Biochemical and physical parameters of the electrical currents measured with the ADP/ATP carrier by photolysis of caged ADP and ATP.
The transport by the mitochondrial ADP/ATP carrier (AAC) has been shown in a preliminary communication to produce electrical capacitive currents on photolysis of caged ATP or ADP with reconstituted AAC liposomes attached to black lipid membranes [Brustovetsky, N., Becker, A., Klingenberg, M., and Bamberg, E. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 664-668]. Here we study the relation of the currents to ADP/ATP fluxes, the interaction of caged ADP and ATP with AAC and other basic facets of this method. Caged ADP and ATP are not transported by the AAC, as shown in mitochondria. Flux measurements with reconstituted AAC show that caged nucleotides are competitive inhibitors (Ki = 5 microM for caged ADP and 1 microM for caged ATP). Caged ATP competes with photolyzed ATP as shown by the dependence of the currents on the caged ATP concentration and on the light intensity. A competition of added ADP with caged ATP on the currents yields Ki = 50 microM for ADP. We conclude that caged ADP and ATP bind tighter to AAC than ADP or ATP, allowing immediate initiation of translocation by in situ photolysis. The caged compounds bind preferentially at the cytosolic side of AAC. With a regenerative hexokinase + glucose system, the currents are stabilized in repetitive flashes and can be used for applying inhibitors etc. during a flash series. The currents are completely inhibited by the combined addition of the AAC inhibitors bongkrekate (BKA) and carboxyatractylate (CAT). The partial inhibition by CAT or BKA is dependent on the number of flash cycles increasing from 60% to 90%, and by replacing chloride with gluconate from only 30% to 90%. The current are increased by a K+ diffusion potential (valinomycin + KCl) and decreased by the permeant anion TPB-. The pH dependence of the currents and of the parallel flux measurements indicates that only the fully charged ATP4- and ADP3- are transported. A strong temperature dependence of the currents with a break at 15 degrees C (EA = 95 and 28 kJ) agrees with former measurements of flux rates in mitochondria. In conclusion, the capacitive currents faithfully reflect AAC transport function and are a powerful tool for investigating the charge transfer in transport.
Broustovetsky
N
N
Institute of Physical Biochemistry, University of Munich, Germany.
Bamberg
E
E
Gropp
T
T
Klingenberg
M
M
D000244:Adenosine Diphosphate / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001278:Atractyloside / Q000031:analogs & derivatives / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D001865:Bongkrekic Acid / Q000494:pharmacology; D004553:Electric Conductivity; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials / Q000187:drug effects; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism; D010782:Photolysis / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013696:Temperature
D013439:Sulfhydryl Reagents; D001865:Bongkrekic Acid; D001278:Atractyloside; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C003853:carboxyatractyloside