Moleculer Organization of Renal Organic Cation Transport

描述（由申请人提供）：有机阳离子转运体（OCTs）影响许多内源性分子的血浆浓度，甚至更广泛的异种化合物（包括药理学和毒理学活性物质）。虽然在许多组织中表达，但通常在屏障上皮中表达水平最高，包括肾脏、肝脏和肠道，它们影响药物的生物利用度；耐药;药物及其代谢产物的排泄；药物毒性;药物的药代动力学和药效学。因此，OCT转运体在健康和疾病的基本细胞过程中起着关键作用，并作为控制药物治疗各个方面的重要介质发挥作用。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特性的了解。在本提案中，我们概述了四个具体目标，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体OCT2的结构：(1)使用一系列拓扑扫描方法定义OCT转运体的膜拓扑；(2)通过定点诱变确定oct的功能区域；(3)构建一个综合的OCT2结构和预测模型，将结构点突变与底物亲和力和转运变化联系起来”。(4)利用选择性光亲和力标签，通过质谱法确定OCT转运体的底物结合域，确定与结合位点结构相关的肽序列。该结果将用于开发影响底物与人类OCT2结合的结构因素模型。通过这些研究获得的信息的应用有望预测潜在的药物相互作用和阳离子外源药物肾脏分泌遗传差异的基础。

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.