Ca2+-induced increased lipid packing and domain formation in submitochondrial particles. A possible early step in the mechanism of Ca2+-stimulated generation of reactive oxygen species by the respiratory chain

Ca2+ and P-i accumulation by mitochondria triggers a number of alterations leading to nonspecific increase in inner membrane permeability [Kowaltowski , A. J., et al. (1996) J. Biol. Chem. 271, 2929-2934]. The molecular nature of the membrane perturbation that precedes oxidative damage is still unkno wn. EPR spectra of spin probes incorporated in submitochondrial particles ( SMP) and in model membranes suggest that Ca2+-cardiolipin (CL) complexation plays an important role. Ca2+-induced lipid domain formation was detected in SMP but not in mitoplasts, in SMP extracted lipids, or in CL-containing liposomes. The results were interpreted in terms of Ca2+ sequestration of C L tightly bound to membrane proteins, in particular the ADP-ATP carrier, an d formation of CE-enriched strongly immobilized clusters in lipid shells ne xt to boundary lipid. The in-plane lipid and protein rearrangement is sugge sted to cause increased reactive oxygen species production in succinate-sup plemented, antimycin A-poisoned SMP, favoring the formation of carbon-cente red radicals, detected by EPR spin trapping. Removal of tightly bound CL is also proposed to cause protein aggregation, facilitating intermolecular th iol oxidation. Lipid peroxidation was also monitored by the disappearance o f the nitroxide EPR spectrum. The decay was faster for nitroxides in a more hydrophobic environment, and was inhibited by butylated hydroxytoluene, by EGTA, or by substituting Mg2+ for Ca2+. In addition, Ca2+ caused an increa se in permeability, evidenced by the release of carboxyfluorescein from res piring SMP. The results strongly support Ca2+ binding to CL as one of the e arly steps in the molecular mechanism of Ca2+-induced nonspecific inner mit ochondrial membrane permeabilization.