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, 29292934]. The molecular nature of the membrane perturbation that precedes oxidative damage is still unknown. 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 CL tightly bound to membrane proteins, in particular the ADP-ATP carrier, and formation of CL-enriched strongly immobilized clusters in lipid shells next to boundary lipid. The in-plane lipid and protein rearrangement is suggested to cause increased reactive oxygen species production in succinate-supplemented, antimycin A-poisoned SMP, favoring the formation of carbon-centered radicals, detected by EPR spin trapping. Removal of tightly bound CL is also proposed to cause protein aggregation, facilitating intermolecular thiol oxidation. Lipid peroxidation was also monitored by the disappearance of 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 increase in permeability, evidenced by the release of carboxyfluorescein from respiring SMP. The results strongly support Ca2+ binding to CL as one of the early steps in the molecular mechanism of Ca2+- induced nonspecific inner mitochondrial membrane permeabilization.38401327913287Zoratti, M., Szabò, I., (1995) Biochim. Biophys. 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