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

• Published in: Cells (Basel, Switzerland) Vol. 11; no. 13; p. 2037
• Main Authors: Martínez-Martínez, Ericka, Sánchez-Vázquez, Víctor Hugo, León-Aparicio, Daniel, Sanchez-Collado, Jose, Gallegos-Gómez, Martín-Leonardo, Rosado, Juan A., Arias, Juan M., Guerrero-Hernández, Agustin
• Format: Journal Article
• Language: English
• Published: Basel MDPI AG 27.06.2022; MDPI
• Subjects: Calcium (intracellular); Calcium channels; Calcium influx; Calcium signalling; Endoplasmic reticulum; Inositol 1,4,5-trisphosphate receptors; Kinases; Mutants; Orai1 protein; Oscillations; phosphomimetic; phosphorylated Orai1; Phosphorylation; PKC; Protein kinase C; Proteins; SOCE; Thapsigargin
• ISSN: 2073-4409; 2073-4409
• DOI: 10.3390/cells11132037

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.