Reddish Florence N, Miller Cassandra L, Gorkhali Rakshya, Yang Jenny J
Department of Chemistry, Center of Diagnostics and Therapeutics (CDT), Georgia State University.
Department of Chemistry, Center of Diagnostics and Therapeutics (CDT), Georgia State University;
J Vis Exp. 2017 May 19(123):55822. doi: 10.3791/55822.
Intracellular calcium (Ca) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of intracellular calcium release are the major intracellular calcium storage organelles, the endoplasmic reticulum (ER) and the more specialized sarcoplasmic reticulum (SR) in muscle cells. The dynamic release of calcium from these organelles is mediated by the ryanodine receptor (RyR) and the inositol 1,4,5-triphosphate receptor (IP3R) with refilling occurring through the sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. A genetically encoded calcium sensor (GECI) called CatchER was created to monitor the rapid calcium release from the ER/SR. Here, the detailed protocols for the transfection and expression of the improved, ER/SR-targeted GECI CatchER in HEK293 and C2C12 cells and its application in monitoring IP3R, RyR, and SERCA pump-mediated calcium transients in HEK293 cells using fluorescence microscopy is outlined. The receptor agonist or inhibitor of choice is dispersed in the chamber solution and the intensity changes are recorded in real time. With this method, a decrease in ER calcium is seen with RyR activation with 4-chloro-m-cresol (4-cmc), the indirect activation of IP3R with adenosine triphosphate (ATP), and inhibition of the SERCA pump with cyclopiazonic acid (CPA). We also discuss protocols for determining the in situ Kd and quantifying basal [Ca] in C2C12 cells. In summary, these protocols, used in conjunction with CatchER, can elicit receptor mediated calcium release from the ER with future application in studying ER/SR calcium related pathologies.
细胞外刺激引发的细胞内钙（Ca）瞬变启动了生物体中的多种生物学过程。细胞内钙释放的核心是主要的细胞内钙储存细胞器，即内质网（ER）和肌肉细胞中更特殊的肌浆网（SR）。这些细胞器中钙的动态释放由兰尼碱受体（RyR）和肌醇1,4,5-三磷酸受体（IP3R）介导，而通过肌浆/内质网钙ATP酶（SERCA）泵进行再填充。一种名为CatchER的基因编码钙传感器（GECI）被创建用于监测内质网/肌浆网中钙的快速释放。在此，概述了在HEK293和C2C12细胞中转染和表达改进的、靶向内质网/肌浆网的GECI CatchER及其在使用荧光显微镜监测HEK293细胞中IP3R、RyR和SERCA泵介导的钙瞬变中的应用的详细方案。选择的受体激动剂或抑制剂分散在腔室溶液中，并实时记录强度变化。通过这种方法，在用4-氯间甲酚（4-cmc）激活RyR、用三磷酸腺苷（ATP）间接激活IP3R以及用环匹阿尼酸（CPA）抑制SERCA泵时，可以观察到内质网钙的减少。我们还讨论了在C2C12细胞中确定原位解离常数（Kd）和定量基础钙浓度[Ca]的方案。总之，这些与CatchER结合使用的方案可以引发内质网中受体介导的钙释放，未来可应用于研究内质网/肌浆网钙相关的病理学。
