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Design and application of a class of sensors to monitor Ca2+ dynamics in high Ca2+ concentration cellular compartments

By Shen Tang, Hing-Cheung Wong, Zhong-Min Wang, Yun Huang, Jin Zou, You Zhuo, Andrea Pennati, Giovanni Gadda, Osvaldo Delbono and Jenny J. Yang

Abstract

Quantitative analysis of Ca2+ fluctuations in the endoplasmic/sarcoplasmic reticulum (ER/SR) is essential to defining the mechanisms of Ca2+-dependent signaling under physiological and pathological conditions. Here, we developed a unique class of genetically encoded indicators by designing a Ca2+ binding site in the EGFP. One of them, calcium sensor for detecting high concentration in the ER, exhibits unprecedented Ca2+ release kinetics with an off-rate estimated at around 700 s−1 and appropriate Ca2+ binding affinity, likely attributable to local Ca2+-induced conformational changes around the designed Ca2+ binding site and reduced chemical exchange between two chromophore states. Calcium sensor for detecting high concentration in the ER reported considerable differences in ER Ca2+ dynamics and concentration among human epithelial carcinoma cells (HeLa), human embryonic kidney 293 cells (HEK-293), and mouse myoblast cells (C2C12), enabling us to monitor SR luminal Ca2+ in flexor digitorum brevis muscle fibers to determine the mechanism of diminished SR Ca2+ release in aging mice. This sensor will be invaluable in examining pathogenesis characterized by alterations in Ca2+ homeostasis

Topics: Biological Sciences
Publisher: National Academy of Sciences
OAI identifier: oai:pubmedcentral.nih.gov:3182728
Provided by: PubMed Central
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