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

Libra是一种检测肌醇1，4，5-三磷酸(IP_3)的荧光生物传感器，它由大鼠III型IP_3受体(IP_3R)的配体结合域与青色和黄色荧光蛋白组成。在本研究中，我们开发了一系列IP3生物传感器，该传感器具有很强的pH稳定性和对IP3的不同亲和力，以及一种定量测量单个活细胞中IP3浓度([IP3]i)的方法。用改进的生物传感器和定量测量方法研究了激动剂诱导的钙振荡过程中的IP_3动力学。我们的结果显示了细胞类型依赖性的IP_3动力学差异；在COS-7细胞和HSY-EA1细胞中，[IP_3]_i和重复性IP_3峰分别在Ca~(2+)振荡过程中无波动地升高。HSY-EA1细胞IP_3峰的大小从10 nm到100 nm不等，第二次及以后的尖峰出现在[IP_3]_i降至静息水平之前。[IP_3]_i在Ca~(2+)振荡开始时表现出明显的波动，而在Ca~(2+)振荡过程中，[IP_3]_i的重复峰逐渐消失。此外，[IP_3]_i尖峰位于Ca^2+尖峰之前。这些观察结果不支持IP_3在Ca~(2+)振荡中波动的要求，也发现Libra及其IP_3不敏感变体应该有助于IP_3Rs配体结合部位的特定激动剂和拮抗剂的检测。我们将该方法应用于IP_3Rs新配体的筛选。在计算对接到I型IP_3R结合域的基础上，我们合成了四个新的代谢稳定的IP_3类似物，其中两个化合物是用Libra计算得到的新的IP_3配体。我们进一步使用带有来自类型I、II或III型的IP_3R的配体结合域的Libra系列来检验腺磷素衍生物的亚型特异性。

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.