The mitochondrial ADP/ATP carrier in the inner mitochondrial membrane imports ADP and exports ATP by switching between two conformational states. In the cytoplasmic state, which can be locked by carboxy-atractyloside, the substrate binding site is accessible to the cytoplasm, whereas in the matrix state, which can be locked by bongkrekic acid, the substrate binding site is open to the mitochondrial matrix. Access to the substrate binding site is regulated by salt bridge networks on either side of the central cavity, called the matrix and cytoplasmic salt bridge network. It has been proposed that during transport the salt bridge networks disrupt and form in an alternating way, opening and closing the binding site to opposite sides of the membrane, but experimental evidence has not been obtained for this mechanism.

Single cysteine mutations were introduced at the cytoplasmic side of the yeast mitochondrial ADP/ATP carrier, and the mutant carriers were expressed in the cytoplasmic membrane of Lactococcus lactis. They were capable of ADP transport and they could be inhibited by carboxy-atractyloside and bongkrekic acid. The complete inhibition by carboxy-atractyloside demonstrated that the carriers were oriented with the cytoplasmic side to the outside of the cells. To probe the accessibility of the single cysteines, the mutant carriers were locked in either the cytoplasmic or matrix state with the two inhibitors and labelled with the membrane-impermeable sulphydryl reagent eosin-5-maleimide. Specific cysteines that were accessible in the cytoplasmic state had become inaccessible in the matrix state. Subsequent experiments showed that ADP and ATP, but not AMP, led to the occlusion of single cysteines, demonstrating that the cytoplasmic side of the ADP/ATP carrier closes as part of the transport cycle. In addition, cross-linking studies combined with mass spectrometry and electron paramagnetic resonance spectroscopy were tried to probe the closure of the cytoplasmic salt bridge network.