Differential functional role of Orai1 variants in constitutive Ca2+ entry and calcification in luminal breast cancer cells

Abstract

Resting cytosolic Ca²⁺ concentration is tightly regulated to fine-tune Ca²⁺-dependent cellular functions. Luminal breast cancer cells exhibit constitutive Ca²⁺ entry mediated by Orai1 and the secretory pathway Ca²⁺-ATPase, SPCA2, which result in mammary microcalcifications that constitute a prognostic marker of mammary lesions. Two Orai1 isoforms have been identified, the full-length Orai1α, consisting of 301 amino acids, and the short variant, Orai1β, lacking the 63 or 70 N-terminal amino acids comprising residues involved in channel inactivation and binding sites with Orai1 partners. We show that only the mammalian-specific Orai1α rescues SPCA2-dependent constitutive Ca²⁺ entry in Orai1-KO MCF7 cells, a widely used luminal breast cancer cell line. FRET analysis and immunoprecipitation revealed that Orai1α shows a greater ability to interact with SPCA2 than Orai1β. Deletion of the first 38 amino acids in Orai1α reduced the interaction with SPCA2 to a similar extent as Orai1β, thus suggesting that the N-terminal 38 amino acids play a relevant role in Orai1α-SPCA2 interaction. Finally, Orai1α, but not Orai1β, rescue the ability of Orai1-deficient cells to form in vitro microcalcifications. These findings provide compelling evidence for a functional role of Orai1α in constitutive Ca²⁺ entry in MCF7 cells, which might be a target to prevent the development of mammary microcalcifications in luminal breast cancer.

Keywords: Orai1α; Orai1β; SPCA2; breast cancer; calcification; constitutive Ca(2+) entry.