A practical guide to the synthesis and use of membrane-permeant acetoxymethyl esters of caged inositol polyphosphates.
A practical guide to the synthesis and use of membrane-permeant acetoxymethyl esters of caged inositol polyphosphates.
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笼中肌醇聚磷酸盐的膜 - 佩氧甲基酯的合成和使用的实用指南。
DOI：
10.1038/nprot.2010.194
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发表时间：
2011-03
期刊：
影响因子：
14.8
通讯作者：
Ellis-Davies, Graham C. R.
中科院分区：
文献类型：
--
作者：
Kantevari, Srinivas;Gordon, Grant R. J.;MacVicar, Brian A.;Ellis-Davies, Graham C. R.
该方案描述了一种有效化学合成肌醇-1，4，5-三磷酸（IP 3）六乙酰氧基甲酯类似物的方法，该类似物在6-羟基位置上具有邻硝基藜芦基光化学笼化基团。这六种酯使探针膜具有渗透性，使得它可以在体外或体内加载到完整的活细胞中。在细胞内，笼状IP 3是惰性的，直到被720 nm的双光子激发激活。光致解放的信号分子可以动员释放的Ca 2+从细胞内储存的内质网。当与荧光Ca 2+指示剂rhod-2共同加载时，一束激光可用于以单细胞分辨率刺激和监测细胞内Ca 2+信号传导。该协议有化学和生物学部分;前者描述了笼中IP 3的有机合成，这需要12天，后者应用于为期一天的研究急性分离的大鼠脑切片中星形胶质细胞调节的神经元功能。由于Ca 2+是细胞内最重要的第二信使，并且许多细胞使用IP 3-Ca 2+信号级联来产生Ca 2+浓度的增加，因此该方法应广泛适用于完整生物系统中各种生理过程的研究。
This protocol describes a method for efficient chemical synthesis of an analog of inositol-1,4,5-trisphosphate (IP3) hexakis acetoxymethyl ester having an ortho-nitroveratryl photochemical caging group on the 6-hydroxyl position. The six esters render the probe membrane permeant, such that it can be loaded into intact living cells in vitro or in vivo. Inside cells, the caged IP3 is inert until activated by two-photon excitation at 720 nm. The photoliberated signaling molecule can mobilize release of Ca2+ from intracellular stores on the endoplasmic reticulum. When co-loaded with the fluorescent Ca2+ indicator rhod-2, one laser can be used for stimulating and monitoring intracellular Ca2+ signaling with single-cell resolution. This protocol has chemistry and biology sections; the former describes the organic synthesis of the caged IP3, which requires 12 d, and the latter an application to a day-long study of astrocyte-regulated neuronal function in living brain slices acutely isolated from rats. As Ca2+ is the single most important intracellular second messenger and the IP3-Ca2+ signaling cascade is used by many cells to produce increases in Ca2+ concentration, this method should be widely applicable for the study of a variety of physiological processes in intact biological systems.
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影响因子：
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作者：
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作者：
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通讯作者：
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