Kinetics of electrogenic transport by the ADP/ATP carrier

The electrogenic transport of ATP and ADP by the mitochondrial ADP/ATP carr ier (AAC) was investigated by recording transient currents with two differe nt techniques for performing concentration jump experiments: 1) the fast fl uid injection method: AAC-containing proteoliposomes were adsorbed to a sol id supported membrane (SSM), and the carrier was activated via ATP or ADP c oncentration jumps. 2) BLM (black lipid membrane) technique: proteoliposome s were adsorbed to a planar lipid bilayer, while the carrier was activated via the photolysis of caged ATP or caged ADP with a UV laser pulse. Two tra nsport modes of the AAC were investigated, ATP(ex)-0(in) and ADP(ex)-0(in). Liposomes not loaded with nucleotides allowed half-cycles of the ADP/ATP e xchange to be studied. Under these conditions the AAC transports ADP and AT P electrogenically. Mg2+ inhibits the nucleotide transport, and the specifi c inhibitors carboxyatractylate (GAT) and bongkrekate (BKA) prevent the bin ding of the substrate. The evaluation of the transient currents yielded rat e constants of 160 s(-1) for ATP and greater than or equal to 400 s(-1) for ADP translocation. The function of the carrier is approximately symmetrica l, i.e., the kinetic properties are similar in the inside-out and right-sid e-out orientations. The assumption from previous investigations, that the d eprotonated nucleotides are exclusively transported by the AAC, is supporte d by further experimental evidence. In addition, caged ATP and caged ADP bi nd to the carrier with similar affinities as the free nucleotides. An inhib itory effect of anions (200-300 mM) was observed, which can be explained as a competitive effect at the binding site. The results are summarized in a transport model.