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, Orai1 alpha, containing 301 amino acids, and the short form, Orai1 beta, which arises from alternative translation initiation from methionines 64 or 71, in Orai1 alpha. 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 Orai1 alpha 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 Orai1 beta to the plasma membrane when expressed individually; by contrast, when Orai1 beta is co-expressed with Orai1 alpha, cell treatment with TG induced rapid trafficking and insertion of compartmentalized Orai1 beta 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.

Automated summary

Orai1 is a subunit of CRAC channels involved in various cellular functions. Ca2+ store depletion leads to the trafficking and insertion of compartmentalized Orai1 alpha in the plasma membrane via a mechanism independent of cytosolic Ca2+ concentration, while Orai1 beta can only translocate to the plasma membrane when co-expressed with Orai1 alpha in response to TG treatment.