The Development of FRET-Based IP₃ Biosensors and Their Use for Monitoring IP₃ Dynamics during Ca²⁺ Oscillations and Ca²⁺ Waves in Non-Excitable Cells

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High resolution imaging of intracellular calcium (Ca²⁺) concentrations ( [Ca²⁺] _i) has revealed that Ca²⁺ signals show diverse responses, such as Ca²⁺ oscillations and Ca²⁺ waves; these signals are exploited to control diverse cellular processes. Ca²⁺ responses in non-excitable cells are primarily regulated by inositol 1,4,5-trisphosphate (IP₃). To elucidate the mechanisms of these diverse Ca²⁺ responses, we have developed a series of fluorescent IP₃ biosensors, the LIBRAv series, using cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) in combination with fluorescence resonance energy transfer (FRET) technologies. These fluorescent IP₃ biosensors are specific to IP₃ and permit the monitoring of IP₃ dynamics in single living cells. Studies conducted with IP₃ biosensors during agonist-induced Ca²⁺ oscillations revealed cell type-specific differences in IP₃ dynamics, non-fluctuating rises in cytosolic IP₃ concentrations ( [IP₃] _i) and repetitive IP₃ spikes. These results provide important experimental information for resolving long-standing questions regarding the mechanisms responsible for Ca²⁺ oscillations. IP₃ biosensors also demonstrated an intracellular gradient of IP₃ and its wave-like expansion during mechanical stimulation-induced Ca²⁺ waves. As such, continued improvements in IP₃ biosensors and the development of additional novel fluorescent biosensors are highly likely to provide a better understanding of the regulatory mechanisms of various forms of Ca²⁺ signals and clarification of their physiologic roles.