Functional differences in agonist-induced plasma membrane expression of Orai1α and Orai1β

Orai1 is the pore-forming subunit of the store-operated Ca²⁺ release-activated Ca²⁺ (CRAC) channels involved in a variety of cellular functions. Two Orai1 variants have been identified, the long form, Orai1α, containing 301 amino acids, and the short form, Orai1β, which arises from alternative translation initiation from methionines 64 or 71, in Orai1α. Orai1 is mostly expressed in the plasma membrane, but a subset of Orai1 is located in intracellular compartments. Here we show that Ca²⁺ store depletion leads to trafficking and insertion of compartmentalized Orai1α in the plasma membrane via a mechanism that is independent on changes in cytosolic free-Ca²⁺ concentration, as demonstrated by cell loading with the fast intracellular Ca²⁺ chelator dimethyl BAPTA in the absence of extracellular Ca²⁺ . Interestingly, thapsigargin (TG) was found to be unable to induce translocation of Orai1β to the plasma membrane when expressed individually; by contrast, when Orai1β is co-expressed with Orai1α, cell treatment with TG induced rapid trafficking and insertion of compartmentalized Orai1β in the plasma membrane. Translocation of Orai1 forms to the plasma membrane was found to require the integrity of the actin cytoskeleton. Finally, expression of a dominant negative mutant of the small GTPase ARF6, and ARF6-T27N, abolished the translocation of compartmentalized Orai1 variants to the plasma membrane upon store depletion. These findings provide new insights into the mechanism that regulate the plasma membrane abundance of Orai1 variants after Ca²⁺ store depletion.