PDF(Pubmed)
Abstract：
ASCT2 (alanine serine cysteine transporter 2), a member of the solute carrier 1 family, mediates Na+-dependent exchange of small neutral amino acids across cell membranes. ASCT2 was shown to be highly expressed in tumor cells, making it a promising target for anticancer therapies. In this study, we explored the binding mechanism of the high-affinity competitive inhibitor L-cis hydroxyproline biphenyl ester (Lc-BPE) with ASCT2, using electrophysiological and rapid kinetic methods. Our investigations reveal that Lc-BPE binding requires one or two Na+ ions initially bound to the apo-transporter with high affinity, with Na1 site occupancy being more critical for inhibitor binding. In contrast to the amino acid substrate bound form, the final, third Na+ ion cannot bind, due to distortion of its binding site (Na2), thus preventing the formation of a translocation-competent complex. Based on the rapid kinetic analysis, the application of Lc-BPE generated outward transient currents, indicating that despite its net neutral nature, the binding of Lc-BPE in ASCT2 is weakly electrogenic, most likely because of asymmetric charge distribution within the amino acid moiety of the inhibitor. The preincubation with Lc-BPE also led to a decrease of the turnover rate of substrate exchange and a delay in the activation of substrate-induced anion current, indicating relatively slow Lc-BPE dissociation kinetics. Overall, our results provide new insight into the mechanism of binding of a prototypical competitive inhibitor to the ASCT transporters.
摘要：
ASCT2（丙氨酸丝氨酸半胱氨酸转运蛋白2），SLC1（溶质载体1）家族的成员，介导跨细胞膜的小中性氨基酸的Na依赖性交换。ASCT2在肿瘤细胞中高表达，使其成为抗癌治疗的有希望的目标。在这项研究中,我们使用电生理学和快速动力学方法,探索了高亲和力竞争性抑制剂Lc-BPE与ASCT2的结合机制.我们的研究表明，Lc-BPE结合需要一个或两个最初以高亲和力与apo转运蛋白结合的Na离子，Na1位点占用对抑制剂结合更为关键。与氨基酸底物结合形式相反，最后,第三个Na+离子不能结合，由于其结合位点（Na2）的扭曲，从而防止易位能力复合体的形成。基于快速动力学分析,Lc-BPE的应用产生了向外瞬态电流，表明,尽管其净中性性质，ASCT2中Lc-BPE的结合是弱电的，很可能是由于抑制剂的氨基酸部分内的不对称电荷分布。与Lc-BPE的预孵育还导致底物交换的转换率降低和底物诱导的阴离子电流的活化延迟，表明Lc-BPE解离动力学相对较慢。总的来说,我们的结果为原型竞争性抑制剂与ASCT转运蛋白的结合机制提供了新的见解.
