Identification of a ryanodine receptor in rat heart mitochondria

Recent studies have shown that, in a wide variety of cells, mitochondria respond dynamically to physiological changes in cytosolic Ca2+ concentrations ([Ca2+](c)). Mitochondrial Ca2+ uptake occurs via a ruthenium red-sensitive calcium uniporter and a rapid mode of Ca2+ uptake. Surprisingly, the molecular identity of these Ca2+ transport proteins is still unknown. Using electron microscopy and Western blotting, we identified a ryanodine receptor in the inner mitochondrial membrane with a molecular mass of approximately 600 kDa in mitochondria isolated from the rat heart. [H-3]Ryanodine binds to this mitochondrial ryanodine receptor with high affinity. This binding is modulated by Ca2+ but not caffeine and is inhibited by Mg2+ and ruthenium red in the assay medium. In the presence of ryanodine, Ca2+ uptake into isolated heart mitochondria is suppressed, In addition, ryanodine inhibited mitochondrial swelling induced by Ca2+ overload. This swelling effect was not observed when Ca2+ was applied to the cytosolic fraction containing sarcoplasmic reticulum. These results are the first to identify a mitochondrial Ca2+ transport protein that has characteristics similar to the ryanodine receptor. This mitochondrial ryanodine receptor is likely to play an essential role in the dynamic uptake of Ca2+ into mitochondria during Ca2+ oscillations.