EVIDENCE FOR THE INVOLVEMENT OF A MEMBRANE-ASSOCIATED CYCLOSPORINE-A-BINDING PROTEIN IN THE CA2-ACTIVATED INNER MEMBRANE PORE OF HEART-MITOCHONDRIA()

Heart and liver mitochondria contain a pore in the inner membrane that is activated by Ca2+ and oxidative stress and that has been implicate d in cell injury. Pore opening is blocked by cyclosporin A (CSA). Foll owing previous indications that the interaction of CSA with the pore i s inhibited by Ca2+ and promoted by ADP, we have investigated how cova lent labelling of heart mitochondria by a photoactive CSA derivative i s influenced by these agents. In situ photolabelling of an 11-22-kDa ( approximately) membrane fraction was selectively increased in the pres ence of ADP and decreased in the presence of Ca2+. This fraction also accounted for all the high affinity [H-3]CSA-binding capacity and cont ained peptidylprolyl cis-trans isomerase activity (PPIase). The membra ne PPIase was extracted using Chaps as detergent, and was purified to a 22-kDa protein (SDS/PAGE). The enzyme was inhibited by CSA (K-i 5 nM ). The major component of the 11-22-kDa fraction, photolabelled in an ADP/Ca2+-sensitive manner, also migrated at 22 kDa on SDS/PAGE; a mino r 11-kDa component was also detected. On the basis of these criteria, it is suggested that the membrane PPIase may be the target for CSA whe n it blocks the pore. The presence of a similar PPIase in the membrane fraction of liver mitochondria was also demonstrated. The implication s of these findings are discussed.