The cyclophilin-D binding protein of the mitochondrial permeability transition pore may not be the adenine nucleotide translocase

The mitochondrial permeability transition pore (MPTP) is a non-specific pore that opens in the inner mitochondrial membrane under conditions of elevated [Ca2+] and oxidative stress, and plays a critical role in reperfusion injury. The molecular composition of the MPTP remains to be firmly established, but current models suggest the involvement of the adenine nucleotide translocase (ANT) and cyclophilin D (CyP-D) as core components. Whilst the lack of a cyclosporin-A (CsA) sensitive MPTP in mitochondria from CyP-D knockout mice has proved the role of CyP-D beyond doubt, the role of the ANT remains uncertain. Phenylarsine oxide (PAO) is a known potent inducer of the MPTP and we have used a PAO affinity column to identify the membrane component(s) of the MPTP. In these studies we confirm that the thiol groups of the ANT could be modified by PAO. However, we found that in the presence of carboxyatractyloside (CAT), a specific inhibitor of ANT that does not prevent PAO from activating the MPTP, most of the ANT did not bind to the column but a residual band at 32 kDa was present. Analysis of this band revealed the presence of the phosphate carrier amongst other proteins. Progress on the use of N-ethylmaleimide and other reagents to investigate its role in the MPTP will be presented.