Developing sensors for real-time measurement of high Ca2+ concentrations.
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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.
published proceedings
Biochemistry
author list (cited authors)
Zou, J., Hofer, A. M., Lurtz, M. M., Gadda, G., Ellis, A. L., Chen, N., ... Yang, J. J.
citation count
41
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
