Search

Ca2+ Measurements in Mammalian Cells with Aequorin-based Probes   

Download PDF How to cite Favorites Q&A Share your feedback Cited by

In this protocol

Original research article

A brief version of this protocol appeared in:
EMBO Molecular Medicine
May 2016

Abstract

Aequorin is a Ca2+ sensitive photoprotein suitable to measure intracellular Ca2+ transients in mammalian cells. Thanks to recombinant cDNAs expression, aequorin can be specifically targeted to various subcellular compartments, thus allowing an accurate measurement of Ca2+ uptake and release of different intracellular organelles. Here, we describe how to use this probe to measure cytosolic Ca2+ levels and mitochondrial Ca2+ uptake in mammalian cells.

Keywords: Ca2+, Aequorin, Probes, Luminescence

Background

Aequorin is a 21 kDa photoprotein isolated from jellyfish Aequorea victoria that emits blue light in the presence of Ca2+. In its active form the photoprotein includes an apoprotein and a covalently bound prosthetic group, called coelenterazine. The apoprotein contains four helix-loop-helix ‘EF-hand’ domains, three of which are Ca2+-binding sites. These domains confer to the protein a particular globular structure forming the hydrophobic core cavity that accommodates the coelenterazine. When Ca2+ ions bind to the three high affinity EF-hand sites, coelenterazine is irreversibly oxidized to coelenteramide, with a concomitant release of CO2 and emission of light (Head et al., 2000).

Aequorin began to be widely used when the cDNA encoding the photoprotein was cloned, thus opening the way to recombinant expression. In particular, recombinant aequorin can be expressed not only in the cytoplasm, but also in single intracellular compartments by including specific targeting sequences in the engineered cDNAs (Hartl et al., 1989). To expand the range of Ca2+ sensitivity that can be monitored, point mutations in the EF-hand motives that lower the affinity for Ca2+ have been introduced (Granatiero et al., 2014a and 2014b). Reconstitution of an active recombinant aequorin in living cells is obtained by simple addition of coelenterazine into the medium. Coelenterazine is highly hydrophobic and permeates cell membranes of various cell types. Different coelenterazine analogues have been synthetized and are now commercially available.

Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Tosatto, A., Rizzuto, R. and Mammucari, C. (2017). Ca2+ Measurements in Mammalian Cells with Aequorin-based Probes. Bio-protocol 7(5): e2155. DOI: 10.21769/BioProtoc.2155.
Q&A

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data including images for the troubleshooting.

You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.