A Regulator of Mitochondrial Calcium Cycling*

Steady-state free Ca2+ concentrations have been measured with a Ca2+ electrode using suspensions of isolated rat liver mitochondria or saponin-treated hepatocytes. Mitochondria, when incubated in the presence of Mg2+ and MgATP2-, maintain a steady-state PCB2+ (-log [Ca2+]) of approximately 6.1 (0.8 FM). Addition of spermine lowered this value to a pCa2+ of 6.6 (0.25 pM). Spermine was the most effective polyamine, giving half-maximal effects at 170 PM and maximal effects at 400 PM. With saponin-permeabilized hepatocytes, spermine addition similarly showed that the mitochondria buffered the steady-state medium-free Ca2+ at a level approximating the cytosolic free Ca2+ concentration of intact hepatocytes. The initial rate of Ca2+ uptake by the mitochondrial Ca2+ uniporter was investigated using Ca2+-depleted mitochondria incubated in the presence of succinate and 0.3 mM free Mg2+. Under control conditions, Ca2+ uptake was not observed at free Ca2’ concentrations below 0.5 p ~ . Spermine (350 PM) increased the rate of Ca2+ uptake at all Ca2+ concentrations below 4.5 FM, but at higher Ca2+ concentrations, it was inhibitory. Spermine also affected mitochondrial Ca2+ efflux by decreasing the apparent K , from 16 to 3.8 nmol of Ca2+/mg of mitochondrial protein with no change of V,,,. Experiments with “Ca2+ confirmed that spermine increased mitochondrial Ca2+ cycling at 0.2 /IM free Ca2+. Hepatic spermine contents are reported to be about 1 pmollg, wet weight, suggesting that this polyamine may have an important physiological role in intracellular calcium homeostasis.