safety.

Despite their promise, several challenges remain for the translational application of CRABs. A fundamental mechanistic boundary of CRABs is that they function as competitive binders that intercept STIM molecules before it engages the ORAI C-terminal tail. Consequently, they are unlikely to inhibit ORAI1 gain-of-function mutations that produce constitutive, STIM-independent channel activity. Safe and effective deployment of CRABs in primary human cells will require targeted gene delivery strategies, and the long-term consequences of sustained SOCE inhibition must be carefully assessed given the essential role of CRAC channels in cellular homeostasis and function. Recent advances in gene delivery strategies, including inducible systems, receptor-targeted viral vectors, and virus-like particles with customized receptor-ligand interactions, offer spatiotemporal and antigen-specific control and could be leveraged to deliver CRABs selectively into disease-relevant cell subsets¹⁴. Inducible CRAB designs further address safety concerns by enabling reversible, stimulus-dependent suppression. Although rapamycin-based systems may be limited by their intrinsic immunosuppressive activity, the FKBP(F36V)/AP20187 module used in our Chemo-CRAB-2 design offers a more clinically compatible alternative.

In summary, CRABs fill a gap in the Ca²⁺ signaling toolkit by providing a programmable, genetically encoded approach for selective inhibition of CRAC channels. They complement existing Ca²⁺ actuators by enabling spatiotemporally precise perturbation of Ca²⁺ signaling, with mechanistic clarity and selectivity where small molecules have proven short. Beyond their utility in basic research, CRABs show therapeutic potential in rare gain-of-function CRAC channelopathies and in immunomodulatory therapies, particularly in CAR-T cell therapies. Therefore, CRABs mark a clear advance in programmable modulation of Ca²⁺ signaling, creating opportunities for both basic discovery and translational applications.

Methods

Ethical statement

We confirm that this research complies with all relevant ethical regulations. The Animal Care and Use Protocol was approved by the Purdue Animal Care and Use Committee (PACUC), adhering to the guidelines for using zebrafish in the NIH Intramural Research Program (protocol number: 1401001018). Human peripheral blood was from anonymous, healthy donors via the Gulf Coast Regional Blood Center (Houston, TX). All experimental protocols followed institutional ethical and biosafety regulations; per Texas A&M University policy, the use of these commercial donor samples did not need formal IRB approval.

Chemicals

Thapsigargin, rapamycin, AP20187 (B/B), phorbol 12-myristate 12-acetate, and ionomycin were purchased from Sigma Aldrich (St. Louis, MO, USA).

Molecular cloning and plasmid construction

Plasmids were generated by using the standard restriction enzyme cloning strategy. KOD Hot Start DNA polymerase (EMD Millipore, MA, USA) was used for PCR amplification. Restriction enzymes and T4 DNA ligase kit were purchased from New England BioLabs (Ipswich, MA, USA). DNA oligos encoding the N- and C-terminal regions of ORAI1-3 genes were synthesized and inserted downstream of mCherry in the pmCherry-N1 vector. Lyn11 was placed between mCherry and the ORAI variants, or the CAAX motif was fused at the C-terminus of the ORAI variants. For mutagenesis of O3CT, primers containing three degenerate “N” nucleotides at specific codons were used to introduce single amino acid substitutions via PCR. The resulting O3CT mutant fragments were cloned into the mCherry-O3CT-CAAX backbone. YFP-SOAR was constructed by inserting human STIM1 residues 343-491 into the pEYFP-N1 vector (Clontech, Mountain View, CA, USA) between XhoI and BamHI. Full-length STIM1-YFP was similarly generated by inserting the entire STIM1 cDNA into pEYFP-N1 using the same restriction sites. Mo-CRAB was created by truncating the CAAX motif in mCherry-CRAB and leaving it with a hexa-lysine tag. Di-CRAB and TD-CRAB were generated from Mo-CRAB by inserting GST or the tetramerization domain of p53 (residues 324-356), synthesized as DNA oligos from Azenta Genewiz (NJ, USA), between mCherry and CRAB. Chemo-CRAB was constructed by inserting FRB-FKBP between mCherry and CRAB in the Mo-CRAB backbone, and Chemo-CRAB-2 was generated by replacing FRB-FKBP with four tandem FKBP(F36V) units. Opto-CRAB was built on Mo-CRAB by inserting CRY2 between mCherry and CRAB. Opto-CRAB-2 was assembled by replacing CRY2 in Opto-CRAB with sspB, then inserting iLID-CAAX (amplified from Addgene #85680) downstream of CRAB and inserting a T2A in between CRAB and iLID-CAAX. For lentiviral packaging, CRAB variants were PCR-amplified and cloned into the pWPXL vector (Addgene #12257) using the BamHI and NdeI restriction sites.

Cell culture and transfection

HeLa cells (ATCC, CCL-2) and HEK293T (ATCC, CRL-3216) cells were purchased from the American Type Culture Collection (ATCC) and cultured at 37 °C with 5% CO₂ in Dulbecco’s Modified Eagle Medium (DMEM, Corning, USA) medium supplemented with 10% Fetal Bovine Serum (Cytiva, USA). For transient transfection, 100–300 ng plasmids were mixed with Lipofectamine 3000 (Thermo Fisher Scientific, USA) in Opti-MEM medium by following the manufacturer’s instructions. For live-cell imaging experiments, cells were seeded in four-chamber 35 mm glass-bottom dishes (Cellvis, Mountain View, CA, USA) at 70–80% confluency then imaged 24 h after transfection. Jurkat-Lucia™ NFAT cells were purchased from InvivoGen (Catalog code: jktl-nfat) and cultured at 37 °C with 5% CO₂ in Roswell Park Memorial Institute 1640 medium (RPMI 1640, Corning, USA) supplemented with 10% FBS. Raji cells (ATCC, CCL-86) and K562 (ATCC, CCL-243) cells were purchased from ATCC and cultured at 37 °C with 5% CO2 in RPMI 1640 supplemented with 10% FBS.

Isolation and transduction of human T Cells

Human peripheral blood was from anonymous, healthy donors via the Gulf Coast Regional Blood Center (Houston, TX). All experimental protocols followed institutional ethical and biosafety regulations; per Texas A&M University policy, the use of these commercial donor samples did not need formal IRB approval. Peripheral blood mononuclear cells (PBMCs) were isolated using SepMate-50 tubes (Stemcell Technologies) and Ficoll-Paque^TM PLUS density gradient media (Cytiva) via density gradient centrifugation. Following erythrocyte removal with ACK lysing buffer (Gibco), the EasySep Human T Cell Isolation Kit (Stemcell Technologies) was used to purify CD3⁺ T cells. Primary T cells were subsequently stimulated with Dynabeads^TM Human T-Activator CD3/CD28 (Thermo Fisher Scientific) at a 1:1 bead-to-cell ratio. Cultures were maintained in CTS^TM OpTmizer^TM T Cell Expansion medium (Gibco) supplemented with 200 IU/mL recombinant human IL-2 (PeproTech) and 2 mM L-glutamine (Gibco, A2916801) at 37 °C and 5% CO₂. Every 2–3 days, media were refreshed to maintain a concentration of 10⁶ cells/mL. For genetic modification, activated cells were treated with lentiviral supernatant and Polybrene Infection/Transfection Reagent (Sigma Aldrich). Transduction efficiency was quantified by measuring mCherry and GFP reporter expression through flow cytometry.

ΔΔG calculation

For ΔΔG predictions, we used the ddg_monomer protocol in Rosetta 3.14 with ref2015 weights³² across 50 iterations. Destabilizing mutations were identified by ΔΔG values of ≥ 1.0 Rosetta Energy Units (REU), neutral mutations were identified by ΔΔG values between 1 and −1 REU, and stabilizing mutations were identified by ΔΔG values ≤ −1 REU^33,89.

Live cell imaging, photostimulation, and image analysis

A Nikon Ti2 inverted epifluorescence microscope equipped with a Yokogawa W1 spinning disk scan-head, a LU-NV solid-state laser system, along with a live-cell culture cage to maintain the temperature at 37 °C with 5% CO₂, was used to acquire fluorescence imaging. 40 × or 60 × oil lens was used for high-resolution imaging. To perform photostimulation with repeated pulses of dark-light cycles, an external blue light source (470 nm, 4 mW/cm²; ThorLabs Inc., Newton, NJ, USA) or the built-in 488-nm laser source (1-5% input of the GFP excitation channel) was used. Data analysis was performed using Nikon NIS-Elements imaging software (version 4.5 1.00).

Colocalization assays

HeLa cells transiently co-expressing YFP-SOAR and indicated O3CT constructs were seeded onto 35-mm glass-bottom dishes (MatTek). Twenty-four hours post-transfection, cells were imaged on a Nikon Ti2 inverted epifluorescence microscope equipped with a 40× oil-immersion objective. Subcellular localization of YFP-SOAR was assessed by quantifying fluorescence intensity at the plasma membrane and cytosol using the “Intensity Line Profile” tool in NIS-Elements AR software (Nikon). The plasma membrane-to-cytosol fluorescence ratio (F_PM/F_cyto) was calculated for each cell. For each condition, 10–20 cells were selected for analysis, and all experiments were independently repeated for at least three times.

Real-time intracellular Ca²⁺ measurements

Ca²⁺ influx was monitored in HeLa cells stably expressing the green Ca²⁺ indicator GCaMP6s and transiently transfected with indicated CRAB variants (except for Opto-CRAB). Time-lapse imaging was taken with 10 s intervals and analyzed using NIS-Elements AR software (version 4.0; Nikon), with manually defined regions of interest (ROIs) for 40–60 cells per condition. To measure store-operated Ca²⁺ entry (SOCE), cells were incubated in Ca²⁺ free HBSS buffer (107 mM NaCl, 7.2 mM KCl, 1.2 mM MgCl₂, 11.5 mM glucose and 20 mM HEPES-NaOH, pH 7.2) 5 minutes prior to imaging, treated with 1 μM Thapsigargin (TG) to passively induce store depletion, followed by addition of CaCl₂ to a final concentration of 2 mM to trigger Ca²⁺ influx. To measure Ca²⁺ influx in cells expressing Opto-CRAB variants, HeLa cells stably expressing the red Ca²⁺ indicator R-GECO1.2 were transiently transfected with Opto-CRAB constructs. A 561-nm laser was used to excite R-GECO1.2 without overlapping the optogenetic activation window (450–490 nm). All experiments were performed three times independently.

NFAT translocation assay

HeLa cells stably expressing NFAT_1-460-GFP were seeded onto 35-mm glass-bottom dishes (MatTek) and transiently transfected with the indicated CRAB constructs using Lipofectamine 3000 (Thermo Fisher) according to the manufacturer’s protocol. Twenty-four hours post-transfection, cells were treated with 1 μM Thapsigargin (TG) to induce store-operated Ca²⁺ entry and activate NFAT signaling. Time-lapse imaging was performed on a Nikon Ti2 inverted epifluorescence microscope equipped with a 40× objective, capturing GFP fluorescence every 30 s for 60 min. Nuclear and cytoplasmic regions of interest (ROIs) were defined manually using NIS-Elements software, and nuclear GFP intensity was quantified over time, all experiments were independently repeated three times.

Bioluminescence resonance energy transfer (BRET) assay

HEK293T cells were seeded in 24-well plates and transfected with NanoLuc-CRAB variants and YFP-SOAR or STIM1-YFP, with a total of 0.5 μg DNA per well. After 24 h, cells were harvested, washed twice with cold PBS, and transferred to opaque 96-well plates. Bioluminescence was initiated using the Nano-Glo® Luciferase Assay System (Promega, N1110), and emission signals were recorded on a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek). Raw BRET signals were calculated as the emission intensity at 520–545 nm divided by the emission intensity at 450–470 nm. Net BRET signals were determined by subtracting the raw BRET signals obtained from cells expressing the NanoLuc donor alone. Data were analyzed using GraphPad Prism.

Electrophysiological measurements

HEK EPC9 USB patch amplifier controlled by Patchmaster software (HEKA Elektronik) was used to record conventional whole-cell CRAC current (I_CRAC) in HEK293T cells and Jurkat cells stably expressing the indicated CRAB variants. Recording protocol was used as previously²⁴, briefly, after achieving a whole-cell configuration mode, a 50 ms step to −100 mV followed by 50 ms ramp from −100 to +100 mV was applied every 2 s, at a holding potential of 0 mV. The internal solution contained (mM): 135 Cs aspartate, 6 MgCl₂, 10 BAPTA and 10 HEPES (pH 7.2 by CsOH). The external solution contained (mM): 130 NaCl, 4.5 KCl, 10 CaCl₂, 10 TEA-Cl, 10 D-glucose, and 5 HEPES (pH 7.4 by NaOH). Junction potential of 10 mV was applied to correct the liquid junction potential between internal and external solutions. Igor Pro 8.04 and GraphPad prism software were used for data analysis.

Zebrafish experiments

The zebrafish experiment was conducted according to internationally accepted standards. The Animal Care and Use Protocol was approved by the Purdue Animal Care and Use Committee (PACUC), adhering to the guidelines for using zebrafish in the NIH Intramural Research Program (protocol number: 1401001018). Zebrafish embryos were injected at the 1-cell stage with 300 ng of mRNAs encoding STIM1-WT and STIM1-R304W as described previously³⁶, together with 300 ng of mRNAs encoding PM-CRAB and Chemo-CRAB. Embryos were well-maintained in E3 embryo medium (in mM: 5 NaCl, 0.17 KCl, 10 HEPES, 0.33 MgSO₄, 0.33 CaCl₂, 60 mg/L penicillin G sodium salt, and 100 mg/L streptomycin sulfate; pH 7.8). Rapamycin (100 nM) was used to activate Chemo-CRAB. To quantify the number of cells with high levels of GFP signals, Tg(CD41:EGFP) embryos were anesthetized with 0.016% tricaine (Bide Pharm) and mounted on a glass culture dish coated with 3% agarose (BioFr). Images were acquired using an OLYMPUS SZX16 microscope equipped with an AxioCam MRm CCD camera and a light source for EGFP excitation (488 nm). Phenotype analysis was performed by categorical scoring of EGFP+ thrombocyte puncta in individual zebrafish embryos. Embryos were classified as normal expression when more than 20 distinct fluorescent puncta were observed. Reduced expression was defined as significant signal depletion, with 5-20 discernible puncta or fragmented fluorescence per embryo. Non-expression was defined as 0-5 detectable puncta per embryo. All scoring was conducted by an investigator blinded to the experimental conditions. Data are shown as mean ± sem from three independent biological replicates, with 20 embryos analyzed per group in each experiment. Images were processed using the ImageJ software (NIH). The data was plotted with GraphPad Prism software.

Lentivirus packaging and transduction

HEK293T cells (ATCC, CRL-3216) were transfected with psPAX2 and pMD2.G together with the PM-CRAB or Chemo-CRAB using the iMFectin DNA transfection reagent (GeneDEPOT). Viral supernatants were collected at 48 and 72 h after transfection and passed through 0.45 μm syringe filters to eliminate cellular debris. For Jurkat cell transduction, the filtered virus was added to 12-well plates together with polybrene (5 μg/ml; EMD Millipore).

NFAT-dependent luciferase reporter assay in Jurkat cells

NFAT-dependent luciferase reporter assay was preformed using Jurkat-Lucia NFAT reporter cells purchased from InvivoGen. PM-CRAB or Chemo-CRAB were transfected by lentivirus. Cells were stimulated with 20 ng/mL PMA and 5 µM ionomycin. Luciferase was measured 18 hours after stimulation. Coelenterazine was used as substrate when measuring luciferase. A Cytation 5 cell imaging multimode reader (BioTek) was used to measure luciferase activity.

ELISA measurements of cytokines

Jurkat cells expressing PM-CRAB or Chemo-CRAB were stimulated with 20 ng/mL PMA and 5 µM Ionomycin, Jurkat cells co-expressing CD19-CAR were stimulated with Raji cells (E:T = 5:1), and human primary T cells were co-cultured with Raji cells (E:T = 5:1). Supernatant was collected from all groups for IL-2, IFN-γ, and TNF-α quantification. Supernatant was diluted at a ratio of 1:20 for IL-2 and IFN-γ measurement, not diluted for TNF-α measurement. Samples were analyzed using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions. A Cytation 5 cell imaging multimode reader (BioTek) was used to measure absorbance.

Cell viability assay

HeLa cells were seeded at a density of 1 × 10⁴ cells per well in 96-well plates. Cells were transfected with mCh-CRAB-CAAX or mCh-CAAX using Lipofectamine 3000 (Thermo Fisher Scientific), and untransfected cells cultured under identical conditions served as controls. At 48 h post-transfection, cell viability was assessed using the Cell Counting Kit-8 (CCK-8; Dojindo) according to the manufacturer’s protocol. Briefly, 10 μL of CCK-8 reagent was added directly to each well containing 100 μL of culture medium (10% v/v final concentration), and cells were incubated at 37 °C for 2 h. Absorbance at 450 nm was measured using a microplate reader (Cytation 5, BioTek). Background absorbance from wells containing medium and CCK-8 reagent without cells was subtracted from all readings. Cell viability was normalized to the untransfected control group, which was set to 100%. For each condition, 3 technical replicates were included per experiment, and experiments were repeated independently three times. Data are presented as mean ± s.e.m.

Fluo-4 AM imaging

HeLa cells were seeded on onto 35-mm glass-bottom dishes (MatTek) and transfected with mCh-CRAB-CAAX or mCh-CAAX using Lipofectamine 3000 according to the manufacturer’s instructions. Cells were imaged 24 h post-transfection. For dye loading, cells were incubated with 2 µM Fluo-4 AM (Thermo Fisher Scientific) in HBSS for 20 min at room temperature, followed by three washes with HBSS. Imaging was performed in HBSS under unstimulated conditions. Transfected cells were identified by mCherry fluorescence. Mean cellular fluorescence intensity was quantified using NIS-Elements. Data represent 49–60 cells pooled from three independent biological replicates.

Statistics and reproducibility

Data are presented as mean ± SEM unless otherwise noted. The sample size for each experiment is indicated in the corresponding figure legends. No data were excluded from experiment analyses. All experiments were not randomized in this study. All representative confocal images were obtained from at least three independent experiments, each producing similar results. Graphs were created using GraphPad Prism version 10.6.0. Half time calculations were performed using the “one-phase decay” function in the software. Figures 1a, 2a, 3a, 4a, 4g, 5a, 6a, 6f, 7d. 7 h and Supplementary Figs. 1a, 4a were created by Biorender.com.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Source data supporting the findings of this study are included within the paper. Key constructs used in the study were also deposited in the nonprofit plasmid repository Addgene (No. 247044, 247045, 247047, 247048). Raw image data are available via Figshare: https://doi.org/10.6084/m9.figshare.31840099⁹⁰. Source data are providing with this paper. Source data are provided with this paper.

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Acknowledgements

The Tg(CD41:GFP) zebrafish line is a generous gift from Dr. Leonard I. Zon (Harvard Stem Cell Institute). We thank Dr. Kai Zhang from University of Illinois Urbana-Champaign for sharing the TrkA-ICD construct, and Dr. Mohamed Trebak for sharing the STIM1/2 double knockout cell line. This work was supported by the Blood Cancer United (to Y.Z.), the Cancer Prevention and Research Institute of Texas (RP250468 to Y.Z.), and the National Institutes of Health (R01GM144986 to Y.Z.; R01HL146652 to F.Z.; R35GM119787 to Q.D.; and R01CA240258, R01DK132286 & R35HL166557 to Y.H.). Portions of this research were conducted with the advanced computing resources provided by Texas A&M High Performance Research Computing.

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These authors contributed equally: Xiaoxuan Liu, Sher Ali, Tien-Hung Lan.

Authors and Affiliations

Center for Translational Cancer Research, Institute of Biosciences and Technology, Texas A&M University, Houston, TX, USA
Xiaoxuan Liu, Sher Ali, Tien-Hung Lan, Brendan McKee, Tatsuki Nonomura, Siyao Liu, Guolin Ma & Yubin Zhou
Department of Biological Sciences, Purdue University, West Lafayette, IN, USA
Decheng Wang & Qing Deng
Department of Biomedical Engineering, College of Engineering, Texas A&M University, College Station, TX, USA
Feng Zhao & Yun Huang
Department of Integrative Biology and Pharmacology, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX, USA
Michael X. Zhu
Center for Epigenetics and Disease Prevention, Institute of Biosciences and Technology, Texas A&M University, Houston, TX, USA
Yun Huang
ORBIT Platform, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
Guolin Ma
Department of Translational Medical Sciences, College of Medicine, Texas A&M University, Houston, TX, USA
Yubin Zhou

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Xiaoxuan Liu
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Sher Ali
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Tien-Hung Lan
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Decheng Wang
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Contributions

Y.Z. and G.M. conceived the ideas and directed the work. G.M., Q.D. and Y.Z. designed the study. G.M., X.L., S.A., T.H.L., D.W. and B.M. performed the experiments. G.M., X.L., S.A., T.H.L., D.W. and B.M. analyzed the results. B.M., F.Z., M.X.Z. and Y.H. provided intellectual inputs and technical supports. Y.Z., T.H.L., X.L., S.A., Q.D. and G.M. wrote the manuscript. All the authors contributed to the discussion and editing of the manuscript.

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Correspondence to Qing Deng, Guolin Ma or Yubin Zhou.

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Competing interests

Y.Z. has submitted a patent application to the United States Patent and Trademark Office pertaining to the design and biomedical application aspect(s) of this work (no. 64/031,929; with Y.Z., X.L., S.L., T.H.L., and G.M. as inventors). The remaining authors declare no competing interests.

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Liu, X., Ali, S., Lan, TH. et al. Engineering of genetically encoded programmable calcium channel inhibitory binders. Nat Commun 17, 3472 (2026). https://doi.org/10.1038/s41467-026-71769-2

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Received: 29 September 2025
Accepted: 30 March 2026
Published: 13 April 2026
Version of record: 13 April 2026
DOI: https://doi.org/10.1038/s41467-026-71769-2