McGuinness, Orla Mary; (1993) A cyclosporin A-sensitive mitochondrial pore. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

Cyclosporin-A is a potent immunosuppressant used to prevent graft rejection. Work in this laboratory has suggested a novel action of cyclosporin, namely protection against the cell injury caused by energy deprivation e.g. during ischaemia by blocking a non-selective, Ca2+-sensitive mitochondrial pore. The long-term aim of this investigation is to reconstitute the Ca2+-sensitive pore and determine which parameters are important in its control. Initially, previous studies were repeated in liver mitochondria mimicking more closely pathophysiological conditions. Isolated rat liver mitochondria exposed to buffered Ca2+ concentrations above 1-2μM, low levels of ATP and either a supraphysiological Pi concentration, or a high oxygen tension cannot maintain a high inner membrane potential on addition of succinate as substrate. [U-14C]-Sucrose entrapment studies confirm that this is due to the reversible opening of a Ca2+-sensitive pore in the inner mitochondrial membrane. Pore closure is induced immediately on chelation of Ca2+ with EGTA. Pore opening, again assayed by the degree of entry and entrapment of [U-14C]-sucrose in the matrix is inhibited by cyclosporin A at the extremely low concentration of 25nM and by physiological concentrations of ATP. However 600nM cyclosporin is needed to prevent proton permeability and allow the maintenance of a high inner membrane a potential. [3H]-Cyclosporin binding studies revealed that cyclosporin A binds to two components in rat liver mitochondria, a soluble matrix component with high affinity and a membrane component with low affinity as well as non-specific binding to mitochondrial phospholipids. There is a good correlation between the total amount of bound cyclosporin yielding maximal pore inhibition and the amount the soluble matrix component estimated in the binding studies. The soluble matrix component was identified as cyclophilin, a ubiquitous protein known to mediate cylosporin's immunosuppressive effects and which has previously been shown to be a peptidyl-prolyl-cis-trans-isomerase. Soluble mitochondrial cyclophilin was purified = 1000x using cation exchange and gel filtration FPLC. Cyclophilin was found to uncouple respiring sub-mitochondrial particles and this effect was inhibited by cyclosporin. These results strongly suggest that cyclosporin interacts with mitochondrial cyclophilin to induce pore blockage. A subsidiary aim of this study was to establish whether or not ADP-ribosylation was involved in modulating pore activity. ADP-ribosylated mitochondrial membrane and soluble proteins were identified using 32 P-NAD and gradient SDS-PAGE.

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