Abstract
Mitochondrial Ca2+ homeostasis is crucial for regulating vital functions such as respiration or apoptosis. Targeted aequorins are excellent probes to measure subcellular Ca2+. Ca2+ concentration in mitochondria ([Ca2+]M) is low at rest (about 10−7 M) and can increase to the micromolar or even approach the millimolar range, upon cell activation. Here we describe a new quantitative luminescent protocol to directly measure mitochondrial Ca2+ uptake, optimized for high throughput. The sensitivity of the method allows detection of changes in either the capacity or the affinity of mitochondrial Ca2+ transport.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Rizzuto R, De Stefani D, Raffaello A, Mammucari C (2012) Mitochondria as sensors and regulators of calcium signalling. Nat Rev Mol Cell Biol 13(9):566–578. http://www.nature.com/nrm/journal/v13/n9/suppinfo/nrm3412_S1.html
Baughman JM (2011) Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter. Nature 476:341–345
De Stefani D, Raffaello A, Teardo E, Szabo I, Rizzuto R (2011) A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter. Nature 476:336–340
Crompton M, Kunzi M, Carafoli E (1977) The calcium-induced and sodium-induced effluxes of calcium from heart mitochondria. Evidence for a sodium-calcium carrier. Eur J Biochem 79:549–558
Gunter TE, Pfeiffer DR (1990) Mechanisms by which mitochondria transport calcium. Am J Physiol 258:C755–C786
Montero M (2000) Chromaffin-cell stimulation triggers fast millimolar mitochondrial Ca2+ transients that modulate secretion. Nature Cell Biol 2:57–61
Rizzuto R, Simpson AW, Brini M, Pozzan T (1992) Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin. Nature 358:325–327
Shimomura O, Johnson FH, Saiga Y (1962) Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol 59:223–239
Manjarres IM, Chamero P, Domingo B, Molina F, Llopis J, Alonso MT, Garcia-Sancho J (2008) Red and green aequorins for simultaneous monitoring of Ca2+ signals from two different organelles. Pflugers Arch 455(5):961–970. doi:10.1007/s00424-007-0349-5
Baubet V, Le Mouellic H, Campbell AK, Lucas-Meunier E, Fossier P, Brulet P (2000) Chimeric green fluorescent protein-aequorin as bioluminescent Ca2+ reporters at the single-cell level. Proc Natl Acad Sci U S A 97(13):7260–7265. doi: 97/13/7260 [pii]
Allen DG, Blinks JR, Prendergast FG (1977) Aequorin luminescence: relation of light emission to calcium concentration—a calcium-independent component. Science 195(4282):996–998
Alonso MT, Villalobos C, Chamero P, Alvarez J, Garcia-Sancho J (2006) Calcium microdomains in mitochondria and nucleus. Cell Calcium 40(5-6):513–525. doi:10.1016/j.ceca.2006.08.013
Patton C, Thompson S, Epel D (2004) Some precautions in using chelators to buffer metals in biological solutions. Cell Calcium 35(5):427–431. doi:10.1016/j.ceca.2003.10.006
Acknowledgments
The authors are grateful to present and past members of the laboratory. We specially thank Dr. Perocchi for her help with the assay and comments on the manuscript. This work was supported by a grant from the Spanish Ministerio de Economía y Competitividad (BFU2014-534698P) and the Instituto de Salud Carlos III (RD16/0011/0003).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Alonso, M.T., Navas-Navarro, P., García-Sancho, J. (2017). A Microplate-Based Bioluminescence Assay of Mitochondrial Calcium Uptake. In: Mokranjac, D., Perocchi, F. (eds) Mitochondria. Methods in Molecular Biology, vol 1567. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6824-4_15
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6824-4_15
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6822-0
Online ISBN: 978-1-4939-6824-4
eBook Packages: Springer Protocols