PKC-Mediated Orai1 Channel Phosphorylation Modulates Ca²⁺ Signaling in HeLa Cells

The overexpression of the Orai1 channel inhibits SOCE when using the Ca²⁺ readdition protocol. However, we found that HeLa cells overexpressing the Orai1 channel displayed enhanced Ca²⁺ entry and a limited ER depletion in response to the combination of ATP and thapsigargin (TG) in the presence of external Ca²⁺. As these effects require the combination of an agonist and TG, we decided to study whether the phosphorylation of Orai1 S27/S30 residues had any role using two different mutants: Orai1-S27/30A (O1-AA, phosphorylation-resistant) and Orai1-S27/30D (O1-DD, phosphomimetic). Both O1-wt and O1-AA supported enhanced Ca²⁺ entry, but this was not the case with O1-E106A (dead-pore mutant), O1-DD, and O1-AA-E106A, while O1-wt, O1-E106A, and O1-DD inhibited the ATP and TG-induced reduction of ER [Ca²⁺], suggesting that the phosphorylation of O1 S27/30 interferes with the IP₃R activity. O1-wt and O1-DD displayed an increased interaction with IP₃R in response to ATP and TG; however, the O1-AA channel decreased this interaction. The expression of mCherry-O1-AA increased the frequency of ATP-induced sinusoidal [Ca²⁺]_i oscillations, while mCherry-O1-wt and mCherry-O1-DD decreased this frequency. These data suggest that the combination of ATP and TG stimulates Ca²⁺ entry, and the phosphorylation of Orai1 S27/30 residues by PKC reduces IP₃R-mediated Ca²⁺ release.