Institute of Physiology, Pathophysiology and Biophysics, Department of Biomedical Sciences, University of Veterinary Medicine, 1210 Vienna, Austria.
Institute of Pharmacology and Toxicology, Rostock University Medical Center, 18057 Rostock, Germany.
Int J Mol Sci. 2021 Mar 2;22(5):2490. doi: 10.3390/ijms22052490.
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. 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.
腺嘌呤核苷酸转位酶（ANT）是一种众所周知的线粒体 ATP 对 ADP 交换体。相比之下，很少有研究表明 ANT 也介导质子穿过线粒体内膜的运输。这些研究的结果存在争议，并导致关于分子运输机制的不同假设。我们假设，ANT 和解偶联蛋白（UCP）介导的 H 转运具有相似的调节模式，可以用脂肪酸循环概念来解释。在平面脂质双层中重新构建纯化的重组 ANT1 后，我们可以在直接施加跨膜电势 ΔΨ 后测量膜电流，这将对应于线粒体状态 III 和 IV。实验结果表明，ANT1 不会导致基础质子泄漏。相反，它仅在存在长链脂肪酸（FA）的情况下介导 H 转运，这与 UCP 已知的情况相同。它取决于 FA 链长和饱和度，这意味着 FA 的运输局限于脂质-蛋白界面。具有 ATP 和 ADP 偏好的嘌呤核苷酸抑制 H 转运。ATP/ADP 转运的特异性抑制剂，羧基三唑啉或苯并呋喃酸，也降低了质子转运。根据荧光相关光谱法确定的 ANT1 浓度计算 H 周转率，等于 14.6 ± 2.5 s。分子动力学模拟显示，在蛋白质/脂质界面处存在一个大的正电荷区，这可能有助于 FA 阴离子穿过膜的运输。ANT 的双重功能-在存在 FA 的情况下的 ADP/ATP 和 H 转运-可能对调节线粒体膜电位以及线粒体中依赖电位的过程很重要。此外，将质子转运调节药物靶标扩展到 ANT1 可能会改善肥胖症、癌症、脂肪变性、心血管和神经退行性疾病的治疗。
