FA Sliding as the Mechanism for the ANT1-Mediated Fatty Acid Anion Transport in Lipid Bilayers.

Mitochondrial adenine nucleotide translocase (ANT) exchanges ADP for ATP to maintain energy production in the cell. Its protonophoric function in the presence of long-chain fatty acids (FA) is also recognized. Our previous results imply that proton/FA transport can be best described with the FA cycling model, in which protonated FA transports the proton to the mitochondrial matrix. The mechanism by which ANT1 transports FA anions back to the intermembrane space remains unclear. Using a combined approach involving measurements of the current through the planar lipid bilayers reconstituted with ANT1, site-directed mutagenesis and molecular dynamics simulations, we show that the FA anion is first attracted by positively charged arginines or lysines on the matrix side of ANT1 before moving along the positively charged protein-lipid interface and binding to R79, where it is protonated. We show that R79 is also critical for the competitive binding of ANT1 substrates (ADP and ATP) and inhibitors (carboxyatractyloside and bongkrekic acid). The binding sites are well conserved in mitochondrial SLC25 members, suggesting a general mechanism for transporting FA anions across the inner mitochondrial membrane.

ANT1 Activation and Inhibition Patterns Support the Fatty Acid Cycling Mechanism for Proton Transport.

Adenine nucleotide translocase (ANT) is a well-known mitochondrial exchanger of ATP against ADP. In contrast, few studies have shown that ANT also mediates proton transport across the inner mitochondrial membrane. The results of these studies are controversial and lead to different hypotheses about molecular transport mechanisms. We hypothesized that the H+-transport mediated by ANT and uncoupling proteins (UCP) has a similar regulation pattern and can be explained by the fatty acid cycling concept. The reconstitution of purified recombinant ANT1 in the planar lipid bilayers allowed us to measure the membrane current after the direct application of transmembrane potential ΔΨ, which would correspond to the mitochondrial states III and IV. Experimental results reveal that ANT1 does not contribute to a basal proton leak. Instead, it mediates H+ transport only in the presence of long-chain fatty acids (FA), as already known for UCPs. It depends on FA chain length and saturation, implying that FA's transport is confined to the lipid-protein interface. Purine nucleotides with the preference for ATP and ADP inhibited H+ transport. Specific inhibitors of ATP/ADP transport, carboxyatractyloside or bongkrekic acid, also decreased proton transport. The H+ turnover number was calculated based on ANT1 concentration determined by fluorescence correlation spectroscopy and is equal to 14.6 ± 2.5 s-1. Molecular dynamic simulations revealed a large positively charged area at the protein/lipid interface that might facilitate FA anion's transport across the membrane. ANT's dual function-ADP/ATP and H+ transport in the presence of FA-may be important for the regulation of mitochondrial membrane potential and thus for potential-dependent processes in mitochondria. Moreover, the expansion of proton-transport modulating drug targets to ANT1 may improve the therapy of obesity, cancer, steatosis, cardiovascular and neurodegenerative diseases.

The Expression of Uncoupling Protein 3 Coincides With the Fatty Acid Oxidation Type of Metabolism in Adult Murine Heart.

The involvement of mitochondrial uncoupling proteins 2 and 3 in the pathogenesis of cardiovascular diseases is widely acknowledged. However, contradictory reports show that the functions of UCP2/UCP3 are still disputed. We have previously described that UCP2 is highly abundant in cells that rely on glycolysis, such as stem, cancer and activated immune cells. In contrast, high amounts of UCP3 are present in brown adipose tissue, followed by heart and skeletal muscles - all known to metabolize fatty acids (FA) to a high extent. Using two different models - mouse embryonic stem cell (mESC) differentiation to cardiomyocytes (CM) and murine heart at different developmental stages - we now tested the concept that the expression ratio between UCP2 and UCP3 indicates the metabolism type in CM. Our results revealed the tight correlation between UCP3 abundance, expression of mitochondrial fatty acid oxidation (FAO) markers and presence of multiple connections between mitochondria and lipid droplets. We further demonstrated that the time course of UCP3 expression neither coincided with the onset of the electrical activity in CM, derived from mESC, nor with the expression of respiratory chain proteins, the observation which rendered protein participation in ROS regulation unlikely. The present data imply that UCP3 may facilitate FAO by transporting FAs into mitochondria. In contrast, UCP2 was highly abundant at early stages of heart development and in mESC. Understanding, that the expression patterns of UCP3 and UCP2 in heart during development reflect the type of the cell metabolism is key to the uncovering their different functions. Their expression ratio may be an important diagnostic criterion for the degree of CM differentiation and/or severity of a heart failure.