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

In mammals, three genes encode IP receptors (IPRs), which are involved in agonist-induced Ca signaling in cells of apparently all types. Using the CRISPR/Cas9 approach for disruption of two out of three IPR genes in HEK-293 cells, we generated three monoclonal cell lines, IP3R1-HEK, IP3R2-HEK, and IP3R3-HEK, with the single functional isoform, IPR1, IPR2, and IPR3, respectively. All engineered cells responded to ACh with Ca transients in an "all-or-nothing" manner, suggesting that each IPR isotype was capable of mediating CICR. The sensitivity of cells to ACh strongly correlated with the affinity of IP binding to an IPR isoform they expressed. Based on a mathematical model of intracellular Ca signals induced by thapsigargin, a SERCA inhibitor, we developed an approach for estimating relative Ca permeability of Ca store and showed that all three IPR isoforms contributed to Ca leakage from ER. The relative Ca permeabilities of Ca 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 signals in ER, engineered cells were ranged by resting levels of stored Ca as IP3R3-HEK ≥ IP3R1-HEK > IP3R2-HEK. The developed cell lines could be helpful for further assaying activity, regulation, and pharmacology of individual IPR isoforms.