Moleculer Organization of Renal Organic Cation Transport

肾脏有机阳离子转运的分子组织

• 批准号: 6893766
• 负责人: STEPHEN H WRIGHT
• 金额: $ 23.18万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-15 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6893766
• 关键词: CHO cells; binding sites; cations; computer simulation; ion transport; mass spectrometry; membrane transport proteins; model design /development; molecular biology; molecular cloning; molecular dynamics; organic chemicals; physical model; point mutation; protein structure function; renal tubular transport; site directed mutagenesis; transfection /expression vector

DESCRIPTION (provided by applicant): Organic Cation Transporters (OCTs) influence the plasma concentration of many endogenous molecules, and of an even wider array of xenobiotic compounds (incl. pharmacologically and toxicologically active agents). Although expressed in many tissues, the highest levels of expression are typically found in barrier epithelia, including the kidney, liver and intestine where they influence drug bioavailability; drug resistance; excretion of drugs and their metabolites; drug toxicity; and drug pharmacokinetics and pharmacodynamics. Thus, OCT transporters play a critical role in fundamental cellular processes in health and disease, and function as important mediators governing all aspects of drug therapy. Despite the apparent physiological and clinical importance of OCT proteins, knowledge of their structure and mechanism of action has lagged far behind the knowledge of these properties of proteins in general. In this proposal, we outline four Specific Aims that will apply a series of experimental and computational approaches to address the structure of a prototypical organic cation transporter, OCT2: (1) Define the membrane topology of OCT transporters using a series of topology scanning approaches; (2) Define the functional regions of OCTs by site-directed mutagenesis; (3) Construct a comprehensive structural and predictive model of OCT2 that can correlate structural point mutations to changes in substrate affinity and transport," and (4) Determine the substrate binding domains of OCT transporters by mass spectrometry employing selective photoaffinity labels to determine peptide sequences structurally associated with the binding site. The results will be used to develop a model of the structural factors that influence binding of substrates to human OCT2. Application of the information gained through these studies holds the promise of predicting potential drug-interactions and the basis of genetic differences in renal secretion of cationic xenobiotics.

描述（由申请人提供）：有机阳离子转运蛋白（OCT）影响许多内源性分子的血浆浓度，甚至影响更广泛的异生物质化合物（包括毒性和毒理学活性剂）。尽管在许多组织中表达，但最高水平的表达通常存在于屏障上皮细胞中，包括肾、肝和肠，在那里它们影响药物生物利用度;耐药性;药物及其代谢物的排泄;药物毒性;以及药物药代动力学和药效学。因此，OCT转运蛋白在健康和疾病的基本细胞过程中起着关键作用，并作为重要的介体调节药物治疗的各个方面。尽管OCT蛋白具有明显的生理和临床重要性，但对其结构和作用机制的了解远远落后于对蛋白质这些性质的了解。在本论文中，我们提出了四个具体的目标，将应用一系列实验和计算方法来解决原型有机阳离子转运蛋白OCT 2的结构：（1）使用一系列拓扑扫描方法来确定OCT转运蛋白的膜拓扑结构;（2）通过定点突变来确定OCT的功能区域;（3）构建OCT 2的综合结构和预测模型，其可以将结构点突变与底物亲和力和转运的变化相关联，（4）采用选择性光亲和标记物通过质谱法测定OCT转运蛋白的底物结合结构域以确定肽序列在结构上与结合位点相关。结果将用于开发影响底物与人OCT 2结合的结构因素模型。通过这些研究获得的信息的应用，预测潜在的药物相互作用的承诺和阳离子外源性物质的肾脏分泌的遗传差异的基础。