The Mechanism of Ion Translocation in Mitochondria. 4. Coupling of K+ Efflux with Ca2+ Uptake

The translocation of Ca2+ across the mitochondrial membrane in the absence of metabolism has been studied. The experimental system consists in coupling the downhill efflux of K+ from rotenone-valinomycin treated mitochondria to the uphill influx of Ca2+. The Vmax of the K+ driven Ca2+ uptake is 4 μg ions μg protein−1μsec−1. The rate of Ca2+ uptake has the following properties: it is insensitive to oligomycin, cyanide or antimycin A; it is dependent on the concentration of Ca2+ and valinomycin, it is dependent on the external K+ and abolished above 13 mM Ko; it shows a saturation kinetics. The K+ driven Ca+ uptake is inhibited by several agents, among which La3+, H+, dinitrophenol, Mg2+ and Na+. Except in the case of Na+ the inhibition is found to be of the competitive type. Apparent Ki were of the order of 50 nM for La3+, 3 μM for dinitrophenol, 0.3 μM for H+ and 4 mM for Mg2+. The inhibition by La3+ appears to be stoichiometric with the protein: 50% inhibition of K+ driven Ca2+ uptake occurs at 50 nmoles La3+μg protein−1. Inhibition of the aerobic Ca2+ uptake and of Ca2+ release require larger amount of La3+. Binding experiments indicate that mitochondria contain La3+ sensitive sites in amount corresponding to 400 nmoles/g protein. The inhibition of Ca2+ binding by La3+ is additive with that caused by dinitrophenol and is not affected by treatments which reduce the amount of surface binding, such as addition of impermeant cations or depletion of phospholipids. At variance with the La3+ sensitive sites, the dinitrophenol sensitive sites are sensitive to depletion of phospholipids and to the addition of impermeant cations. A model for the translocation of Ca2+ across the membrane is discussed which assumes that the fluxes of K+ and Ca2+ are coupled together through the operation of a common chemical translocator. The operation of the carrier is suggested also to be responsible for the metabolism linked uptake of Ca2+.