title: Design and application of a class of sensors to monitor Ca2+ dynamics in high Ca2+ concentration cellular compartments.
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.
authors.affiliation: Department of Chemistry, Center for Drug Design and Biotechnology, Georgia State University, Atlanta, GA 30303, USA.
authors.forename: Shen
authors.initials: S
authors.lastname: Tang
authors.forename: Hing-Cheung
authors.initials: HC
authors.lastname: Wong
authors.forename: Zhong-Min
authors.initials: ZM
authors.lastname: Wang
authors.forename: Yun
authors.initials: Y
authors.lastname: Huang
authors.forename: Jin
authors.initials: J
authors.lastname: Zou
authors.forename: You
authors.initials: Y
authors.lastname: Zhuo
authors.forename: Andrea
authors.initials: A
authors.lastname: Pennati
authors.forename: Giovanni
authors.initials: G
authors.lastname: Gadda
authors.forename: Osvaldo
authors.initials: O
authors.lastname: Delbono
authors.forename: Jenny J
authors.initials: JJ
authors.lastname: Yang
mesh_terms: D000367:Age Factors; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002451:Cell Compartmentation; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D013347:Subcellular Fractions / Q000378:metabolism*
chemical_list: D002118:Calcium