Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey, Newark, NJ, USA.
Biophys J. 2010 Nov 17;99(10):3224-33. doi: 10.1016/j.bpj.2010.09.025.
Cationic amino acid transporters are highly selective for L-enantiomers such as L-arginine (L-Arg). Because of this stereoselectivity, little is known about the interaction of these transporters with D-isomers. To study whether these compounds can provide information on the molecular mechanism of transport, inward currents activated by L-Arg with low apparent affinity were measured in whole-cell voltage-clamped cardiomyocytes as a function of extracellular L-Arg and D-Arg concentrations. D-Arg inhibited L-Arg currents in a membrane-potential (V(M))-dependent competitive manner, indicating the presence of D-Arg binding sites in the carrier. Analysis of these steady-state currents showed that L- and D-Arg binding reactions dissipate a similar small fraction of the membrane electric field. Since D-Arg is not transported, these results suggest that enantiomer recognition occurs at conformational transitions that initiate amino acid translocation. The V(M) dependence of maximal current levels suggests that inward currents arise from the slow outward movement of negative charges in the unliganded transporter. Translocation of the L-Arg-bound complex, on the other hand, appears to be electroneutral. D-Arg-dependent transient charge movements, also detected in these cells, displayed a V(M)-dependent charge distribution and kinetics that are consistent with amino acid binding in an ion well in a shallow, water-filled extracellular binding pocket.
阳离子氨基酸转运体对 L-对映体（如 L-精氨酸）具有高度选择性。由于这种立体选择性，人们对这些转运体与 D-对映体的相互作用知之甚少。为了研究这些化合物是否能为转运的分子机制提供信息，我们在全细胞膜片钳电压钳心肌细胞中测量了与低表观亲和力的 L-精氨酸激活的内向电流，作为细胞外 L-精氨酸和 D-精氨酸浓度的函数。D-精氨酸以膜电位（Vm）依赖性竞争性方式抑制 L-精氨酸电流，表明载体中存在 D-精氨酸结合位点。对这些稳态电流的分析表明，L-和 D-精氨酸的结合反应会消耗膜电场的相似小部分。由于 D-精氨酸不能被转运，这些结果表明，对映体识别发生在启动氨基酸转运的构象转变中。最大电流水平的 Vm 依赖性表明，内向电流源自未配体转运体中负电荷的缓慢外向运动。另一方面，L-精氨酸结合复合物的转运似乎是电中性的。在这些细胞中还检测到的 D-精氨酸依赖性瞬态电荷移动，显示出与离子阱中氨基酸结合一致的 Vm 依赖性电荷分布和动力学，在离子阱中，氨基酸结合在浅的、充满水的细胞外结合口袋中。
