Proton leak through Adenine Nucleotide Translocase
The adenine nucleotide translocator (ANT, ATP/ADP carrier) is the most abundant protein in the inner mitochondrial membrane and its dis-function is associated with severe myopathies or ophthalmoplegia. In addition to its main transport function - the exchange of ATP against ADP - ANT is thought to transport protons, contributing to the inhibitor-sensitive proton leak, JH, in mitochondria. In contrast to the uncoupling protein 1 (UCP1)-mediated JH, which is crucial for nonshivering thermogenesis, neither significance, nor extent, nor regulation of JH mediated by ANT are understood. We will reconstitute purified ANT family members from different tissues (ANT1, ANT2, and ANT3), as well various ANT-mutants into planar bilayer membranes. This provides us with the unique opportunity to selectively measure their proton conductance while applying transmembrane potentials that are typical for mitochondria in states III and IV. We will discriminate between two hypotheses according to which protons either leak through the central cavity of ANT in the presence or absence of long chain fatty acids (FA) or are transported by FA, whereas ANT may transport the FA anion in the opposite direction - similar as has been proposed for UCP1. Therefore, we will (i) measure H+ transport rates of ANT1-ANT3 and ANT mutants with modified proton binding capabilities, (ii) identify activators and inhibitors of ANT-mediated H+ transport, (iii) investigate how membrane lipid composition, transmembrane potential and pH affect ANT-mediated H+ conductance, and (iv) elucidate the role of H+ transport for ATP/ADP exchange. Comparing ANT’s single moleculeproton turnover number with that of UCPs and taking into account protein´s relative abundances allows assessment of ANT’s importance for JH. The detailed understanding of ANT’s proton transportmechanism and its contribution to JH opens new perspectives for drug design and therapeutic applications, for example against obesity, cancer and neurodegenerative diseases.