"Allosteric regulation" of calcium-uptake in rat liver mitochondria

During investigations of calcium uptake by rat liver mitochondria, at a buffered free calcium concentration of 2 microM, a considerable acceleration of calcium uptake was occasionally observed. From the following experiments it can be concluded that the acceleration occurred when mitochondria had become anaerobic, and hence deenergized, because they had been stored in the refrigerator for a while. Mitochondria which had become transitorily deenergized by blocking the respiratory chain with KCN, rotenone or antimycin showed an accelerated calcium uptake when the membrane potential necessary for calcium uptake was regenerated. This acceleration of calcium uptake was also seen when a potassium diffusion potential was induced by valinomycin in previously deenergized mitochondria. The velocity of calcium uptake in transitorily deenergized mitochondria increased irrespective of the presence of magnesium in the incubation medium. The activation of the Ca uniporter was reversible, and both processes, activation and deactivation, were time-dependent and developed within a time span of minutes. Oligomycin strongly inhibited the deactivation of the uniporter by ATP, hence the membrane potential is intrinsically effective and does not act via ATP. The altered kinetics of the Ca uniporter were responsible for the acceleration of calcium uptake which was measured at low calcium concentration with previously deenergized mitochondria. The dependence of the rate of calcium uptake on the concentration of calcium in the medium is hyperbolic in transitorily deenergized mitochondria [Km = 6.7 microM; V = 455 nmol/(min X mg protein)] and sigmoidal in normal ones. It is additionally independent of the presence of magnesium ions. We found Hill coefficients of 3.47 and 2.94 in experiments with and without magnesium, respectively. Correspondent kinetics, hyperbolic in deenergized and sigmoidal in normal mitochondria, were obtained when calcium uptake was not driven by the system of respiratory chain, but by the potassium diffusion potential induced by valinomycin. The alteration in the kinetics of the Ca uniporter has consequences in the range of physiological calcium levels, but mainly in pathological states of liver cells. These points are discussed.