Brustovetsky N, Klingenberg M
Institute for Physical Biochemistry, University of Munich, Federal Republic of Germany.
Biochemistry. 1996 Jul 2;35(26):8483-8. doi: 10.1021/bi960833v.
Single-channel current measurements of excised patches with reconstituted purified mitochondrial ADP/ATP carrier (AAC) indicates the presence of a large low cation selective (PK+/PCl- = 4.3 +/- 0.6) channel. The channel conductance has multiple sublevels and varies from 300 to 600 pS. It has low probability of current fluctuations at Vhold up to 80-100 mV of both signs and is reversibly gated at Vhold > 150 mV. The opening of the channel is Ca(2+)-dependent (1 mM Ca2+) and can be reversibly closed on removal of Ca2+. It is strongly pH dependent and closes completely at pHex 5.2. The AAC-specific inhibitor bongkrekate inhibits the channel partially and completely in combination with ADP, whereas carboxyatractylate did not affect the conductance. The effects of these AAC-specific ligands prove that the channel activity belongs to AAC. The AAC-linked conductance can clearly be differentiated from the porin channel, rarely detected in our preparations. The properties of the AAC-linked channel coincide with the mitochondrial permeability transition pore (MTP), which is also affected by the AAC ligands [Hunter, D. R., & Haworth, R. A. (1979) Arch. Biochem. Biophys. 195, 453-459] and resembles the mitochondrial "multiconductance channel" [Kinnally, K. W., Campo, M. L., & Tedeschi, H. T. (1989) J. Bioenerg. Biomembr. 21, 497-506] or "megachannel" [Petronilli, V., Szabo, I., & Zoratti, M. (1989) FEBS Lett. 259, 137-143]. Therefore we conclude that the AAC, when converted into a large unselective channel, is a key component in the MTP and thus is involved in the ischemia-reperfusion damage and cytosolic Ca2+ oscillations. The channel opening in AAC is proposed to be caused by binding of Ca2+ to the cardiolipin, tightly bound to AAC, thus releasing positive charges within the AAC which open the gate.
对重构纯化的线粒体 ADP/ATP 载体（AAC）进行切除膜片的单通道电流测量，结果表明存在一个大的低阳离子选择性通道（PK+/PCl- = 4.3 ± 0.6）。该通道电导具有多个亚水平，范围在 300 至 600 pS 之间。在 ±80 - 100 mV 的保持电压下，电流波动概率较低，且在保持电压 > 150 mV 时可逆门控。通道开放依赖于 Ca(2+)（1 mM Ca2+），去除 Ca2+后可逆转关闭。它强烈依赖于 pH，在细胞外 pH 为 5.2 时完全关闭。AAC 特异性抑制剂邦克里酸单独作用时部分抑制该通道，与 ADP 联合使用时则完全抑制，而羧基苍术苷不影响电导。这些 AAC 特异性配体的作用证明通道活性属于 AAC。与 AAC 相关的电导可明显区别于我们制备过程中很少检测到的孔蛋白通道。与 AAC 相关的通道特性与线粒体通透性转换孔（MTP）相符，MTP 也受 AAC 配体影响[Hunter, D. R., & Haworth, R. A. (1979) Arch. Biochem. Biophys. 195, 453 - 459]，且类似于线粒体“多电导通道”[Kinnally, K. W., Campo, M. L., & Tedeschi, H. T. (1989) J. Bioenerg. Biomembr. 21, 497 - 506]或“大通道”[Petronilli, V., Szabo, I., & Zoratti, M. (1989) FEBS Lett. 259, 137 - 143]。因此我们得出结论，当 AAC 转变为一个大的非选择性通道时，它是 MTP 的关键组成部分，从而参与缺血再灌注损伤和胞质 Ca2+振荡。有人提出 AAC 中的通道开放是由于 Ca2+与紧密结合在 AAC 上的心磷脂结合，从而释放 AAC 内的正电荷，进而打开门控。
