Molecular mechanisms of drug transport across cell membrane

负责有机物跨膜转运的转运体的显著特征之一是其在底物识别中的多特异性，其分子机制尚不清楚。即使用晶体学方法解决了转运蛋白的三维结构，但仍难以理解其在底物识别中多特异性的结构基础。因此，在本研究中，我们使用分子药理学分析进行了结构-活性分析。中性氨基酸转运蛋白LAT1 （l型氨基酸转运蛋白1）不仅运输天然存在的l -氨基酸，也运输与氨基酸相关的药物。为了获得稳定的横切细胞系，我们将人LAT1 cDNA横切到来自过表达SV40大T抗原的转基因小鼠肾近端小管S2段的S2细胞上。然后，我们检测了这些化合物对人类lat1稳定转染细胞系摄取放射性标记苯丙氨酸的抑制作用。我们证明了苯丙氨酸的摄取被芳香族氨基酸相关化合物如左旋多巴、α -甲基多巴、三碘去甲状腺原氨酸、甲状腺素竞争性地抑制。另一方面，苯丙氨酸-甲基lester、n -甲基苯丙氨酸和多巴胺对lat1介导的转运没有影响。计算分析结果表明，芳香疏水侧链以及游离羧基和游离氨基的存在是与LAT1底物结合位点相互作用所必需的。结果表明，分子药理学分析与计算分析相结合是揭示转运体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.