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Proc Natl Acad Sci U S A. 2011 Sep 27;108(39):16265-70. doi: 10.1073/pnas.1103015108. Epub 2011 Sep 13.

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

Author information

1
Department of Chemistry, Center for Drug Design and Biotechnology, Georgia State University, Atlanta, GA 30303, USA.

Abstract

Quantitative analysis of Ca(2+) fluctuations in the endoplasmic/sarcoplasmic reticulum (ER/SR) is essential to defining the mechanisms of Ca(2+)-dependent signaling under physiological and pathological conditions. Here, we developed a unique class of genetically encoded indicators by designing a Ca(2+) binding site in the EGFP. One of them, calcium sensor for detecting high concentration in the ER, exhibits unprecedented Ca(2+) release kinetics with an off-rate estimated at around 700 s(-1) and appropriate Ca(2+) binding affinity, likely attributable to local Ca(2+)-induced conformational changes around the designed Ca(2+) binding site and reduced chemical exchange between two chromophore states. Calcium sensor for detecting high concentration in the ER reported considerable differences in ER Ca(2+) 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 Ca(2+) in flexor digitorum brevis muscle fibers to determine the mechanism of diminished SR Ca(2+) release in aging mice. This sensor will be invaluable in examining pathogenesis characterized by alterations in Ca(2+) homeostasis.

PMID:
21914846
PMCID:
PMC3182728
DOI:
10.1073/pnas.1103015108
[Indexed for MEDLINE]
Free PMC Article

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