STUDIES ON THE DYNAMICS OF PHOSPHOINOSITIDE PATHWAYS AND THE DEVELOPMENT OF FLUORESCENT BIOSENSORS

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LIBRA is a fluorescent biosensor for inositol 1,4,5-trisphosphate (IP_3), which is composed of the ligand-binding domain of the rat type III IP_3 receptor (IP_3R) and cyan and yellow fluorescent proteins. In the present study, we developed a series of IP_3 biosensors that exhibit strong pH stability and varying affinities for IP_3, as well as a method for the quantitative measurement of cytosolic concentrations of IP_3([IP_3]_i] in single living cells. Improved biosensors and the method for the quantitative measurement were used to elucidate IP_3dynamics during agonist-induced Ca2+ oscillations. Our result demonstrated cell type-dependent differences in IP_3dynamics; a non-fluctuating rise in [IP_3]_i and repetitive IP_3spikes during Ca^2+ oscillations in COS-7cells and HSY-EA1cells, respectively. The size of IP_3spikes in HSY-EA1cells varied from10to100nM, and the second and later spikes were initiated before the decline of [IP_3]_i to resting levels. While [IP_3]_i showed clear fluctuations at the beginning of Ca^2+ oscillations, repetitive spikes of [IP_3]_i gradually obscured during Ca^2+ oscillations. In addition, [IP_3]_i spike peaks were preceded by Ca^2+ spike peaks. These observations do not support the requirement of IP_3fluctuations in Ca^2+ oscillations.It is also found that LIBRA and its IP_3-insensitive variant should facilitate the detection of specific agonists and antagonists of the ligand-binding site of IP_3Rs. We applied this method for the screening of novel ligand for IP_3Rs. On the basis of computational docking of the ligands to the binding domain of the type I IP_3R, we synthesis of four novel metabolically stabilized analogues of IP_3. Of these analogues, two compounds were novel IP_3 ligands using LIBRA. We further examined the subtype specificity of adenophostin derivatives using series of LIBRA with ligand-binding domains of IP_3Rs from type I, II, or III.