Abstract
We have studied the induction of the mitochondrial cyclosporin A-sensitive permeability transition pore (PTP) by the bifunctional SH group reagent phenylarsine oxide (PhAsO). Addition of nanomolar concentrations of the electroneutral H(+)-K+ ionophore nigericin to nonrespiring mitochondria in sucrose medium determines a dramatic increase of the time required for PTP induction by PhAsO, while no effect of nigericin is apparent in KCl medium. Using mitochondria loaded with the internal pH indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, we show that the effect of nigericin is mediated by the ionophore-induced acidification of matrix pH. Indeed, experimental manipulation of pHi by a number of treatments indicates that PTP induction is directly related to matrix pH, in that the PTP induction process becomes slower as pHi decreases at constant pHo. PTP induction by PhAsO in respiration-inhibited mitochondria is stimulated by Ca2+ and inhibited by a series of divalent cations. Since PhAsO induces the PTP even in the presence of excess EGTA and in the absence of respiration (Lenartowicz, E., Bernardi, P., and Azzone, G.F. (1991) J. Bioenerg. Biomembr. 23, 679-688), we have been able to study the Ca2+ dependence of the induction process. We show that the apparent Km for Ca2+ activation is about 10(-5) M and that Ca2+, cyclosporin A, and inhibitory Me2+ ions behave as if they were competing for the same binding site(s) on the pore. Since similar results are obtained from patch-clamp experiments on the mitochondrial megachannel (Szabó, I., Bernardi, P., and Zoratti, M. (1992) J. Biol. Chem. 267, 2940-2946), we suggest that (i) the PTP and the mitochondrial megachannel are the same molecular structures and (ii) the same factors affect both the process of pore induction and its open-closed orientation.
Highlights
We have studied the induction of the mitochondrial synthesis and carrier-mediated ion and metabolite transport cyclosporin A-sensitive permeability transition pore [1, 2]
Ex- cause a Ca2+-dependenitncrease of mitochondrial permeabilperimental manipulation of pHi by a number of treat- ity to ions and solutes with molecular masses up to 1200 ments indicates that PTPinduction is directly related daltons, leading to loss of the homeostatic mechanisms of to matrix pH, in that the PTP induction process be- volume control with matrix swelling and uncoupling of oxicomes slower as pHi decreases at constant pH
PTP even in the presence of excess EGTA and in the transition has been widely considered as a “damaging”effect absence of respiration
Summary
Strong support for the pore hypothesis came with the discovery that cyclosporin A inhibits the Energy conservation by a chemiosmotic mechanism implies a very low permeability of the innermitochondrial membrane to ions and solutes, allowing the buildup of a proton electrochemical gradient (ALH)’of 200 mVthat can be used for ATP permeability transition [31,32,33] butnot phospholipase A2 activity [33] Both pathways maybeinvolved in parallel and/or cooperate in maintaining the PTP in an “open” conformation [14,34], adding complexityto the interpretation of a phenomenon that in thepresence of Ca2+can.
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