Agonist-Induced Ca²⁺ Signaling in HEK-293-Derived Cells Expressing a Single IP₃ Receptor Isoform

Area of Science:

• Cellular Biology
• Molecular Biology
• Biochemistry

Background:

• Inositol trisphosphate receptors (IP3Rs) mediate cellular calcium (Ca2+) signaling, crucial for various cell functions.
• Three IP3R genes (IP3R1, IP3R2, IP3R3) exist in mammals, but their individual roles in Ca2+ signaling are not fully elucidated.

Purpose of the Study:

• To generate and characterize HEK-293 cell lines expressing single IP3R isoforms (IP3R1, IP3R2, or IP3R3).
• To investigate the functional contribution of each IP3R isoform to agonist-induced Ca2+ transients and Ca2+ store permeability.

Main Methods:

• CRISPR/Cas9 gene editing was used to create monoclonal HEK-293 cell lines lacking two of the three IP3R genes.
• Acetylcholine (ACh) stimulation was used to induce Ca2+ transients.
• A mathematical model and the genetically encoded sensor R-CEPIA1er were employed to assess Ca2+ store properties.

Main Results:

• All engineered cell lines exhibited "all-or-nothing" Ca2+ responses to ACh, indicating each IP3R isoform can mediate Ca2+ induced Ca2+ release (CICR).
• Cellular sensitivity to ACh correlated with the IP3 binding affinity of the expressed IP3R isoform.
• Relative Ca2+ permeabilities of the Ca2+ stores were determined as 1:1.75:0.45 for IP3R1, IP3R2, and IP3R3, respectively.
• Resting Ca2+ levels in the endoplasmic reticulum (ER) were ranked as IP3R3-HEK ≥ IP3R1-HEK > IP3R2-HEK.

Conclusions:

• Individual IP3R isoforms are capable of mediating Ca2+ signaling and contribute to Ca2+ leakage from the ER.
• The developed cell lines provide a valuable tool for dissecting the specific roles, regulation, and pharmacology of each IP3R isoform.