Does ATP synthase form the PTP in pea stem mitochondria?

The mitochondrial Permeability Transition (PT) is mediated by the opening of a mitochondrial channel that has been named Permeability Transition Pore (PTP). Recently, it has been demonstrated that the PTP is a channel formed by dimers of F-ATP synthase in mammals, yeast and Drosophila. Since plants possess all the components needed for PTP formation, this work was undertaken to verify if plant F-ATP synthase possesses features similar to those already seen in other species, as this would provide a significant contribution to understand evolution of the channel function of F-ATP synthases. Pea stem mitochondria showed a substrate-dependent electrical potential formation. In the presence of the Ca2+ ionophore ETH129, mitochondria were able to accumulate Ca2+ up to 0.2 mM, which was followed by an abrupt collapse of the membrane potential that was delayed by Cyclosporin A (CsA) and Pi. The Ca2+ retention capacity was also evaluated, which showed a trend similar to that of membrane potential. These results suggest that plant mitochondria, when permeabilized to Ca2+, can undergo a PT. We detected F-ATP synthase activity after blue-native (BN)-PAGE and the active bands, corresponding to the dimers, were eluted from the gel. The active protein bands were then analyzed for the presence of F-ATP synthase components, and for the presence of Cyclophylin D (CyPD), which associates to mammalian mitochondria. The dimers were also inserted into an artificial bilayer, and we will report our preliminary results on their channel activity in electrophysiology experiments. Supported by MIUR grant PRIN 2010CSJX4F.