Carbenoxolone induces permeability transition pore opening in rat mitochondria via the translocator protein TSPO and connexin43

Abstract

Ca2+-induced permeability transition pore (mPTP) opening in isolated rat brain mitochondria is promoted through targeting of connexin43. After a threshold Ca2+ load, mitochondrial membrane potential drops and efflux of accumulated Ca2+ from the mitochondrial matrix occurs, indicating the mPTP opening. Specific antibodies were used to assess the role of the translocator protein (18 kDa; TSPO) and connexin43 in swelling of isolated rat liver and brain mitochondria induced by carbenoxolone and the endogenous TSPO ligand protoporphyrin IX. Mitochondrial membrane potential, Ca2+ transport and oxygen consumption were determined using selective electrodes. All the parameters were detected simultaneously in a chamber with the selective electrodes. The phosphorylation state of mitochondrial protein targets was assessed. We report that Ca2+-induced mitochondrial swelling was strengthened in the presence of both carbenoxolone and protoporphyrin IX. The carbenoxolone- and protoporphyrin IX-accelerated mPTP induction in brain mitochondria was completely prevented by antibodies specific for the mitochondrial translocator protein (TSPO). The anti-TSPO antibodies were more effective than anti-connexin43 antibodies. Moreover, carbenoxolone-stimulated phosphorylation of mitochondrial proteins was inhibited by anti-TSPO antibodies. Taken together, the data suggests that, in addition to acting via connexion43, carbenoxolone may exert its effect on mPTP via mitochondrial outer membrane TSPO.

TSPO ligand residence time influences human glioblastoma multiforme cell death/life balance

“…-induced MPT pore opening. The same effect was evidenced using PIGA at micromolar concentration (2.5 lM), accordingly to data obtained using other TSPO reversible ligands [72][73][74].…”

“…Highly debated data in TSPO literature regards the ability of TSPO ligands to modulate MPT pore [72][73][74][75][76][77][78][79][80]. In the present work, the TSPO ligand modulatory activity on MTP pore functioning was investigated on U87MG cell suspension with digitonin-permeabilized plasmatic membrane at different times.…”

The changing landscape in translocator protein (TSPO) function

“…In addition, different classes of TSPO-binding chemicals bind to different regions in the TSPO structure with variable specificity. Nevertheless, these effects were considered to be specific, as it was demonstrated that anti-TSPO antibodies could block the MPT [23] induced by the endogenous TSPO-binding heme precursor protoporphyrin IX [18,54]. However, recent experiments using TSPO À/À cells indicate that the effects of both the endogenous protoporphyrin IX and the synthetic PK11195 and Ro5-4864 TSPO ligands were not mediated through TSPO [52], as shown previously [54], and that the mechanism by which the outer mitochondrial membrane regulates the MPT did not involve TSPO [52].…”

“…Although a role for TSPO was not discussed in the new model, it remains to be explained how TSPO-binding chemicals could modulate cell death processes. The pharmacological evidence supporting a TSPO link to the MPTP appears well documented [22][23][24][25]. Developments in the field of the MPT were extensively described in a recent review on this topic [26].…”

Effect of the CRAC Peptide, VLNYYVW, on mPTP Opening in Rat Brain and Liver Mitochondria

“…The existence of the CRAC domain in BAX may promote the protein incorporation into the cholesterol-enriched membrane [19]. The CRAC motif is also found in transmembrane domains of connexin43 (LLIQWYIY), which is present in mitochondria, where it might interact with TSPO [20]. Interestingly, proteins shown to bind cholesterol, e.g., VDAC and TSPO, or having the CRAC motif, e.g., TSPO and Bax, participate in the regulation of permeability of the outer mitochondrial membrane.…”