title: Developing sensors for real-time measurement of high Ca2+ concentrations.
abstract: Ca2+ regulates numerous biological processes through spatiotemporal changes in the cytosolic Ca2+ concentration and subsequent interactions with Ca2+ binding proteins. The endoplasmic reticulum (ER) serves as an intracellular Ca2+ store and plays an essential role in cytosolic Ca2+ homeostasis. There is a strong need to develop Ca2+ sensors capable of real-time quantitative Ca2+ concentration measurements in specific subcellular environments without using natural Ca2+ binding proteins such as calmodulin, which themselves participate as signaling molecules in cells. In this report, a strategy for creating such sensors by grafting a Ca2+-binding motif into chromophore sensitive locations in green fluorescence protein is described. The engineered Ca2+ sensors exhibit large ratiometric fluorescence and absorbance changes upon Ca2+ binding with affinities corresponding to the Ca2+ concentrations found in the ER (Kd values range from 0.4 to 2 mM). In addition to characterizing the optical and metal binding properties of the newly developed Ca2+ sensors with various spectroscopic methods, we also examined the kinetic properties using stopped-flow spectrofluorimetry to ensure accurate monitoring of dynamic Ca2+ changes. The developed Ca2+ sensor was successfully targeted to the ER of mammalian cell lines to monitor Ca2+ changes occurring in this compartment in response to stimulation with agonists. We envision that this class of Ca2+ sensors can be modified further to measure the Ca2+ concentration in other cellular compartments, providing tools for studying the contribution of these compartments to cellular Ca2+ signaling.
authors.affiliation: Department of Chemistry and Biology, Center for Drug Design and Biotechnology, Georgia State University, Atlanta, Georgia 30303, USA.
authors.forename: Jin
authors.initials: J
authors.lastname: Zou
authors.forename: Aldebaran M
authors.initials: AM
authors.lastname: Hofer
authors.forename: Monica M
authors.initials: MM
authors.lastname: Lurtz
authors.forename: Giovanni
authors.initials: G
authors.lastname: Gadda
authors.forename: April L
authors.initials: AL
authors.lastname: Ellis
authors.forename: Ning
authors.initials: N
authors.lastname: Chen
authors.forename: Yun
authors.initials: Y
authors.lastname: Huang
authors.forename: Angela
authors.initials: A
authors.lastname: Holder
authors.forename: Yiming
authors.initials: Y
authors.lastname: Ye
authors.forename: Charles F
authors.initials: CF
authors.lastname: Louis
authors.forename: Kristy
authors.initials: K
authors.lastname: Welshhans
authors.forename: Vincent
authors.initials: V
authors.lastname: Rehder
authors.forename: Jenny J
authors.initials: JJ
authors.lastname: Yang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006706:Homeostasis; D006801:Humans; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D002118:Calcium