Abstract
The current optogenetic toolkit lacks a robust single-component Ca2+-selective ion channel tailored for remote control of Ca2+ signaling in mammals. Existing tools are either derived from engineered channelrhodopsin variants without strict Ca2+ selectivity or based on the stromal interaction molecule 1 (STIM1) that might crosstalk with other targets. Here, we describe the design of a light-operated Ca2+ channel (designated LOCa) by inserting a plant-derived photosensory module into the intracellular loop of an engineered ORAI1 channel. LOCa displays biophysical features reminiscent of the ORAI1 channel, which enables precise optical control over Ca2+ signals and hallmark Ca2+-dependent physiological responses. Furthermore, we demonstrate the use of LOCa to modulate aberrant hematopoietic stem cell self-renewal, transcriptional programming, cell suicide, as well as neurodegeneration in a Drosophila model of amyloidosis.
Highlights
The current optogenetic toolkit lacks a robust single-component Ca2+-selective ion channel tailored for remote control of Ca2+ signaling in mammals
After testing four reported constitutively active ORAI1 (caORAI1) mutants[32,33,34] with LOV2 inserted in the intracellular loop connecting TM2 and TM3, we discovered that the ORAI1(P245T)-LOV2 hybrid construct showed the largest light-induced Ca2+ influx (Fmax/F0: ~1.5; Fig. 1b)
By optimizing the LOV2 insertion site within the intracellular loop (Fig. 1c,d), we further enhanced the dynamic range of Ca2+ changes from 1.5 to 2.1, with the best construct (LOV2 insertion at Site 6 between residues S163 and P164; Fig. 1c–e) named as LOCa2
Summary
The current optogenetic toolkit lacks a robust single-component Ca2+-selective ion channel tailored for remote control of Ca2+ signaling in mammals. Other optogenetic tools capable of inducing transient intracellular Ca2+ mobilization include light-activated chimeric G protein-coupled receptors (GPCRs) and receptor tyrosine kinases[20,21,22,23] These engineered receptors, could lead to the co-activation of diacylglycerol (DAG)-mediated signaling to initiate non-Ca2+-related physiological responses. Attempts have been made to generate channelrhodopsin-2 (ChR2) variants with increased selectivity for Ca2+ over other cations[24], but they generally fail to match the exceptional Ca2+ selectivity seen in CRAC channels (PCa/PNa > 1000)[2] To overcome these hurdles, we set out to engineer a singlecomponent light-operated Ca2+ (LOCa) channel by inserting the light-oxygen-voltage domain (LOV2) of Avena sativa phototropin 1 into various regions of ORAI125, a four-pass transmembrane (TM) protein that constitutes the pore-forming subunit of the CRAC channel[1,2,3,4]. Rational design, randomized mutations via error-prone PCR, and high-throughput fluorescence-based screening assays were employed to generate LOCa as a light-gated Ca2+ channel that could reversibly mediate Ca2+ influx without the need for exogenous cofactors
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