Introduction

Cystic fibrosis transmembrane conductance regulator (CFTR) is essential for epithelial ion and fluid secretion. While CFTR is primarily activated via cAMP/PKA signaling, the role of intracellular Ca²⁺ signaling—particularly through store-independent mechanisms—remains incompletely understood.

Aims & Methods

We aimed to elucidate how store-independent Ca²⁺ influx via Orai1 regulates basal CFTR activity in primary polarized epithelial cells.
To investigate this we used mouse and human pancreatic, hepatic and airway organoids, we combined RNA-sequencing, immunofluorescence, Fura-2 and MQAE dye-based Ca²⁺/Cl⁻ imaging, FLIM-FRET, dSTORM microscopy, and in vivo fluid secretion assays. Genetic silencing and pharmacological inhibition (CM5480) were employed to dissect Orai1-dependent signaling pathways.

Results

We discovered that Orai1 mediates a constitutive, store-independent Ca²⁺ influx sustained by SPCA2. This influx occurs independently of ER Ca²⁺ depletion but requires STIM1. Super-resolution imaging revealed Orai1 clustering into discrete apical membrane nanodomains that colocalize with SPCA2, STIM1, PKA, and CFTR. Functional assays confirmed that this localized signaling complex maintains basal CFTR activity. Inhibiting Orai1 reduced basal Cl⁻ and HCO₃⁻ secretion, disrupted mitochondrial ATP production, and suppressed pancreatic ductal fluid secretion in vitro and in vivo. Further, the stimulatory effect of this Ca²⁺ influx on CFTR was mediated via Ca²⁺/calmodulin-sensitive adenylyl cyclases (AC1, 3, 8), which were co-clustered with Orai1 and CFTR. Their knockdown phenocopied Orai1 inhibition, and no additive effect was observed when both pathways were blocked.

Conclusion

Our results reveal a previously unrecognized SPCA2–STIM1–Orai1 nanodomain that drives store-independent Ca²⁺ influx, activating CFTR via local cAMP production in the absence of neurohormonal stimuli. This regulatory module is conserved across secretory epithelia and represents a key mechanism for maintaining basal fluid secretion and epithelial homeostasis.