Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin

INTRODUCTION of Ca2+ indicators (photoproteins, fluorescent dyes) that can be trapped in the cytosolic compartment of living cells has yielded major advances in our knowledge of Ca2+ homeostasis1,2. Ca2+ however regulates functions not only in the cytosol but also within various organelles3,4 where indicators have not yet been specifically targeted. Here we present a novel procedure by which the free Ca2+ concentration of mitochondria, [Ca2+]m, can be monitored continuously at rest and during stimulation. The complementary DNA for the Ca2+ sensitive photoprotein aequorin was fused in frame with that encoding a mitochondrial presequence. The hybrid cDNA was transfected into bovine endothelial cells and stable clones were obtained expressing variable amounts of mitochondrially targeted apoaequorin. The functional photopro-tein could be reconstituted in intact cells by incubation with purified coelenterazine and [Ca2+]m could thus be monitored in situ. This allowed the unprecedented direct demonstration that agonist-stimulated elevations of cytosolic free Ca2+, [Ca2+]i, (measured in parallel with Fura-2) evoke rapid and transient increases of [Ca2+]m, which can be prevented by pretreatment with a mitochondrial uncoupler. The possibility of targeting aequorin to cellular organelles not only offers a new and powerful method for studying aspects of Ca2+ homeostasis that up to now could not be directly approached, but might also be used in the future as a tool to report in situ a variety of apparently unrelated phenomena of wide biological interest.