![]() Inspired by successful design of a light-activatable viral potassium channel designated BLINK1 26, we set out to engineer a more challenging target, the ORAI1 channel made of four-pass TM domains and assembled as a hexamer in the plasma membrane 27, 28. i Representative confocal images showing blue light-triggered Ca 2+ response and changes in subcellular localization of NFAT 1-460-GFP. The construct showing the least dark activation and high Ca 2+ response in the lit-condition was designated LOCa3, which bears two mutations in ORAI1: H171D and P245T. h Biplot showing the degrees of NFAT nuclear translocation in HeLa cells expressing evolved mutants, either in the dark ( x-axis) or under photo-illumination ( y-axis). g The experimental flow for high-throughput screening of evolved LOCa constructs. f Blue light modulated Ca 2+ entry (top) and NFAT nuclear translocation (bottom) in HeLa cells expressing LOCa2. n = 24 cells from three independent assays. Blue bar, photo-illumination at 470 nm with a power density of 40 µW/mm 2. The domain architecture of the construct is shown above the curve. Two cycles of light stimulation were applied. e Light-induced changes in cytosolic Ca 2+ reported by GCaMP6m in HeLa cells transfected with LOCa2. n = 22-66 cells from three independent assays. S6 showed the highest light-dependent changes and was named as LOCa2. d Comparison of light-induced Ca 2+ changes after inserting LOV2 into the indicated positions of the ORAI1-P245T variant. TM3 and the second extracellular loop regions targeted for randomized mutagenesis are highlighted in green. The LOV2 insertion sites tested in the study are indicated as arrowheads. ![]() ![]() c Snake-like diagram of the ORAI1 Ca 2+ channel. The mutant P245T showed the most notable light-induced changes in intracellular Ca 2+ (designated LOCa1). LOV2 was inserted between residues R167 and M168. b The fold-change of photo-induced Ca 2+ responses reported by GCaMP6m in HeLa cells expressing LOV2–ORAI1 hybrid variants. Upon photosimulation at 470 nm, conformational changes within LOV2 trigger allosteric activation of engineered ORAI1 to evoke Ca 2+ flux across the plasma membrane. The LOV2 domain is inserted into the intracellular loop of a constitutively active ORAI1 (caORAI1), which maintains the hybrid channel in a largely closed state in the dark. a Schematic depiction of photo-switchable Ca 2+ influx through an engineered ORAI1 Ca 2+ channel. Rational design, randomized mutations via error-prone PCR, and high-throughput fluorescence-based screening assays were employed to generate LOCa as a light-gated Ca 2+ channel that could reversibly mediate Ca 2+ influx without the need for exogenous cofactors.ĭata are shown as mean ± s.e.m. We envision that photon-induced conformational changes in LOV2 could trigger allosteric ORAI1 gating (Fig. To overcome these hurdles, we set out to engineer a single-component light- operated Ca 2+ (LOCa) channel by inserting the light-oxygen-voltage domain (LOV2) of Avena sativa phototropin 1 into various regions of ORAI1 25, a four-pass transmembrane (TM) protein that constitutes the pore-forming subunit of the CRAC channel 1, 2, 3, 4. Furthermore, attempts have been made to generate channelrhodopsin-2 (ChR2) variants with increased selectivity for Ca 2+ over other cations 24, but they generally fail to match the exceptional Ca 2+ selectivity seen in CRAC channels ( P Ca/ P Na > 1000) 2. These engineered receptors, nonetheless, could lead to the co-activation of diacylglycerol (DAG)-mediated signaling to initiate non-Ca 2+-related physiological responses. Other optogenetic tools capable of inducing transient intracellular Ca 2+ mobilization include light-activated chimeric G protein-coupled receptors (GPCRs) and receptor tyrosine kinases 20, 21, 22, 23. ![]() However, STIM1-based GECAs have two intrinsic drawbacks: the absolute requirement of endogenous ORAI channels and the potential side effects arising from crosstalk with other STIM1-associated targets, such as transient receptor potential (TRP) channels and voltage-gated Ca 2+ (Ca V) channels 16, 17, 18, 19. Recently, plant-derived photosensitive modules have been engineered into STIM1 to generate genetically encoded Ca 2+ actuators (GECA) 5, 8, 9, 10, 11, thereby enabling remote and noninvasive control of CRAC channel-mediated Ca 2+ entry into cells 12, 13, 14, 15. Aberrant STIM1–ORAI1 signaling has been intimately linked to immunoinflammatory disorders, myopathy, tumorigenesis, and neurodegenerative diseases, making the CRAC channel a potential therapeutic target 5, 6, 7. The prototypical Ca 2+ release-activated Ca 2+ (CRAC) channel, composed of ORAI1 and the stromal interaction molecule 1 (STIM1), constitutes an important Ca 2+ entry route in mammalian cells 1, 2, 3, 4.
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