Heart mitochondria utilize multiple Ca 2؉ transport mechanisms. Among them, the mitochondrial ryanodine receptor provides a fast Ca 2؉ uptake pathway across the inner membrane to control "excitation and metabolism coupling." In the present study, we identified a novel ryanodine-sensitive channel in the native inner membrane of heart mitochondria and characterized its pharmacological and biophysical properties by directly patch clamping mitoplasts. Four distinct channel conductances of ϳ100, ϳ225, ϳ700, and ϳ1,000 picosiemens (pS) in symmetrical 150 mM CsCl were observed. The 225 pS cation-selective channel exhibited multiple subconductance states and was blocked by high concentrations of ryanodine and ruthenium red, known inhibitors of ryanodine receptors. Ryanodine exhibited a concentration-dependent modulation of this channel, with low concentrations stabilizing a subconductance state and high concentrations abolishing activity. The 100, 700, and 1,000 pS conductances exhibited different channel characteristics and were not inhibited by ryanodine. Taken together, these findings identified a novel 225 pS channel as the native mitochondrial ryanodine receptor channel activity in heart mitoplasts with biophysical and pharmacological properties that distinguish it from previously identified mitochondrial ion channels.
Mitochondrial Ca2ϩ transport plays critical roles in the regulation of mitochondrial function under both physiological and pathological conditions (1-3). An increase of matrix Ca 2ϩ within the physiological range facilitates ATP production (4) by stimulating the activity of Ca 2ϩ -dependent dehydrogenases (5, 6) in the tricarboxylic acid cycle and the ATP synthase (7). Dysregulation of mitochondrial Ca 2ϩ handling and subsequent alterations in bioenergetics contribute to contractile dysfunction in heart failure (8, 9). Moreover, mitochondrial Ca 2ϩ overload during ischemia/reperfusion induces opening of the mitochondrial permeability transition pore (mPTP), 2 mitochondrial swelling, and an increase of reactive oxygen species generation, all of which lead to cardiac cell death (10, 11). Several Ca 2ϩ transport pathways have been identified. The mitochondrial Ca 2ϩ uniporter (MCU) is a large capacity, low affinity system that provides a slow Ca 2ϩ uptake mechanism (12). A highly Ca 2ϩ selective, inwardly rectifying conductance with biophysical properties similar to those expected of the MCU was characterized in elegant patch clamp studies of mitoplasts isolated from Cos-7 cells (13) and human cardiomyocytes (14). Recently, MICU1, a gene encoding an EF hand protein that regulates MCU, was identified (15). However, heart mitochondria are also known to exhibit rapid Ca 2ϩ uptake and efflux pathways that possess Ca 2ϩ sensitivity and pharmacology different from those of the MCU (3).We previously reported that a ryanodine-sensitive, fast Ca 2ϩ uptake pathway co-exists with MCU in heart mitochondria (16, 17). High affinity binding of [ 3 H]ryanodine, immunogold staining, and Western blot analysis revealed th...