European J. Biochem. 8 (1969) 473-481 Correlation between Anion Uptake and the Movement of K+ and H+ across the Mitochondria1 Membrane zyx F. PALMIERI and E. QUAOLIARIELLO Istituto zyxwvut di Chimica Biologica, Universiti di Bari (Received November l3,1968/January 13,1969) The relationship between the H+ and K+ movement across the mitochondrial membrane and the distribution of anions between the intra- and extramitochondrial space has been analyzed in the presence of uncouplers, nigericin and nonactin. 1. Mitochondria accumulate added anions in the presence of respiratory inhibitors and oligo- mycin. This accumulation is accompanied by the exit of endogenous anions. Respiration or ATP hydrolysis does not, in itself, increase anion accumulation, but only in as much as it gives rise to proton ejection and cation uptake. zyxwvu 2. In sucrose medium anion uptake, found in the presence of respiratory inhibitors and oligomycin, is inhibited by uncouplers, nigericin and nonactin, all of which cause H+ uptake and K+ release. The ratio of the intra- to the extramitochondrial concentration of endogenous anions is also strongly diminished by uncouplers, nigericin and nonactin. 3. With nonactin-pretreated mitochondria, uncouplers, even a t very high concentrations, do not inhibit anion uptake. 4. With nonactin, as the external K+ concentration is increased, the uptake of H+ is pro- gressively decreased and similarly the anion uptake inhibition is diminished. 5. The 2,4-dinitrophenol-induced uptake of H+ is unaffected by the presence of Kf in the medium UD to a concentration of zyxwvu 125 mM. The inhibition of anion untake and the efflux of endogenois anions, induced by dinitrophenol, are equal in the presenie or absence of 100 mM KC1. 6. In the presence of an external concentration of 100 mM KC1, with nigericin the uptake of H+ is strongly diminished and similarly the anion uptake inhibition and the efflux of endog- enous anions are greatly decreased. With nonactin, I€+ ejection occurs accompanied by stimu- lation of anion uptake. zyxwvuts 7. The ratio of the intra- to the extramitochondrial concentration of succinate increases as the external pH decreaaes. This increase of anion uptake by lowering the pH is inhibited by dinitrophenol. 8. It is concluded that inhibition of anion uptake and efflux of endogenous anions are de- pendent upon the uptake of H+ by the mitochondria and vice versa H+ ejection is responsible for stimulation of anion uptake. Various mechanisms of anion uptake by mito- chondria have been proposed recently [l-61. The concentration of anions in respiring mito- chondria was found to be generally greater than that in the suspending medium [7]. During the uptake of cations (Ca++ or K+ plus valinomycin) the accu- mulation of anions was further increased [7- 151. Chance and Mela[l6] found that the pH gradient established across the mitochondrial membrane by Ca++ uptake collapsed on addition of anions. Purther- Unwrual Abbreviations. TMPD, tetramethyl-p-phenylene- diamine; DNP, 2-4 dinitrophenol; FCCP, p-trifluoromethoxy- carboxylcyanide phenylhydrazone; [H+]i, proton activity in the inner mitochondrial phme; [H+l0, proton activity in the outer mitochondrial phase. 32. more, Azzone zyxw et zyxwv d. [10,14] observed that the anion influx caused a decrease of the H+ : cation ratio during both Ca++and K+ uptake, The concentration ratio of added anions between the intra- and the extramitochondrial space was decreased by un- couplers of oxidative phosphorylation [7,11- 131. Pressman in 1958 [17] found that the mitochondrial content of endogenous inorganic phosphate was de- creased by dinitrophenol zyxw (cf. also [18]) and increased by the cationic reagent guanidine. Recently, un- couplers have been found to stimulate the efflux of internally produced anions such as a-oxoglutarate [19] (from glutamate plus arsenite) and malate [7] (from succinate plus rotenone). Nigericin was also shown to inhibit succinate uptake [12,20].