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

Abstract

In mammals, three genes encode IP₃ receptors (IP₃Rs), which are involved in agonist-induced Ca²⁺ signaling in cells of apparently all types. Using the CRISPR/Cas9 approach for disruption of two out of three IP₃R genes in HEK-293 cells, we generated three monoclonal cell lines, IP3R1-HEK, IP3R2-HEK, and IP3R3-HEK, with the single functional isoform, IP₃R1, IP₃R2, and IP₃R3, respectively. All engineered cells responded to ACh with Ca²⁺ transients in an "all-or-nothing" manner, suggesting that each IP₃R isotype was capable of mediating CICR. The sensitivity of cells to ACh strongly correlated with the affinity of IP₃ binding to an IP₃R isoform they expressed. Based on a mathematical model of intracellular Ca²⁺ signals induced by thapsigargin, a SERCA inhibitor, we developed an approach for estimating relative Ca²⁺ permeability of Ca²⁺ store and showed that all three IP₃R isoforms contributed to Ca²⁺ leakage from ER. The relative Ca²⁺ permeabilities of Ca²⁺ 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 Ca²⁺ signals in ER, engineered cells were ranged by resting levels of stored Ca²⁺ as IP3R3-HEK ≥ IP3R1-HEK > IP3R2-HEK. The developed cell lines could be helpful for further assaying activity, regulation, and pharmacology of individual IP₃R isoforms.

Keywords: CRISP/Cas9; Ca2+ imaging; Ca2+ signaling; IP3 receptor; R-CEPIA1er.