Developing sensors for real-time measurement of high Ca2+ concentrations.
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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.
author list (cited authors)
Zou, J., Hofer, A. M., Lurtz, M. M., Gadda, G., Ellis, A. L., Chen, N., ... Yang, J. J.
complete list of authors
Zou, Jin||Hofer, Aldebaran M||Lurtz, Monica M||Gadda, Giovanni||Ellis, April L||Chen, Ning||Huang, Yun||Holder, Angela||Ye, Yiming||Louis, Charles F||Welshhans, Kristy||Rehder, Vincent||Yang, Jenny J