Molecular mechanisms of drug transport across cell membrane

负责有机物质跨膜转运的转运体的显著特征之一是其在底物识别方面的多重专一性，其分子机制仍有待阐明。即使用结晶学方法解决了转运蛋白的三维结构问题，仍然很难理解底物识别中多特异性的结构基础。因此，在本研究中，我们利用分子药理分析方法进行了构效关系分析。中性氨基酸转运蛋白LAT1(L型氨基酸转运蛋白1)不仅可以转运天然产生的L氨基酸坚果，还可以转运氨基酸相关药物。为了建立稳定表达SV40大T抗原的转基因小鼠肾小管上皮细胞株，我们将人LAT1基因克隆到表达SV40大T抗原的转基因小鼠肾小管S2段的S2细胞中。然后，我们检测了这些化合物对人LAT1稳定转染细胞系摄取放射性标记苯丙氨酸的抑制作用。我们发现芳香族氨基酸相关化合物如L多巴、α-甲基多巴、三碘甲腺原氨酸、甲状腺激素以竞争性方式抑制苯丙氨酸摄取。苯丙氨酸甲酯、N-甲基苯丙氨酸和多巴胺对LAT1介导的转运无影响。计算分析结果表明，芳香族疏水侧链的存在以及自由羧基和游离氨基的存在是LAT1与底物结合部位所必需的。分子药理分析和计算分析相结合是揭示转运蛋白底物识别结构基础的良好手段。

One of the remarkable features of the transporters responsible for the transmembrane transport of organic substances is their multispecificity in the substrate recognition, whose molecular mechanisms remain to be clarified. Even after the three-dimensional structures of the transporter proteins are solved by crystallography, it would be still difficult to understand the structural basis for the multispecific nature in the substrate recognition. Therefore, in the present investigation, we have performed structure-activity analysis using molecular pharmacological analysis.Neutral amino acid transporter LAT1 (L-type amino acid transporter 1) transports not only naturally-occurring L-amino acids nut also amino acid related drugs. In order to generate stably transected cell lines, we transected human LAT1 cDNA to S2 cells derived from mouse renal proximal tubule S2 segment of transgenic mice overexpressing SV40 large T antigen. We, then, examined the inhibitory effects of the compounds on the uptake of radiolabeled phenylalanine by the human LAT1-stable transfected cell line. We demonstrated that the phenylalanine uptake was inhibited by aromatic amino acid-related compounds such as L-dopa, alpha-methyldopa, triiodethyronine, thyroxine in a competitive manner. On the other hands phenylalanine-methylester, N-methylphenylalanine and dopamine had no effects on the LAT1-mediated transport. Computational analysis results indicated that presence of aromatic hydrophobic side chains as well as free carboxyl and free amino groups is essential to interacted with substrate binding site of LAT1.It was proved that the combination of the molecular pharmacological analysis and the cpmput ational analysis is the excellent means to reveal the structural basis of the substrate recognition of transporters.