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.