“Ca2+-dependent regulation of the proton permeability of the inner membrane of lupine cotyledon mitochondria”

Abstract

change in volume, and proton permeability of the inner membrane of mitochondria isolated from cotyledons of etiolated seedlings of narrow-leaved lupine (Lupinus angustifolius L.) was studied. The mitochondria used in the work were characterized by a strong coupling of oxidation and phosphorylation processes. The high functional activity of mitochondria was confirmed by their ability to generate a transmembrane gradient of protons on the inner membrane (membrane potential or ∆Ψ) during the oxidation of succinate and also sustainably maintain it for a long time , both due to the operation of the electron transport chain and due to ATP hydrolysis under conditions of anaerobiosis. It was shown that the presence of 60–120 µM CaCl2 in the mitochondrial incubation medium had no significant effect on the rate of succinate oxidation and the parameters of oxidative phosphorylation; however, it induced ∆Ψ dissipation under conditions of oxygen depletion in the incubation medium. The most complete removal Ca2+ from the medium in the presence of chelators (EGTA, EDTA) prevented the membrane potential dissipation. Ca2+-dependent depolarization of the inner membrane was inhibited by dithiothreitol, suggesting involvement in this process of reactive oxygen species. The reset of the membrane potential was not accompanied by swelling of mitochondria and was not sensitive to cyclosporine A. Using metallochromic Ca2+-indicator arsenazo III, it was shown that the mitochondria of lupine cotyledons are able to actively absorb exogenous Ca2+ and store it in the matrix. Ca2+-induced dissipation of ∆Ψ under conditions of anaerobiosis was accompanied by the release of Ca2+ from mitochondria, the rate of which sharply increased in the presence of calcium ionophore A23 (A23187). It is assumed that the accumulation of Ca2+ and an increase in the level of reactive oxygen species in the matrix induces reversible permeabilization of the inner mitochondrial membrane of lupine cotyledons under conditions of anaerobiosis, which is due to the opening of a pore of nonspecific permeability in a state of low conductivity permeable to protons and, possibly, to other small cations (Na+, K+, Ca2+).