Broustovetsky N, Bamberg E, Gropp T, Klingenberg M
Institute of Physical Biochemistry, University of Munich, Germany.
Biochemistry. 1997 Nov 11;36(45):13865-72. doi: 10.1021/bi971578x.
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.
线粒体ADP/ATP载体（AAC）的转运在一份初步报告中显示，当用附着于黑色脂质膜的重组AAC脂质体对笼锁ATP或ADP进行光解时，会产生电容性电流[Brustovetsky, N., Becker, A., Klingenberg, M., and Bamberg, E. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 664 - 668]。在此，我们研究了电流与ADP/ATP通量的关系、笼锁ADP和ATP与AAC的相互作用以及该方法的其他基本方面。如在线粒体中所示，笼锁ADP和ATP不能被AAC转运。对重组AAC进行通量测量表明，笼锁核苷酸是竞争性抑制剂（笼锁ADP的Ki = 5 microM，笼锁ATP的Ki = 1 microM）。电流对笼锁ATP浓度和光强度的依赖性表明，笼锁ATP与光解的ATP存在竞争。添加的ADP对电流与笼锁ATP之间的竞争得出ADP的Ki = 50 microM。我们得出结论，笼锁ADP和ATP比ADP或ATP与AAC结合更紧密，通过原位光解可立即启动转运。笼锁化合物优先结合在AAC的胞质侧。利用再生己糖激酶 + 葡萄糖系统，电流在重复闪光中得以稳定，可用于在一系列闪光期间施加抑制剂等。AAC抑制剂邦克雷酸（BKA）和羧基苍术苷（CAT）联合添加可完全抑制电流。CAT或BKA的部分抑制取决于闪光循环次数，从60%增加到90%，并且用葡萄糖酸盐替代氯化物时，抑制率仅从30%增加到90%。K⁺扩散电位（缬氨霉素 + KCl）会增加电流，而渗透性阴离子TPB⁻会降低电流。电流以及平行通量测量的pH依赖性表明，只有完全带电的ATP⁴⁻和ADP³⁻被转运。电流具有强烈的温度依赖性，在15℃时有一个转折点（EA = 95和28 kJ），这与之前线粒体中通量速率的测量结果一致。总之，电容性电流忠实地反映了AAC的转运功能，是研究转运过程中电荷转移的有力工具。
