guanosine-triphosphate and carboxyatractyloside

The electrophoretic export of ATP against the import of ADP in mitochondria bridges the intra- versus extramitochondrial ATP potential gap. Here we report that the electrical nature of the ADP/ATP exchange by the mitochondrial ADP/ATP carrier (AAC) can be directly studied by measuring the electrical currents via capacitive coupling of AAC-containing vesicles on a planar lipid membrane. The currents were induced by the rapid liberation of ATP or ADP with UV flash photolysis from caged nucleotides. Six different transport modes of the AAC were studied: heteroexchange with either ADP or ATP inside the vesicles, initiated by photolysis of caged ATP or ADP; homoexchange with ADPex/ADPin or ATPex/ATPin; and caged ADP or ATP with unloaded vesicles. The heteroexchange produced the largest currents with the longest duration in line with the electrical charge difference ATP4- versus ADP3-. Surprisingly, also in the homoexchange and with unloaded vesicles, small currents were measured with shorter duration. In all three modes with caged ATP, a negative charge moved into the vesicles and with caged ADP it moved out of the vesicles. All currents were completely inhibited by a mixture of the inhibitors of the AAC, carboxyatractyloside and hongkrekate, which proves that the currents are exclusively due to AAC function. The observed charge movements in the heteroexchange system agree with the prediction from transport studies in mitochondria and reconstituted vesicles. The unexpected charge movements in the homoexchange or unloaded systems are interpreted to reveal transmembrane rearrangements of charged sites in the AAC when occupied with ADP or ATP. The results also indicate that not only ATP4- but also ADP3- contribute, albeit in opposite direction, to the electrical nature of the ADP/ATP exchange, which is at variance with former conclusions from biochemical transport studies. These measurements open up new avenues of studying the electrical interactions of ADP and ATP with the AAC.

Topics: Adenine Nucleotides; Adenosine Triphosphate; Animals; Atractyloside; Binding Sites; Biological Transport; Bongkrekic Acid; Cattle; Electric Conductivity; Guanosine Triphosphate; Membrane Proteins; Membranes, Artificial; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Models, Biological; Ultraviolet Rays

We have reconstituted a partially purified extract from rat liver endoplasmic reticulum membrane proteins into phosphatidylcholine liposomes. The resulting proteoliposomes, of an average diameter of 58 nm, transport intact ATP into their lumen in a temperature-dependent manner; transport was saturable (apparent Km = 0.72 microM) and highly specific: CMP-sialic acid and GTP were transported very slowly or not at all. Transport of ATP was inhibited by DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) but not by carboxyatractyloside. Previously, we showed that vesicles derived from rat liver and dog pancreas endoplasmic reticulum translocate ATP into their lumen in vitro but in these studies, following incubations with ATP, most of the phosphate was transferred to proteins because of the many kinases, endogenous acceptors for phosphorylation, and ATP binding proteins present in the vesicle membranes and lumen. This reconstituted system, which yielded a highly functional ATP transporter, can be used for further characterization and purification of this and probably other nucleotide transporters of the endoplasmic reticulum membrane. Previously used reconstitution protocols which were successful for Golgi membrane nucleotide transporters did not yield a functional endoplasmic reticulum ATP transporter.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenosine Triphosphate; Animals; Atractyloside; Biological Transport; Cytidine Monophosphate N-Acetylneuraminic Acid; Dogs; Endoplasmic Reticulum; Guanosine Triphosphate; Kinetics; Liposomes; Liver; Male; Membrane Proteins; Microscopy, Electron; Pancreas; Phosphorylation; Proteolipids; Rats; Rats, Sprague-Dawley; Substrate Specificity