Orai1 mediates ICRAC and SOCE in human placental extravillous trophoblasts and contributes to cell viability, migration, and invasion.

A healthy pregnancy depends on proper placental development, which requires tightly regulated calcium (Ca2+) signaling. In non-excitable cells, store-operated Ca2+ entry (SOCE) is mediated by Ca2+ release-activated Ca2+ (CRAC) channels, composed of Orai1, Orai2 and Orai3 subunits and activated by stromal interaction molecules (STIM1 and STIM2). In this study, we investigated the potential role of CRAC channels in extravillous trophoblast (EVT) function. Using the human placenta-derived EVT cell line HTR8/SVneo, we combined CRISPR/Cas9, immunocytochemistry, confocal microscopy, whole-cell patch-clamp electrophysiology, Ca2+ imaging, pharmacological CRAC channel inhibitors, and functional assays of cell proliferation, migration, and invasion to determine the contribution of Orai1 to the regulation of key trophoblast functions. In human primary EVTs, STIM1-2 and Orai1-3 mRNA expression was analyzed from single-cell RNA-sequencing data (Cambridge Trophoblast Atlas). Similar to human primary EVT, HTR8/SVneo cells expressed all STIM and Orai isoforms, and Orai1 is the predominant pore-forming ion channel subunit. Targeted deletion of Orai1 or its pharmacological inhibition with BTP2 or GSK-7975A significantly reduced ICRAC, SOCE, and impaired EVT cell viability, migration, and invasion. In conclusion, our findings demonstrate the role of Orai1 in mediating ICRAC, SOCE, and SOCE-dependent EVT functions and suggest its potential involvement in EVT (patho)physiology.