SOCE: Store-Operated Calcium Entry; GECA: Genetically Encoded Calcium Channel Actuators; STIM1: Stromal Interaction Molecule 1; CRAC: Calcium Release-Activated Calcium; ChR2: Channel Protein-2; LOCa: Light-Operated Calcium channel; LOV2: Light-Oxygen-Voltage domain 2; NFAT: Nuclear Factor of Activated T cells; HSPCs: Hematopoietic Stem and Progenitor Cells; Tet2: Ten-eleven Translocation 2; LSK: Lin^- c-Kit⁺ Sca-1⁺ population; NFAT-RE: NFAT Response Element; CaRROT: Ca²⁺-Responsive Transcriptional Reprogramming Tool; AD: Alzheimer's Disease; Aβ42: Amyloid beta 42

Optogenetics utilizes the fusion of naturally occurring light-sensing proteins with target proteins to induce conformational changes, oligomerization, or dissociation in response to illumination of light with specific wavelengths. This technique allows precise spatiotemporal control of intracellular signal transduction, offering new insights into the physiological and pathological mechanisms underlying health and diseases [1]. The calcium release-activated calcium (CRAC) channel, composed of stromal interaction molecule 1 (STIM1) and ORAI1, stands out as one of the most selective calcium channels known to date, playing a crucial role in mediating store-operated calcium entry (SOCE) in mammalian cells [2-4]. Aberrant STIM1-ORAI1 signaling is intricately linked to immune-inflammatory diseases, skeletal disorders, tumorigenesis, and neurodegenerative diseases, thus making the CRAC channel an attractive therapeutic target [5].

Genetically encoded calcium channel actuators (GECA), as gene-encoded optogenetic tools, offer a promising approach for remotely activating Ca²⁺ signals with merits including rapid reversibility, superior spatiotemporal resolution, and non-invasiveness [6]. The microbial opsin-based channelrhodopsin-2 (ChR2) enables researchers to precisely and remotely modulate intracellular calcium levels using light. However, it should be noted that ChR2's ion selectivity is not exclusive to calcium ions, as it is also permeable to other cationic ions. Even its mutant variant CatCh exhibits higher Ca²⁺ permeability but lacks the high selectivity for Ca²⁺ compared to CRAC [3,7]. Further improvements have been made by several laboratories to directly incorporate blue light-dependent photosensitive modules such as cryptochrome 2 (CRY2) or light-oxygen-voltage domain 2 (LOV2) into STIM1[6,8,9]. These engineered STIM1 was then utilized to remotely optically activate Ca²⁺ entry signals through ORAI channels. However, it is essential to consider that these optogenetic tools based on STIM1 may have the potential to interfere with other STIM1-related signaling targets, such as transient receptor potential (TRP) channels and voltage-gated Ca²⁺ (CaV) channels [10-12], potentially resulting in side effects. Moreover, the absolute requirement for endogenous ORAI1 channels may limit the applications of these tools in cells with no or low levels of ORAI [8,9]. Careful assessment and validation of the specificity and selectivity of these tools will be crucial to ensure their safe and effective use in research and potential therapeutic applications.

Notably, the lack of Ca²⁺ selectivity or crosstalk with other signaling pathways may raise the possibility of side effects. As research progresses, addressing these challenges will be crucial to fully realize the potential of optogenetic-based calcium modulators in various biological systems. In the work described in "Engineering of a bona fide light-operated calcium channel" [13], we successfully designed a single-component light-operated calcium channel (LOCa) that allows reversible induction of cellular Ca²⁺ influx, independent of exogenous cofactors. This was achieved by inserting the light-oxygen-voltage domain 2 (LOV2) of Avena sativa phototropin 1 into the intracellular loop of a constitutively active ORAI1 mutant, resulting in a blue-light-gated Ca²⁺ channel. Remarkably, LOCa exhibits pharmacological and biophysical characteristics similar to those of the native ORAI1 channel. Compared to other optogenetic GECAs, LOCa demonstrates relatively low basal activity, high Ca²⁺ selectivity, independence of endogenous STIM or ORAI expression, and unique kinetic properties. This tool opens new possibilities for precise manipulation of intracellular calcium dynamics in various cellular contexts.

To design LOCa, we initially attempted to insert LOV2 into different loop regions known to be crucial for ORAI1 channel activation [14,15]. However, these fusion or insertion attempts of LOV2 failed to induce light-controlled intracellular Ca²⁺ changes, possibly due to the insufficient free energy (~3.8 kcal/mol) generated by the conformational switch of LOV2 to open the ORAI1 Ca²⁺ channel [16,17]. To overcome this limitation, we utilized a constitutively active mutant variant of ORAI1 (P245T) as an engineering template to reduce energy costs [18]. Furthermore, by optimizing the LOV2 insertion site within the intracellular loop of ORAI1, we significantly increased light-induced Ca²⁺ influx with the construct LOCa2. However, when examining responses of cells expressing LOCa2, we observed a biphasic light-induced changes in cytoplasmic Ca²⁺ levels. The downstream Ca²⁺-responsive transcription factor, nuclear factor of activated T cells (NFAT), also exhibited pre-activation evidenced by nuclear localization before light stimulation, indicating that the engineered LOV2-ORAI1 construct was not entirely sequestered in the dark state. To overcome this issue, we proceeded with the third round of optimization using an error-prone PCR strategy and high-throughput fluorescence-based screening assays. Given the close correlation between TM3-TM4 helix coupling and STIM1-induced ORAI1 channel gating [19], we introduced additional mutations in TM3 and the second extracellular loop through random mutagenesis. After a rigorous selection process using NFAT nuclear entry and Ca²⁺ signal as readouts, we identified a construct with dual mutations H171D/P245T (referred to as LOCa3) that displayed minimal activation in the dark. LOCa3 was confirmed to exhibit a higher dynamic range of light-activated Ca²⁺ influx, along with good reversibility and faster activation kinetics. Notably, the degree of Ca²⁺ influx could be modulated by adjusting the intensity of input light. Importantly, we demonstrated that LOCa3 enables precise temporal and spatial control of Ca²⁺ signaling in mammalian cells, irrespective of the presence of endogenous ORAI and STIM expression. Comprehensive pharmacological and biophysical studies on LOCa3 verified that the engineered construct maintains pharmacological characteristics similar to ORAI1, and LOV2 insertion into ORAI1 does not compromise its Ca²⁺ selectivity. These results showcase LOCa3 as a promising tool for optogenetic-based manipulation of calcium signaling in various biological systems.

Next, we explored the potential of LOCa3 in regulating Ca²⁺-dependent cellular functions within the cells. We first employed LOCa3 and its light-induced calcium influx to regulate the cell fate of hematopoietic stem cells (HSCs). Hematopoietic stem and progenitor cells (HSPCs) from wild-type (WT) and Ten-eleven Translocation 2 knockout (Tet2-KO) mice were isolated and transduced with a retrovirus encoding LOCa3. The in vitro self-renewal capacity of HSPCs was assessed in the absence or presence of light. It is known that HSPCs exhibit enhanced HSC self-renewal and increased hematopoietic capacity when the Tet2 gene is disrupted, while increased Ca²⁺ influx leads to the suppression of HSC self-renewal [20,21]. Tet2-KO LSK cells (Lin^- c-Kit⁺ Sca-1⁺ population) showed a significant reduction in the presence of light, whereas normal HSPCs showed only a modest, nonsignificant decrease in LSK cells, indicating that light-induced Ca²⁺ entry indeed suppressed the self-renewal of HSPCs. This observation suggests that light-induced Ca²⁺ influx through LOCa3 may potentially rescue the abnormal self-renewal of Tet2-deficient HSCs. Next, we demonstrated that LOCa3-induced Ca²⁺ entry can be utilized to activate downstream Ca²⁺-responsive NFAT response elements, leading to the expression of luciferase reporter or necroptosis-inducing genes. As expected, there was an increase in luciferase activity or cell death in the light-treated cells. Moreover, by combining LOCa3 with CaRROT (Ca²⁺-responsive transcriptional reprogramming tool) and utilizing sgRNA targeting the MYOD1 promoter region, light stimulation can induce the expression of the target gene [22]. Therefore, LOCa3 can be used for optogenetic transcriptional programming in mammalian cells. This highlights the potential of LOCa3 for optogenetic transcriptional programming in mammalian cells. Overall, our findings indicate that LOCa3 can serve as a powerful tool for precise manipulation of Ca²⁺ signaling, enabling researchers to explore various Ca²⁺-dependent cellular functions and their potential implications in disease mechanisms and therapeutic applications.

In addition to its applications in cellular studies, LOCa3 has been successfully utilized for in vivo optogenetic intervention. Impaired calcium influx through SOCE has been implicated in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD) [23]. Evidence suggests that enhancing SOCE activity may ameliorate AD symptoms. To explore this, we generated AD fly models with a neuron-specific expression of Aβ42 and employed LOCa3 to stimulate neuronal calcium signaling, aiming to rescue impaired neuronal SOCE activity [24]. Remarkably, the LOCa3 transgenic flies exposed to pulsed blue light displayed an accelerated climbing ability compared to the dark group. This observation suggests that LOCa3 has the ability to rescue the neurodegenerative phenotype in a light-dependent manner. Our findings provide evidence for the feasibility of using optogenetic modulation of Ca²⁺ signaling for potential intervention of neurodegeneration in vivo. These results underscore the potential of LOCa3 as a versatile tool for studying and manipulating calcium signaling in vivo, providing valuable insights into its therapeutic applications in neurodegenerative diseases and beyond. This innovative approach opens new avenues for targeted interventions and treatments in various disease contexts.

Overall, the single-component GECA LOCa3, with its advantageous features of relatively low basal activity and high Ca²⁺ selectivity, has demonstrated the potential to precisely control cytosolic Ca²⁺ concentration and physiological processes through blue light stimulation. Its independence from STIM or ORAI expression makes it compatible with various cell types, while its lack of crosstalks with other signaling pathways reduces the risk of side effects. Consequently, we believe that LOCa3 represents a new generation of GECA with broad applications in both in vitro and in vivo studies in the future. However, there is still room for improvement, such as the dark activity, kinetics, and Ca²⁺ dynamic range. Our ultimate goal is to develop an ideal LOCa with no dark activity, fast kinetics, and a broad range of calcium dynamics. One potential solution is to explore novel LOV2 domains from other species. By utilizing various LOV2 domains, we hope to introduce diverse response times and conformational change abilities to ORAI, thus enhancing the performance of LOCa in future applications. Additionally, our ORAI1-LOV2 hybrid strategy can be extended to other types of ion channels, enabling precise photo manipulation for a wide range of ion channel-related studies. Through ongoing research and innovations, we anticipate that LOCa and related GECA technologies will continue to evolve, providing researchers with powerful tools for investigating calcium signaling and its impact on cellular functions and disease mechanisms. With further refinement, LOCa and similar optogenetic tools hold great promise in advancing our understanding of biology and offering potential therapeutic applications in the future.

In conclusion, LOCa3 represents a significant advancement in the field of optogenetics, providing researchers with a powerful and precise tool to control cytosolic Ca²⁺ concentration and study various biological processes related to disease mechanisms. As a single-component, optically switchable, and Ca²⁺-selective channel, LOCa3 offers versatility in its applications. Its unique features, including relatively low basal activity, high Ca²⁺ selectivity, and independence from STIM or ORAI expression, make LOCa3 compatible with a wide range of cell types. Its ability to achieve temporal and spatial control over Ca²⁺ signaling holds immense potential for personalized immune or neural modulation, paving the way for future treatments of immune or neurodegenerative diseases. With ongoing research, further improvements in LOCa3 and related optogenetic tools, we can look forward to more insights into cellular processes and disease pathways. LOCa3's contribution to personalized therapeutic approaches and disease intervention may hold promise for addressing various health challenges in the future.

WL wrote the original draft, LH and YW reviewed and edited the manuscript with help from LW.

The authors declare no competing interests.

This work was supported by the Ministry of Science and Technology of China (2019YFA0802104 for WY), the National Natural Science Foundation of China (32301222 for LH, 91954205 for WY).