BPS2026 – High-throughput screening for ryanodine receptor modulators using a novel green ER Ca2+ sensor protein

Ca 2+ release channels on the ER/SR membrane, such as ryanodine receptors (RyRs) and inositol triphosphate receptors (IP3Rs), play important roles in various cell signaling pathways. Therefore, their inhibitors and activators of these channels are highly sought after for therapeutic development and research purposes. We previously screened RyR inhibitors and activators in 96-well plates using HEK293 cells expressing RyR and the red-fluorescent ER Ca 2+ sensor protein R-CEPIA1er (Murayama et al., Mol Pharmacol. 2018; Takenaka et al., Molecular Pharmacol . 2023). Here, we modified this method and established a high-throughput screening (HTS) system compatible with 384-well plates. We used HEK293 cells expressing a bright green-fluorescent ER Ca 2+ sensor protein G-CEPIA3er with a high signal-to-noise ratio and disease-linked mutant RyRs. Using this method, we screened a library consisting of 9,600 compounds for RyR2-specific modulators and identified several hit compounds. We further characterized the binding domains of the hit compounds using RyR1-RyR2 chimeric proteins. This HTS system is useful for large-scale screening of modulators of ER Ca 2+ regulatory proteins, including the ER Ca 2+ release channels.