Agonist-Induced Ca 2+ Signaling in HEK-293-Derived Cells Expressing a Single IP 3 Receptor Isoform.

In mammals, three genes encode IP ₃ receptors (IP ₃ Rs), which are involved in agonist-induced Ca ²⁺ signaling in cells of apparently all types. Using the CRISPR/Cas9 approach for disruption of two out of three IP ₃ R genes in HEK-293 cells, we generated three monoclonal cell lines, IP3R1-HEK, IP3R2-HEK, and IP3R3-HEK, with the single functional isoform, IP ₃ R1, IP ₃ R2, and IP ₃ R3, respectively. All engineered cells responded to ACh with Ca ²⁺ transients in an "all-or-nothing" manner, suggesting that each IP ₃ R isotype was capable of mediating CICR. The sensitivity of cells to ACh strongly correlated with the affinity of IP ₃ binding to an IP ₃ R isoform they expressed. Based on a mathematical model of intracellular Ca ²⁺ signals induced by thapsigargin, a SERCA inhibitor, we developed an approach for estimating relative Ca ²⁺ permeability of Ca ²⁺ store and showed that all three IP ₃ R isoforms contributed to Ca ²⁺ leakage from ER. The relative Ca ²⁺ permeabilities of Ca ²⁺ stores in IP3R1-HEK, IP3R2-HEK, and IP3R3-HEK cells were evaluated as 1:1.75:0.45. Using the genetically encoded sensor R-CEPIA1er for monitoring Ca ²⁺ signals in ER, engineered cells were ranged by resting levels of stored Ca ²⁺ as IP3R3-HEK ≥ IP3R1-HEK > IP3R2-HEK. The developed cell lines could be helpful for further assaying activity, regulation, and pharmacology of individual IP ₃ R isoforms.