Cell Lines with a Single Functional Isoform of the IP₃ Receptor

Many agonists regulate cellular functions through surface receptors coupled by the phosphoinositide cascade to Са²⁺ mobilization. In unexcitable cells, the generation of Са²⁺ signals relies largely on Са²⁺ release from из Са²⁺ store localized in the endoplasmic reticulum (ER). In this system, IP₃ receptors (IP₃Rs), which are intracellular IP₃-gated Са²⁺- channels, mediate controllable Ca²⁺ release from Ca²⁺ store in response to cell stimulation. A variety of factors embarrass both the analysis of a specific role for IP₃Rs in cell physiology and detailing their regulatory mechanisms. Firstly, three genes encode IP₃Rs, while cells usually express two or even all of them. Moreover, different IP₃R isoforms are under control of distinct mechanisms. Yet, isoform-specific antagonists of IP₃Rs have not been identified yet. Cell lines that express a single isoform appear to be an effective cellular model for studying regulatory mechanisms, pharmacology, and physiological role of I-P₃R subtypes. Here we employed the CRISPR/Cas9 technology to inactivate genes of IP₃Rs in cells of the HEK-293 line, which express all three isoforms. Several monoclones of genetically modified HEK-293 were obtained, and those containing biallelic inactivation mutation in two IP₃R genes were identified. Finally, three monoclonal cell lines, each containing the only functional IP₃R isoform, were generated. These cellular models can be used to evaluate a role for IP₃R of a particular subtype in agonist-induced Ca²⁺ signaling as well as for the analysis of their regulatory mechanisms.