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

Ericka Martínez-Martínez1, Víctor Hugo Sánchez-Vázquez1, Daniel León-Aparicio1,2

  • 1Department of Biochemistry, Cinvestav, Mexico City 07360, Mexico.

Cells
|July 9, 2022
PubMed

Insights

Phosphorylation of Orai1 channels by protein kinase C (PKC) influences calcium (Ca2+) entry and release. This study reveals how Orai1 phosphorylation impacts calcium signaling, particularly its interaction with IP3 receptors.

Area of Science:

  • Cell Biology
  • Molecular Physiology
  • Calcium Signaling

Background:

  • Store-operated calcium entry (SOCE) is a critical process regulated by the Orai1 channel.
  • Previous studies indicated Orai1 overexpression inhibits SOCE under specific conditions.
  • The role of Orai1 phosphorylation in modulating calcium entry and release remains incompletely understood.

Purpose of the Study:

  • To investigate the impact of Orai1 phosphorylation at S27/S30 residues on calcium (Ca2+) entry and endoplasmic reticulum (ER) Ca2+ levels.
  • To elucidate the functional consequences of Orai1 phosphorylation on its interaction with inositol trisphosphate receptors (IP3Rs).
  • To determine the effect of Orai1 phosphorylation status on agonist-induced Ca2+ oscillations.

Main Methods:

  • Utilized wild-type (wt) Orai1 and mutant channels (O1-AA, O1-DD, O1-E106A) in HeLa cells.
  • Stimulated cells with ATP and thapsigargin (TG) in the presence of external Ca2+.
  • Measured Ca2+ entry, ER Ca2+ depletion, Orai1-IP3R interaction, and intracellular Ca2+ ([Ca2+]i) oscillations.

Main Results:

  • Orai1 overexpression enhanced Ca2+ entry and limited ER depletion when stimulated with ATP and TG.
  • Phosphorylation-resistant (O1-AA) and wild-type (O1-wt) Orai1 supported enhanced Ca2+ entry, unlike phosphomimetic (O1-DD) or dead-pore mutants.
  • Orai1 phosphorylation at S27/S30 was found to interfere with IP3R activity, reducing Ca2+ release, and altered Orai1-IP3R interaction and Ca2+ oscillation frequency.

Conclusions:

  • The combination of ATP and TG stimulates Ca2+ entry via Orai1.
  • Phosphorylation of Orai1 at S27/S30 residues by PKC plays a crucial role in modulating Ca2+ signaling.
  • Orai1 phosphorylation negatively impacts IP3R-mediated Ca2+ release and alters the dynamics of Ca2+ oscillations.
Keywords:
PKCSOCEphosphomimeticphosphorylated Orai1

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