(7) THE MOLECULAR STRUCTURE OF MITOCHONDRIAL CONTACT SITES. THEIR ROLE IN REGULATION OF ENERGY METABOLISM AND PERMEABILITY TRANSITION

Dieter Brdiczka, Gisela Beutner, Alexander Rück
Faculty of Biology, University of Konstanz, D-78457 Konstanz, Germany

Contact sites between the outer- and peripheral inner membrane of mitochondria are involved in protein precursor uptake and energy transfer. Hexokinase and mitochondrial creatine kinase could be attributed by different techniques to the energy transfer contacts. Kinetic analyses suggested a functional interaction between the kinases, outer membrane pore protein, and inner membrane adenylate translocator (ANT). This suggestion was strongly supported by isolation of hexokinase and creatine kinase complexes that were constituted of kinase oligomers, porin and ANT. Phospholipid vesicles carrying reconstituted kinase-porin-ANT complexes enclosed internal ATP in contrast to vesicles containing free porin only. This indicated that unspecific transport through porin was regulated by its interaction with a specific antiporter, the ANT. A direct interaction between porin and ANT in the hexokinase complex conferred the reconstituted system with permeability properties reminiscent of the mitochondrial permeability transition (PT) pore. In the creatine kinase complex this interaction between porin and ANT was replaced by contact of both with the kinase octamer. Thus PT-pore-like functions were not observed unless the creatine kinase octamer was dissociated, suggesting that the ANT was locked in the antiporter state by interaction with the octamer. Indeed, reconstituted pure ANT showed PT-pore-like properties concerning Ca2+ sensitivity. However, as cyclophilin was missing, sensitivity against cyclosporin (CsA) was not observed. The CsA sensitivity was however installed by addition of cyclophilin D during reconstitution of the ANT. The presence of cyclophilin furthermore led to 10 times higher sensitivity of the PT-pore for Ca2+. It is thus postulated that the ANT can adopt a structure in which it forms an unspecific uniporter with PT-pore like properties. PTP opening is involved in myocardial cells injury during ischemia and reperfusion. The creatine analogues cyclo-creatine and guanidino propionic acid such as CsA have been shown to be potent inhibitors of such damage in cardiac tissue. As cyclo-creatine and guanidino propionic acid turnover by creatine kinase is very slow it may lock the creatine kinase ANT complex in the antiporter conformation. On the other hand this role of creatine kinase is further emphasised by recent observations that the enzyme is the main target of reactive oxygen species in cardiac myofibrils and the re-association of the mitochondrial creatine kinase is suppressed by peroxynitrate. In general the findings support the view that the mitochondrial creatine kinase structure in muscle and brain tissue may play an important role in PTP regulation.