Molecular Organization of Renal Organic Cation Transport

肾脏有机阳离子转运的分子组织

• 批准号: 7623693
• 负责人: STEPHEN H WRIGHT
• 金额: $ 23.03万
• 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 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7623693
• 关键词: Address; Alkaloids; Behavior; Bile fluid; Binding; Blood; Carrier Proteins; Cations; Cells; Charge; Cleaved cell; Computing Methodologies; Cysteine; Development; Drug Design; Drug Interactions; Drug Kinetics; Excretory function; Family; Genetic Polymorphism; Grant; Helix (Snails); Hepatic; Hepatocyte; Heterocyclic Compounds; Homology Modeling; Human; Kidney; Ligands; Liver; Mass Spectrum Analysis; Mediating; Membrane; Methods; Modeling; Molecular; Molecular Conformation; Mutagenesis; Numbers; Organic Cation Transporter; Orthologous Gene; POU2F1 gene; POU2F2 gene; Pharmaceutical Preparations; Photoaffinity Labels; Physiological; Play; Population; Prevalence; Process; Program Development; Protein Conformation; Protein Region; Proteins; Proteomics; Proximal Kidney Tubules; Range; Research; Role; Series; Single Nucleotide Polymorphism; Site; Site-Directed Mutagenesis; Structure; Structure-Activity Relationship; Surface; Testing; Therapeutic; Tissues; Toxic Environmental Substances; Transport Process; Tubular formation; Validation; Work; Xenobiotics; apical membrane; base; basolateral membrane; computer studies; crosslink; ear helix; inhibitor/antagonist; insight; interest; member; model development; molecular dynamics; novel; predictive modeling; preempt; programs; protein structure; research study; solute; uptake

The liver and kidney excrete from the body a wide array of positively charged organic molecules of physiological, pharmacological and toxicological significance. The first step in the transepithelial secretion of the ¿organic cations¿ (OCs) by tissues in the kidney and liver involves mediated OC uptake from the blood into cells, across the basolateral membrane. This process, the entry step in OC secretion, is mediated by members of the SLC22A family of transport proteins: OCT2 (in the kidney), and OCT1 (in the liver). OCTs are sites of clinically important drug-drug interactions, and genetic polymorphisms of these transporters have been shown to influence both the efficacy and pharmacokinetics of selected drugs. Development of programs for rational drug design will require an understanding of the structure of these proteins. During the course of the current grant cycle of this continuing research program we developed a homology model of OCT2 structure, based upon crystal structures of several related transporters from the Major Facilitator Superfamily of transport proteins. This model, along with models of other SLC22A transport proteins, has provided novel insights into relationships between transporter structure and function. However, confidence in the accuracy of these models will remain modest, at best, until structural and functional predictions based upon these models receive rigorous testing and validation. This proposal describes four sets of related studies that will continue our ongoing examination of the structure and function of the human ortholog of the organic cation transporter, OCT2. (1) Using site-directed mutagenesis and the substituted cysteine accessibility method (SCAM), we will identify points of transition between transmembrane helices and adjacent loop segments that will establish the topology of this protein. (2) Additional SCAM analyses and cross-linking studies will determine the accessibility of residues in the proposed cleft region of the protein, organization of helices associated with the binding cleft, and will test if conformational shifts of the transporter change the binding surface of the cleft. (3) Proteomic methods (photoaffinity labeling and mass spectrometry) will identify regions in the cleft to which OC substrates bind. (4) The results of these experiments will be integrated with the use of computational methods, including molecular dynamics simulations, to refine the OCT2 model, assess its quality and stability, predict conformational changes associated with the transport process, and characterize ligand interactions with putative binding surfaces. These studies will be essential for development of models that accurately predict and, ideally, preempt unwanted interactions of cationic drugs in both the kidney and liver.

肝脏和肾脏从体内排泄出大量带正电的有机分子 生理、药理和毒理学意义。跨上皮细胞分泌的第一步 肾和肝组织的有机阳离子(OCS)涉及从血液到 细胞，穿过基底膜。这个过程，即OC分泌的入门步骤，是由 SLC22A转运蛋白家族的成员：OCT2(在肾脏)和OCT1(在肝脏)。OCT是 临床上重要的药物-药物相互作用的部位和这些转运蛋白的遗传多态已经被 对选定药物的疗效和药代动力学都有影响。为以下项目开发计划 合理的药物设计需要了解这些蛋白质的结构。在这个过程中， 这一持续研究计划的当前拨款周期我们开发了OCT2结构的同源模型， 基于主要促进剂超家族中几个相关转运蛋白的晶体结构 蛋白质。这个模型，以及其他SLC22A运输蛋白的模型，提供了对 转运蛋白结构与功能的关系。然而，对这些数据准确性的信心 充其量，模型将保持适度，直到基于这些模型的结构和功能预测收到 严格的测试和验证。该提案描述了四组相关研究，这些研究将继续我们的 正在进行的有机阳离子转运蛋白的人类同源基因的结构和功能的检查， OCT2.(1)使用定点突变和替代半胱氨酸可及性方法，我们将 确定跨膜螺旋和相邻环段之间的过渡点，这将建立 这种蛋白质的拓扑结构。(2)其他诈骗分析和交叉链接研究将确定可获得性 蛋白质建议的裂隙区域的残基，与结合裂隙相关的螺旋的组织， 并将测试转运蛋白的构象变化是否改变了裂隙的结合表面。(3)蛋白质组学 方法(光亲和标记和质谱学)将识别裂隙中OC底物所在的区域 捆绑。(4)这些实验的结果将与计算方法的使用相结合，包括 分子动力学模拟，以提炼OCT2模型，评估其质量和稳定性，预测 与转运过程相关的构象变化，并表征配体与 假定的结合表面。这些研究对于开发准确预测的模型是必不可少的 而且，理想的情况是，抢先阻止阳离子药物在肾脏和肝脏中不必要的相互作用。