Mitochondrial Permeability Transition Pore (mPTP) Formation Requires the Participation of c-Subunit of ATP-Synthase, Polyhydroxybutyrate (PHB) and Inorganic Polyphosphate (polyP)

Mitochondrial Permeability Transition Pore( mPTP) is a channel in the mitochondrial inner membrane. Opening of mPTPduring acute stress conditions following ischemia-reperfusion is the principal molecular event leading to cell death and tissue damage. We demonstrated previously that a highly purified mitochondrial fraction containing c-subunit of ATP synthase(c-subunit), polyP and PHB possesses channel activity with properties resembling mPTPas seen in native mitochondrial membranes. Importantly, we have been able to purify this fraction mainly from mitochondria with calcium-activated mPTP. When mitochondria were exposed to the mPTPblocker Cyclosporine A, the components of the channel forming fraction were reduced to control levels. Here we investigate the molecular details of the interactions between components of the channel-forming complex. Using immunoblot and mass spectrometry approaches we demonstrate that c-subunit purified from intact mitochondria is closely associated with PHB. Furthermore, we are able to reconstitute mPTP-like channel activity in artificial lipid bilayers by combining purified mammalian c-subunit with synthetic polyP in the presence of calcium. We propose that the c-subunit-PHB-polyP complex is sufficient and essential for formation of the mPTPchannel. According to our hypothesis, during cellular stress conditions, calcium induces formation of the complex between the c-subunit, PHB and polyP. This is a critical event required for mPTPactivation.