A Quartet of Native Orai Channel Isoforms Orchestrates Graded NFAT Activation and Transcription

The Ca²⁺ release-activated Ca²⁺ (CRAC) channel mediates store-operated calcium entry (SOCE), a ubiquitous pathway essential for many cell types, including immune cells. Three Orai (Orai1/2/3) proteins constitute the plasma membrane pore-forming units of CRAC channels that are activated by the endoplasmic reticulum (ER) Ca²⁺-sensing STIM1/2 proteins when ER Ca²⁺ stores are depleted. Orai1/2/3 are differentially expressed across primary cells with discernible differences in their structures and biophysical properties. Further, Orai1 has two alternatively translated isoforms: long mammalian-specific Orai1α and the 63-residue shorter Orai1β, which is evolutionarily older and conserved across vertebrates. Whether Orai1α/1β/2/3 produce unique cytosolic Ca²⁺ signatures that bias transcriptional responses through effectors like NFAT is unclear. Here, we used HEK293 cells engineered to express one native Orai isoform and show that all Orai isoforms couple to NFAT1/4 induction. The magnitude of NFAT1/4 induction for each Orai isoform matches that of SOCE, with the following profile: Orai1β>Orai1α>>Orai2>Orai3. Near-native re-expression of either Orai1α or Orai1β in primary murine Orai1 ^-/- CD4⁺ T cells restored SOCE, NFAT activation, cytokine production and promoted near identical transcriptional responses enriched for immune activation pathways. An analysis of genetic and clinical data of human individuals showed that homozygous null mutations selectively abolishing Orai1α are not associated with disease resembling CRAC channelopathy. Primary T cells from individuals homozygous or heterozygous for an Orai1α null mutation showed enhanced, rather than impaired, SOCE and NFAT induction. Our data indicate that NFAT activation and transcriptional outputs are primarily driven by the graded strength of SOCE mediated by each isoform of the Orai quartet.