Involvement of Cyclophilin D in the Activation of A mitochondrial Pore by Ca2+ and Oxidant Stress

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Abstract
Heart and liver mitochondria contain a structure that is able to form a large non‐selective pore in the inner membrane under conditions of high matrix Ca2+ and oxidant stress. The pore is blocked by cyclo‐sporin A (CSA). In this study, rat liver mitochondria were covalently labelled with a photoactive CSA derivative in the presence and absence of the pore ligands Ca2+ and ADP Photolabelling of a 21‐kDa protein was selectively depressed by Ca2+ in a manner reversed by ADP. The protein exhibited peptidyl‐prolyl cis‐trans isomerase (PPIase) activity and was inhibited by CSA (Ki, 8 nM). The PPIase was associated with the outside of sonicated submitochondrial particles but dissociated in 0.5 M NaCl. When mitochondria were treated with increasing concentrations of digitonin, the 21‐kDa PPIase fractionated with the matrix marker enzyme, malate dehydrogenase. A second PPIase of 18 kDa fractionated with the intermembrane‐space marker, adenylate kinase. Photolabelling of the 18‐kDa PPIase was unaffected by Ca2+ or ADP. The 21‐kDa PPIase was digested with endoproteinase Asp‐N and 11 of the peptides were N‐terminally sequenced. The sequences were most similar to those of human cyclophilin‐D, and it is concluded that this protein is probably the CSA receptor during pore blockade by CSA. The implications of these findings are discussed.