Tanimura Akihiko, Nezu Akihiro, Morita Takao, Turner R James, Tojyo Yosuke
Department of Dental Pharmacology, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan.
J Biol Chem. 2004 Sep 10;279(37):38095-8. doi: 10.1074/jbc.C400312200. Epub 2004 Jul 22.
The second messenger inositol 1,4,5-trisphosphate (IP(3)) plays a central role in the generation of a variety of spatiotemporally complex intracellular Ca(2+) signals involved in the regulation of many essential physiological processes. Here we describe the development of "LIBRA", a novel ratiometric fluorescent IP(3) biosensor that allows for the quantitative monitoring of intracellular IP(3) concentrations in single living cells in real time. LIBRA consists of the IP(3)-binding domain of the rat type 3 IP(3) 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 IP(3) and unaffected by concentrations of Ca(2+) 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 IP(3) and Ca(2+) concentrations in SH-SY5Y cells following acetylcholine stimulation.
第二信使肌醇1,4,5 - 三磷酸（IP(3)）在产生各种时空复杂的细胞内Ca(2+)信号中起着核心作用，这些信号参与调控许多重要的生理过程。在此，我们描述了“天秤座”（LIBRA）的开发，这是一种新型的比率荧光IP(3)生物传感器，可实时定量监测单个活细胞内的IP(3)浓度。LIBRA由大鼠3型IP(3)受体的IP(3)结合结构域组成，融合于荧光共振能量转移对的青色荧光蛋白和黄色荧光蛋白之间，并在前面加上膜靶向信号。我们表明，LIBRA荧光信号对IP(3)具有高度选择性，不受生理范围内Ca(2+)和ATP浓度的影响。此外，LIBRA可原位校准。我们通过监测乙酰胆碱刺激后SH - SY5Y细胞内IP(3)和Ca(2+)浓度反应之间的时间关系，证明了LIBRA的实用性。
