Fluorescent biosensor for quantitative real-time measurements of inositol 1,4,5-trisphosphate in single living cells

Abstract

The second messenger inositol 1,4,5-trisphosphate (IP3) plays a central role in the generation of a variety of spatiotemporally complex intracellular Ca2+ signals involved in the regulation of many essential physiological processes. Here we describe the development of "LIBRA", a novel ratiometric fluorescent IP3 biosensor that allows for the quantitative monitoring of intracellular IP3 concentrations in single living cells in real time. LIBRA consists of the IP3-binding domain of the rat type 3 IP3 receptor fused between the fluorescence resonance energy transfer pair cyan fluorescent protein and yellow fluorescent protein and preceded by a membrane-targeting signal. We show that the LIBRA fluorescent signal is highly selective for IP3 and unaffected by concentrations of Ca2+ and ATP in the physiological range. In addition, LIBRA can be calibrated in situ. We demonstrate the utility of LIBRA by monitoring the temporal relationship between the responses intracellular IP3 and Ca2+ concentrations in SH-SY5Y cells following acetylcholine stimulation.