Calcium store refilling and STIM activation in STIM- and Orai-deficient cell lines

Area of Science:

• Cellular Biology
• Molecular Biology
• Physiology

Background:

• Store-operated calcium entry (SOCE) is a fundamental cellular mechanism involving STIM proteins and Orai channels for calcium signal regulation.
• The complexity of multiple STIM and Orai gene homologs complicates the understanding of their specific roles in calcium signaling.
• CRISPR/Cas9 gene editing enables precise genetic manipulation to elucidate the functions of these proteins.

Purpose of the Study:

• To investigate the essentiality of SOCE for immediate and long-term endoplasmic reticulum (ER) calcium homeostasis.
• To determine the role of STIM1's plasma membrane (PM) translocation and clustering in SOCE activation.
• To delineate the specific contributions of STIM and Orai homologs in mediating calcium signals.

Main Methods:

• Generation of STIM or Orai knockout (KO) cell lines using CRISPR/Cas9 gene editing.
• Monitoring of ER calcium stores and calcium signals in engineered cell lines.
• Förster resonance energy transfer (FRET) analysis to study STIM1 translocation and oligomerization.
• Expression of truncated STIM1 variants in KO cells to assess functional rescue of SOCE.

Main Results:

• SOCE-null KO cells retained significant ER calcium stores (50-70% of wildtype) in unstimulated conditions.
• Acute refilling of ER calcium stores was completely blocked in KO cells after store depletion, but stores were refilled within 24 hours.
• STIM1's plasma membrane tethering and clustering at ER-PM junctions facilitate but are not essential for its conformational switch, oligomerization, and Orai channel activation.

Conclusions:

• SOCE is critical for the immediate replenishment of ER calcium stores but is dispensable for maintaining long-term ER calcium homeostasis.
• STIM1 oligomerization and clustering at ER-PM junctions are important for efficient SOCE activation but not strictly required for Orai channel gating.
• Understanding the distinct roles of STIM and Orai homologs is crucial for deciphering complex calcium signaling pathways.