Proton leak through Adenine Nucleotide Translocase

The adenine nucleotide translocator (ANT) is the most abundant protein in the inner mitochondrial membrane. In addition to its main transport function - the exchange of ATP from the mitochondrial matrix against ADP from the intermembrane space - ANT was proposed to transport protons, contributing to the inhibitor-sensitive proton leak in mitochondria. In contrast to UCP1-mediated proton leak, which is crucial for the non-shivering thermogenesis and is regulated by long chain fatty acids (FA) and purine nucleotides, the significance, extent and regulation mechanisms of proton leak mediated by ANT are not understood. In this project we aimed to investigate (i) FA structure requirements for ANT-mediated proton transport activation and FA binding site in ANT, (ii) role of purine nucleotides in modulation of proton leak, (iii) effect of membrane lipid composition, transmembrane potential and pH on the proton conductance of membranes reconstituted with ANT and (iv) interplay between ATP/ADP exchange and H+ transport. ANT relative contribution to the inhibitor-sensitive membrane proton leak will be evaluated by comparison of its single molecule proton conductance with those of mitochondrial uncoupling proteins. For the first time highly purified recombinant ANT1 and ANT1-mutants will be investigated in planar bilayer membranes that provide the unique possibility to directly apply the transmembrane potentials typical for mitochondria in state III and IV. The ultimate outcome will be a detailed understanding of the proton transporting pathway in ANT, its role in the total proton leak in mitochondria with new perspectives for the therapeutic applications.

The main function of mitochondria is the production of the cell's energy store - ATP. While ATP is produced by the proteins of the respiratory chain, the adenine nucleotide transporter (ANT) exchanges ATP for ADP. ANT is the most abundant protein in the inner mitochondrial membrane, and its malfunction leads to severe myopathies or ophthalmoplegia. In addition to its main transport function, ANT presumably transports protons and thus contributes to proton leakage in the mitochondria. In contrast to proton transport mediated by uncoupling protein 1, which is essential for non-shivering thermogenesis, neither the significance, extent nor regulation of proton transport mediated by ANT is known. In this project we investigated the protonophoric activity of ANT1 and mutants incorporated into planar bilayer membranes. Comparison of the proton turnover number of ANT with that of UCPs and consideration of the proteins relative abundance showed its importance for proton leak. The main aim of the project was to distinguish between two hypotheses. According to the first hypothesis, protons escape through the central cavity of ANT in the presence or absence of long-chain fatty acids (FA). The second hypothesis postulates that protonated FA are spontaneously transported, while ANT transports the FA anion in the opposite direction. To test these hypotheses, we (i) measured the proton transport rates of ANT1 and ANT mutants with altered proton-binding capabilities, (ii) identified ANT activators and inhibitors, and (iii) investigated how membrane lipid composition affects ANT-mediated membrane conductance. To test the mechanism by which ANT1 transports protons, we combined conductance measurements of bilayer membranes with molecular dynamics simulations. We have shown that a polar FA head slides along the outer, positively charged protein surface, while FA acyl chains move in the hydrophobic core of the bilayer. ATP binding to positively charged amino acids inside the protein (arginine 79) reduces the positively charged electric field and thereby inhibits FA anion transport. We have demonstrated that arginine 79 is also crucial for the competitive binding of ADP, ATP and inhibitors (carboxyatractyloside and bongkrekic acid). The results we obtained with ANT are important for other proteins, as the putative binding sites are conserved among the mitochondrial SLC25 members. This points to a general mechanism for the transport of FA anions across the inner mitochondrial membrane. Our results provide an explanation for the contribution of ANT to the uncoupling effect in mitochondria and are essential for understanding (i) the proton transport mechanism mediated by other proteins and (ii) lipid/fatty acid transport across biological membranes mediated by flippases and scramblases. The detailed understanding of the proton transport mechanism of ANT and its contribution to mitochondrial proton flux opens new perspectives for drug development and therapeutic applications, e.g. against obesity, cancer and neurodegenerative diseases.