Molucular analysis of tissue distribution and structure-function relationship of transporters responsible for the regulation of drug transport

本研究对参与药物吸收和排泄的膜转运蛋白的组织分布和结构与功能的关系进行了研究，获得了以下结果：1. H^+偶联肽转运蛋白的结构与功能关系表达大鼠肽转运蛋白PEPT 1和PEPT 2的稳定转染细胞经组氨酸修饰剂DEPC处理后，两种转染细胞对甘氨酰肌氨酸的摄取均降低。通过分析二肽与β-内酰胺类抗生素的相互作用，发现β-内酰胺类抗生素的α-氨基部分可能与组氨酸残基相互作用，参与底物降解。2.肾脏有机离子转运蛋白的组织分布及功能特征。（1）有机阴离子转运蛋白OAT-K1：RT-PCR检测肾单位沿着节段的mRNA表达，OAT-K1 mRNA主要表达于肾单位近端直小管。Western印迹分析显示OAT-K1仅在肾小管刷状缘膜中表达。在非甾体抗炎药（NSAID）存在下，稳定表达OAT-K1的转染子的甲氨蝶呤转运受到显著抑制。（2）有机阳离子转运蛋白OCT 2：由于OCT 2转运四乙铵不依赖于H^+-梯度，且受膜电位的影响，推测OCT 2为基底外侧型有机阳离子转运蛋白。通过构建稳定表达OCT 1或OCT 2的转染子，进一步分析转运特性，然后明确OCT 1和OCT 2都是具有广泛底物特异性的基底外侧型有机阳离子转运蛋白，并且彼此具有相似的底物识别。

Tissue distribution and structure-function relationship of membrane transporters responsible for absorption and excretion of drugs have been studied, and then following results were obtained.1.Structure-function relationship of H^+-coupled peptide transportersWhen stable transfectans expressing rat peptide transporters PEPT1 and PEPT2 were treated with DEPC, a histidine-modifying agent, glycylsarcosine uptake by both transfectants were decreased. Interactions of dipeptides and beta-lactam antibiotics were analyzed, and then it became clear that alpha-amino moiety of beta-lactam antibiotics should interact with histidine residues, and may participate in substrate recognition.2.Tissue distribution and functional characteristics of renal organic ion transporters.(1)Organic anion transporter, OAT-K1 : Expression of mRNA along nephron segments was analyzed by RT-PCR.OAT-K1 mRNAs were mainly expressed in proximal straight tubules of superficial and juxtamedullary nephrons. Western blot analysis revealed that OAT-K1 is expressed only in brush-border membranes of renal tubules. Methotrexate transport by the transfectants stably expressing OAT-K1 was significantly inhibited in the presence of nonsteroidal anti-inflammatory drugs (NSAID).(2)Organic cation transporter, OCT2 : Because tetraethylammonium transport by OCT2 was H^+-gradient independent and was affected by membrane potential, OCT2 is deduced to be basolateral-type organic cation transporter. By constructing stable transfectants expressing OCT1 or OCT2, transport characteristics were further analyzed, and then it became clear that both OCT1 and OCT2 are basolateral-type organic cation transporters with broad substrate specificity and have similar substate recognition in each other.