CHAOTROPIC AGENTS AND INCREASED MATRIX VOLUME ENHANCE BINDING OF MITOCHONDRIAL CYCLOPHILIN TO THE INNER MITOCHONDRIAL-MEMBRANE AND SENSITIZE THE MITOCHONDRIAL PERMEABILITY TRANSITION TO [CA2+]

Binding of mitochondrial cyclophilin (CyP) to the inner mitochondrial membrane is induced by treatment of mitochondria with thiol reagents o r oxidative stress and correlates with a sensitization to [Ca2+] of th e cyclosporin A-sensitive mitochondrial permeability transition pore ( MTP) [Connern, C. P., & Halestrap, A. P. (1994) Biochem. J. 303, 321-3 24]. Here we show that detection of the bound CyP by western blotting is greatly enhanced by fixing the CyP to the blotting membrane with gl utaraldehyde. CyP binding was only observed when mitochondria were inc ubated and then frozen in KSCN medium before preparation of the membra ne fraction, but not when KCl medium was used. However, incubation of mitochondria (energized or deenergized) in KCl medium followed by KSCN addition immediately prior to freezing did allow CyP binding to be de tected. The action of KSCN could be mimicked by guanidinium chloride, implying that the chaotropic action of these agents stabilized the bou nd complex. The sensitivity to [Ca2+] of the MTP in deenergized mitoch ondria was greatly enhanced in KSCN medium as compared to KCI medium. Binding of CyP to the mitochondrial membrane was increased by treatmen t with tert-butylhydroperoxide, phenylarsine oxide, and diamide and by hypoosmotic KCI medium. These conditions all increased the sensitivit y of the MTP to [Ca2+]. Conditions known to increase the mitochondrial NADH/NAD(+) ratio decreased CyP binding, In contrast, the effects of mitochondrial membrane potential, matrix pH, and adenine nucleotide tr anslocase conformation on the sensitivity of the MTP to [Ca2+] were no t associated with a change in CyP binding. Our data imply that there m ap be two independent mechanisms of altering the Ca2+ sensitivity of t he MTP, one brought about by CyP binding which is stabilized by chaotr opic agents and another involving additional regulatory sites on the p ore complex.