Effect of cations and anions on the steady state kinetics of energy-dependent Ca2+ transport in rat liver mitochondria

Abstract

The divalent cation ionophore A23187 has been used to investigate the kinetics of energy-dependent Ca2+ uptake by rat liver mitochondria under steady state conditions. During A23187-induced cyclic Ca2+ flux, the free Ca2+ concentration is adjusted using [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) buffers. The rate of Ca2+ transport, which is inferred from the rate of succinate oxidation, is a function of the free Ca2+ concentration in the medium. The kinetics are sigmoidal with the free Ca2+ concentration at half-maximal respiratory stimulation (K0.5) equal to 3.1 +/- 0.4 muM at 25 degrees. The maximal Ca2+-stimulated respiratory rate (Vmax) is a function of the ionic composition of the medium. Magnesium and Mg2+ plus phosphate produced a parallel stimulation of the maximal respiration rate whether activated by Ca2+ uptake or by the uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP). In the absence of A23187, Ca:O rations of 4.0 were obtained under most experimental conditions. Magnesium is a potent competitive-like inhibitor, increasing the K0.5 for Ca2+ to 30.0 muM at 2.0 mM MgCl2. Magnesium dramatically decreases the apparent affinity for Ca2+ but does not appear to alter the kinetic mechanism. In contrast, the alkali metal cations are weak inhibitors, at most doubling the K0.5 for Ca2+; however, they antagonized Mg2+ inhibition with an order of effectiveness Li+ greater than or equal to Na+ greater than K+ greater than Rb+ =Cs+. Phosphate and acetate increased the Vmax slightly without altering the K0.5 for Ca2+. Phosphate did not influence the inhibitory effects of Mg2+ or Mg2+ plus K+. This study suggests that during steady state conditions, the maximal rate of Ca2+ accumulation is primarily electron transport-limited. The results are also discussed in terms of a possible physiological role for Mg2+ and K+ in the intracellular regulation of energy-dependent mitochondrial Ca2+ transport in liver.