Tanaka Kiwamu, Choi Jeongmin, Stacey Gary
Divisions of Plant Science and Biochemistry, University of Missouri, Columbia, MO, USA.
Methods Mol Biol. 2013;1043:45-54. doi: 10.1007/978-1-62703-532-3_5.
Heterotrimeric GTP-binding proteins (G-proteins) and G-protein-coupled receptors are important signaling components in eukaryotes. In plants, the G-proteins are involved in diverse physiological processes, some of which are exerted via changes in the level of cytosolic free calcium concentration ([Ca(2+)]cyt). Various techniques have been developed to measure the change of [Ca(2+)]cyt, e.g., calcium-sensitive microelectrodes, chemical fluorescent dyes, and biosensors based on luminescent or fluorescent indicators. In this chapter, we describe a protocol for in vivo [Ca(2+)]cyt measurement in G-protein mutants expressing aequorin, a luminescent-based calcium biosensor, to extend our knowledge about G-protein mediated Ca(2+) signaling. This method is also applicable to other early signaling events that are mediated by changes in [Ca(2+)]cyt levels.
异源三聚体GTP结合蛋白（G蛋白）和G蛋白偶联受体是真核生物中重要的信号传导成分。在植物中，G蛋白参与多种生理过程，其中一些是通过胞质游离钙浓度（[Ca(2+)]cyt）水平的变化来实现的。已经开发出各种技术来测量[Ca(2+)]cyt的变化，例如钙敏感微电极、化学荧光染料以及基于发光或荧光指示剂的生物传感器。在本章中，我们描述了一种在表达水母发光蛋白（一种基于发光的钙生物传感器）的G蛋白突变体中进行体内[Ca(2+)]cyt测量的方案，以扩展我们对G蛋白介导的Ca(2+)信号传导的认识。该方法也适用于其他由[Ca(2+)]cyt水平变化介导的早期信号事件。
