ORAI1 Knockout Cell Line (HEK293)

Store-operated calcium entry (SOCE) provided through channels formed by ORAI proteins is a major regulator of several cellular processes. In immune cells, it controls fundamental processes such as proliferation, cell adhesion, and migration, while in cancer, SOCE and gene expression are dysregulated and lead to abnormal migration and/or cell proliferation. In the present study, we used the CRISPR/Cas9 technique to delete the gene and to identify its role in proliferative and migrative properties of the model cell line HEK-293. We showed that ORAI1 deletion greatly reduced SOCE. Thereby, we found that this decrease and the absence of ORAI1 protein did not affect HEK-293 proliferation. In addition, we determined that ORAI1 suppression did not affect adhesive properties but had a limited impact on HEK-293 migration. Overall, we showed that ORAI1 and SOCE are largely dispensable for cellular proliferation, migration, and cellular adhesion of HEK-293 cells. Thus, despite its importance in providing Ca entry in non-excitable cells, our results indicate that the lack of SOCE does not deeply impact HEK-293 cells. This finding suggests the existence of compensatory mechanism enabling the maintenance of their physiological function.

ORAI1 and stromal interaction molecule 1 (STIM1) are the critical mediators of store-operated Ca entry by acting as the pore subunit and an endoplasmic reticulum-resident signaling molecule, respectively. In addition to Ca signaling, STIM1 is also involved in regulation of the type I IFN (IFN-I) response. To examine their potential role in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we generated and knockout human HEK293-angiotensin-converting enzyme 2 cells and checked their responses. knockout cells showed strong resistance to SARS-CoV-2 infection as a result of enhanced IFN-I response. On the contrary, deletion induced high susceptibility to SARS-CoV-2 infection. Mechanistically, knockout cells showed reduced homeostatic cytoplasmic Ca concentration and severe impairment in tonic IFN-I signaling. Transcriptome analysis showed downregulation of multiple antiviral signaling pathways in knockout cells, likely because of reduced expression of the Ca-dependent transcription factors of the AP-1 family and Accordingly, modulation of homeostatic Ca concentration by pretreatment with ORAI1 blocker or agonist could influence baseline expression and resistance to SARS-CoV-2 infection in a human lung epithelial cell line. Our results identify a novel role of ORAI1-mediated Ca signaling in regulating the tonic IFN-I levels, which determine host resistance to SARS-CoV-2 infection.

The role of Ca release-activated Ca (CRAC) channels mediated by ORAI isoforms in calcium signalling has been extensively investigated. It has been shown that the presence or absence of different isoforms has a significant effect on store-operated calcium entry (SOCE). Yoast et al. (2020) showed that, in addition to the reported narrow-spike oscillations (whereby cytosolic calcium decreases quickly after a sharp increase), ORAI1 knockout HEK293 cells were able to oscillate with broad-spike oscillations (whereby cytosolic calcium decreases in a prolonged manner after a sharp increase) when stimulated with a muscarinic agonist. This suggests that Ca influx through ORAI-mediated CRAC channels negatively regulates the duration of Ca oscillations. We hypothesise that, through the activation of protein kinase C (PKC), ORAI1 negatively regulates phospholipase C (PLC) activity to decrease inositol 1,4,5-trisphosphate (IP) production and limit the duration of agonist-evoked Ca oscillations. Based on this hypothesis, we construct a new mathematical model, which shows that the formation of broad-spike oscillations is highly dependent on the absence of ORAI1. Predictions of this model are consistent with the experimental results.