Authors
Masayuki Ikeda, Masahiro Takeda, and Tohru Yoshioka
Summary
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 CaCl2 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.Further details
The protocol was published on Protocol Exchange on 21 June 2013. To see the entire protocol, click on the source link.Advertisement
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