Thermal dynamics of metabotropic glutamate receptor signaling revealed by subtraction microcalorimetric measurements on live cells
Thermal dynamics of metabotropic glutamate receptor signaling revealed by subtraction microcalorimetric measurements on live cells
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通过活细胞减法微量热测量揭示代谢型谷氨酸受体信号传导的热动力学
DOI：
10.1038/protex.2013.061
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Takeda M & Yoshioka T
中科院分区：
文献类型：
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作者：
Ikeda M;Takeda M & Yoshioka T
等温滴定量热法(ITC)是一种用于分析分子间相互作用的通用方法，主要发生在非生物系统中。在这里，我们成功地将ITC应用于活细胞中受体-配体相互作用的表征。在本研究中，CHO细胞或表达代谢型谷氨酸受体-1α(CHO-mGluR1α)的细胞被悬浮在含有蛋白酶抑制剂的低CaCl2缓冲盐水中，并用激动剂(DHPG)滴定。在最佳记录条件下，DHPG的滴定产生的热的时间常数在野生型和CHO-mGluR1α细胞中不同。这些信号的减去显示了mGluR1α依赖的双峰反应，如代谢性受体信号的热成像所表明的那样，具有快速的热吸收和连续的热产生。用滴定曲线表征了mGluR1DHPG与α的相互作用和每个活细胞的结合位点数。该技术在理论上也适用于其他受体表达模型和配体。该方案需要一天的时间来完成成对测量和单减法研究。
Isothermal titration calorimetry (ITC) is a versatile method used to analyze molecule-to-molecule interactions primarily occurring in non-living systems. Here, we successfully applied ITC for the characterization of receptor-ligand interactions in live cells. In this study, CHO cells or those expressing metabotropic glutamate receptor-1α (CHO-mGluR1α) were suspended in low CaCl 2 buffered-saline containing protease inhibitors at incremented temperatures and titrated with agonist (DHPG). Under the optimal recording conditions, titration of DHPG produced heat with time constants that differed between wild type-and CHO-mGluR1α cells. Subtraction of these signals revealed mGluR1α-dependent bimodal responses with rapid heat absorption and successive heat production, as suggested by the thermal imaging of metabotropic receptor signaling. Using the titration curve, mGluR1α-DHPG interactions and the number of binding sites per live cell were characterized. This technique is theoretically applicable for other receptor expression models and ligands. This protocol requires a day to complete the pairwise measurements and single subtraction study.