Green genetically encoded IP ₃ biosensor for hierarchical analysis of its signaling network

Inositol 1,4,5-trisphosphate (IP ₃ ) is a key second messenger that regulates diverse physiological processes. Visualization of IP ₃ dynamics in living cells is therefore important for understanding its signaling processes. In this study, we developed genetically encoded green fluorescent IP ₃ biosensors named Green iPenguins with distinct half-maximal effective concentrations (EC ₅₀ ) for IP ₃ , enabling detection of IP ₃ signals over a range of concentrations. The biosensors displayed more than a 4-fold increase in fluorescence intensity upon IP ₃ and showed high specificity for IP ₃ over structurally related molecules. When expressed in HEK293T cells, the biosensors enabled visualization of IP ₃ dynamics involved in different signaling pathways. They were also compatible with dual-color imaging, allowing simultaneous monitoring of IP ₃ together with cAMP or Ca ²⁺ signals. In addition, the hierarchical relationship between IP ₃ and Ca ²⁺ signaling was visualized, providing insight into the temporal relationship between these two second messengers. The biosensors are expected to facilitate future studies of physiological processes involving IP ₃ signaling networks.