ATP Gradient Across the Innermitochondrial Membrane

Abstract

Adenosine 5′-triphosphate (ATP) is one of the most important biological molecules, playing roles as an energy-currency and also as an intracellular and extracellular signal transducer of the cells. In spite of its importance, however, how ATP distribute inside a cell is not clear, because cellular ATP is usually measured after disruption of numbers of cells. Recently, we have developed genetically-encoded Förster resonance energy transfer (FRET)-based indicators specific to ATP, which are called ATeams. These indicators enabled us to monitor ATP concentrations at any desired place inside living cells. To investigate whether there is any biased ATP distribution inside living cells, we compare ATP level of cytoplasm, nucleus and mitochondria using ATeams. ATP levels of cytoplasm and nucleus were almost the same, suggesting that ATP can freely pass through nucleic pores. On the other hand, we observed that ATP level of mitochondria matrix was significantly lower than those of cytoplasm and nucleus. Therefore, there is a gradient of ATP across inner mitochondrial membrane. When the loss of membrane potential of mitochondria was induced by an uncoupling reagent, CCCP, mitochondrial ATP level elevated and the gradient of ATP across the inner mitochondrial membrane disappeared. In addition, loss of membrane potential of mitochondria during apoptosis also resulted in the elevation of mitochondrial ATP. Our results suggest that membrane potential-dependent unidirectional exchange of ATP and ADP by ATP:ADP carrier proteins occurs very rapidly, enough to keep intramitochondrial ATP lower than outside.