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(20) CHARACTERIZATION OF CYCLOSPORIN A SENSITIVE AND INSENSITIVE PERMEABILITY TRANSITION PORES IN RAT LIVER MITOCHONDRIA L. He, S.A. Franklin and J.J.
Lemasters
BACKGROUND: The mitochondrial permeability transition (MPT) mediates both necrotic and apoptotic cell death, but the composition of the PT pore remains poorly understood. Cyclosporin A inhibits the MPT through its binding to cyclophilin D (CypD). However, earlier studies showed that CsA-insensitive PT pores may also form in mitochondria. Our AIM was to characterize CsA sensitive and insensitive PT pores. METHODS: The MPT in rat liver mitochondria was monitored spectrophotometrically at 540 nm. PT pore size was estimated by the effect of polyethylene glycol (PEG) of various molecular weights on swelling. CypD of mitochondrial membrane and matrix fractions was detected by Western blot without chaotrope treatment. Results: HgCl2 (5 µM), phenylarsine oxide (100 nM), mastoparan (1 µM), and alamethicin (50 nM) all induced a calcium-dependent MPT that was blocked by CsA, Mg2+, and EGTA. At higher doses, these same agents induced a calcium-independent MPT that was no longer blocked by CsA, Mg2+ or EGTA. PT pores conducted PEG up to 1450 to 2000 Da, but pore size was similar at low and high concentrations of all inducers. CypD associated with mitochondrial membranes in the absence of a MPT inducer. This association did not increase after incubation with MPT inducers. However, CsA consistently released CypD from the membrane, even when CsA did not block the MPT at high inducer concentrations. Conclusion: As the concentration of MPT inducers increases, the PT pore switches from a Ca2+-dependent, Mg2+-regulated and CsA-sensitive form to a divalent cation-independent and CsA-insensitive form. Both the exogenous pore-forming peptides, mastoparan and alamethicin, and the oxidant chemicals, HgCl2 and phenylarsine oxide, produced regulated and unregulated PT pores. We propose that PT pores form from aggregation of amphipathic transmembrane proteins, including endogenous integral membrane proteins that become misfolded by oxidative stress. Pores formed from these amphiphiles associate with an as yet unidentified regulatory factor in the mitochondrial matrix that inhibits pore conductance. CsA-sensitive association of CypD with this complex causes pore opening. When pore number exceeds the concentration of regulatory factor, unregulated PT pores form that do not require CypD for an open conductance state. The unregulated CsA-insensitive PT pore may explain why CsA fails to protect in some models of apoptotic and necrotic cell killing in which the MPT seems to play a role.
For further information contact...Carmen Mannella: carmen@wadsworth.org |
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