Moleculer Organization of Renal Organic Cation Transport

描述（由申请人提供）：肝脏和肾脏从体内排泄大量带正电的有机分子，具有生理、药理学和毒理学意义。肾脏和肝脏组织经上皮分泌“有机阳离子”（OCs）的第一步涉及到从血液中介导的有机阳离子通过基底外膜进入细胞。这个过程是OC分泌的进入步骤，是由SLC22A转运蛋白家族的成员：OCT2（在肾脏中）和OCT1（在肝脏中）介导的。oct是临床上重要的药物-药物相互作用位点，这些转运体的遗传多态性已被证明会影响选定药物的疗效和药代动力学。开发合理的药物设计程序需要了解这些蛋白质的结构。在这个持续的研究项目的当前拨款周期中，我们基于运输蛋白主要促进者超家族的几个相关转运蛋白的晶体结构，开发了OCT2结构的同源模型。该模型与其他SLC22A转运蛋白模型一起，为转运蛋白结构和功能之间的关系提供了新的见解。然而，在基于这些模型的结构和功能预测得到严格的测试和验证之前，对这些模型的准确性的信心最多将保持适度。本提案描述了四组相关研究，这些研究扩展了我们正在进行的有机阳离子转运体OCT2的人体同源物的结构和功能的研究。(1)利用位点定向诱变和替代半胱氨酸可及性扫描方法（SCAM），我们将验证转运体方向的改变（从内向到外向）会改变易位途径中氨基酸残基谱的假设。(2)蛋白质组学方法（光亲和标记和质谱）将确定OC底物结合的裂口区域。(2)这些实验结果将整合到平行研究中，采用计算方法，包括应用3D-QSAR方法和分子动力学模拟，来完善OCT2模型，预测与传输过程相关的构象变化，并表征配体与假定结合表面的相互作用。(4)荧光共振能量转移（FRET）将用于验证质膜中OCT2的寡聚化改变转运蛋白对底物的亲和力的假设。这些研究对于准确预测模型的发展至关重要，并且在理想情况下，可以预防阳离子药物在肾脏和肝脏中的不良相互作用。公共卫生相关性：肾脏和肝脏积极地从体内分泌许多药物，在这些器官的分泌部位发生不必要的药物-药物相互作用是大量发病率和死亡率的来源。有机阳离子转运体OCT1和OCT2分别介导人体肝脏和肾脏中阳离子药物分泌的第一步。在当前的研究项目周期中，我们生成了OCT2的3D结构模型，该模型已被用于帮助识别这种蛋白质中影响药物分子结合方式的特定位点。在本提案中，我们概述了验证和完善该模型的实验，并探讨了与运输过程本身相关的这种蛋白质的结构变化。这些研究的结果将有助于预测，并在理想情况下，预防肾脏和肝脏中不必要的药物-药物相互作用，并有望帮助开发合理的药物设计方案。

DESCRIPTION (provided by applicant): 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 insight 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 extend and expand 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 scanning method (SCAM), we will test the hypothesis that changes orientation of the transporter (from inward- to outward-facing) changes the profile of amino acid residues within the translocation pathway. (2) Proteomic methods (photoaffinity labeling and mass spectrometry) will identify regions in the cleft to which OC substrates bind. (2) The results of these experiments will be integrated into parallel studies employing computational methods, including the application of 3D-QSAR methods and molecular dynamics simulations, to refine the OCT2 model, predict conformational changes associated with the transport process, and characterize ligand interactions with putative binding surfaces. (4) Fluorescence Resonance Energy Transfer (FRET) will be used to test the hypothesis that oligomerization of OCT2 in the plasma membrane changes the affinity of the transporter for substrates. 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. PUBLIC HEALTH RELEVANCE: The kidney and liver actively secrete many drugs from the body, and unwanted drug-drug interactions at the sites of secretion in these organs are the source of substantial morbidity and mortality. The organic cation transporters, OCT1 and OCT2, mediate the first step in secretion of cationic drugs in the human liver and kidney, respectively. During the current cycle of the research program, we generated a model of the 3D structure of OCT2 that has been used to help identify specific sites in this protein that influence how drug molecules bind. In this proposal we outline experiments to validate and refine this model, and probe the structural changes of this protein associated with the transport process itself. The results of these studies will help predict and, ideally, preempt unwanted drug-drug interactions in both the kidney and liver, and can be expected to assist in development of programs of rational drug design.