Bisindolylpyrrole Induces a Cpr3- and Porin1/2-Dependent Transition in Yeast Mitochondrial Permeability in a Low Conductance State via the AACs-Associated Pore.

Although the mitochondrial permeability transition pore (PTP) is presumably formed by either ATP synthase or the ATP/ADP carrier (AAC), little is known about their differential roles in PTP activation. We explored the role of AAC and ATP synthase in PTP formation in Saccharomyces cerevisiae using bisindolylpyrrole (BP), an activator of the mammalian PTP. The yeast mitochondrial membrane potential, as indicated by tetramethylrhodamine methyl ester signals, dissipated over 2-4 h after treatment of cells with 5 µM BP, which was sensitive to cyclosporin A (CsA) and Cpr3 deficiency and blocked by porin1/2 deficiency. The BP-induced depolarization was inhibited by a specific AAC inhibitor, bongkrekate, and consistently blocked in a yeast strain lacking all three AACs, while it was not affected in the strain with defective ATP synthase dimerization, suggesting the involvement of an AAC-associated pore. Upon BP treatment, isolated yeast mitochondria underwent CsA- and bongkrekate-sensitive depolarization without affecting the mitochondrial calcein signals, indicating the induction of a low conductance channel. These data suggest that, upon BP treatment, yeast can form a porin1/2- and Cpr3-regulated PTP, which is mediated by AACs but not by ATP synthase dimers. This implies that yeast may be an excellent tool for the screening of PTP modulators.

Bisindolylpyrrole triggers transient mitochondrial permeability transitions to cause apoptosis in a VDAC1/2 and cyclophilin D-dependent manner via the ANT-associated pore.

Bisindolylpyrrole at 0.1 µM is cytoprotective in 2% FBS that is counteracted by cyclosporin-A (CsA), an inhibitor of cyclophilin-D (CypD). We hypothesized that the cytoprotective effect might be due to transient mitochondrial permeability transition (tPT). This study tested the hypothesis that bisindolylpyrrole can trigger tPT extensively, thereby leading to cell death under certain conditions. Indeed, CsA-sensitive tPT-mediated apoptosis could be induced by bisindolylpyrrole at > 5 µM in HeLa cells cultured in 0.1% FBS, depending on CypD and VDAC1/2, as shown by siRNA knockdown experiments. Rat liver mitochondria also underwent swelling in response to bisindolylpyrrole, which proceeded at a slower rate than Ca2+-induced swelling, and which was blocked by the VDAC inhibitor tubulin and the ANT inhibitor bongkrekate, indicating the involvement of the ANT-associated, smaller pore. We examined why 0.1% FBS is a prerequisite for apoptosis and found that apoptosis is blocked by PKC activation, which is counteracted by the overexpressed defective PKCε. In mitochondrial suspensions, bisindolylpyrrole triggered CsA-sensitive swelling, which was suppressed selectively by pretreatment with PKCε, but not in the co-presence of tubulin. These data suggest that upon PKC inactivation the cytoprotective compound bisindolylpyrrole can induce prolonged tPT causing apoptosis in a CypD-dependent manner through the VDAC1/2-regulated ANT-associated pore.

The yeast mitochondrial permeability transition is regulated by reactive oxygen species, endogenous Ca2+ and Cpr3, mediating cell death.

We investigated the properties of the permeability transition pore (PTP) in Saccharomyces cerevisiae in agar-embedded mitochondria (AEM) and agar-embedded cells (AEC) and its role in yeast death. In AEM, ethanol-induced pore opening, as indicated by the release of calcein and mitochondrial membrane depolarization, can be inhibited by CsA, by Cpr3 deficiency, and by the antioxidant glutathione. Notably, the pore opening is inhibited, when mitochondria are preloaded by EGTA or Fluo3 to chelate matrix Ca2+, or are pretreated with 4-Br A23187 to extract matrix Ca2+, prior to agar-embedding, or when pore opening is induced in the presence of EGTA; opened pores are re-closed by sequential treatment with CsA, 4-Br A23187 plus EGTA and NADH, indicating endogenous matrix Ca2+ involvement. CsA also inhibits the pore opening with low conductance triggered by exogenous Ca2+ transport with ETH129. In AEC, the treatment of tert-butylhydroperoxide, a pro-oxidant that triggers transient pore opening in high conductance in AEM, induces yeast death, which is also dependent on CsA and Cpr3. Furthermore, AEMs from mutants lacking three ADP/ATP carrier (AAC) isoforms and with defective ATP synthase dimerization exhibit high and low conductance pore openings with CsA sensitivity, respectively. Collectively, these data show that the yeast PTP is regulated by Cpr3, endogenous matrix Ca2+, and reactive oxygen species, and that it is involved in yeast death; furthermore, ATP synthase dimers play a key role in CsA-sensitive pore formation, while AACs are dispensable.