Moleculer Organization of Renal Organic Cation Transport

性状（由申请方提供）：肝脏和肾脏从体内分泌大量具有生理学、药理学和毒理学意义的带正电荷的有机分子。肾脏和肝脏组织经上皮分泌“有机阳离子”（OC）的第一步涉及介导的OC从血液穿过基底外侧膜进入细胞。这一过程是OC分泌的进入步骤，由转运蛋白SLC 22 A家族的成员介导：OCT 2（肾脏）和OCT 1（肝脏）。OCT是临床上重要的药物相互作用的位点，这些转运蛋白的遗传多态性已被证明会影响所选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。在这个持续的研究计划的当前资助周期的过程中，我们开发了一个OCT 2结构的同源模型，该模型基于来自主要促进剂转运蛋白超家族的几种相关转运蛋白的晶体结构。这个模型，沿着与其他SLC 22 A转运蛋白的模型，提供了新的洞察转运蛋白的结构和功能之间的关系。然而，这些模型的准确性的信心将保持适度，充其量，直到基于这些模型的结构和功能预测接受严格的测试和验证。该提案描述了四组相关研究，这些研究扩展和扩展了我们正在进行的对有机阳离子转运蛋白OCT 2的人类直系同源物的结构和功能的研究。(1)使用定点诱变和取代的半胱氨酸可及性扫描方法（SCAM），我们将测试的假设，改变方向的转运（从内向外）改变的易位途径内的氨基酸残基的配置文件。(2)蛋白质组学方法（光亲和标记和质谱）将确定OC底物结合的裂缝区域。(2)这些实验的结果将被整合到并行研究采用计算方法，包括应用3D-QSAR方法和分子动力学模拟，以完善OCT 2模型，预测与运输过程相关的构象变化，并表征配体与假定的结合表面的相互作用。(4)荧光共振能量转移（FRET）将用于检验质膜中OCT 2寡聚化改变转运蛋白对底物亲和力的假设。这些研究将是必不可少的模型，准确预测，理想情况下，先发制人的不必要的相互作用的阳离子药物在肾脏和肝脏的发展。公共卫生相关性：肾脏和肝脏从体内主动分泌许多药物，并且在这些器官中的分泌部位处的不希望的药物-药物相互作用是大量发病率和死亡率的来源。有机阳离子转运蛋白OCT 1和OCT 2分别介导人肝脏和肾脏中阳离子药物分泌的第一步。在目前的研究计划周期中，我们生成了OCT 2的3D结构模型，该模型已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。在这个建议中，我们概述了实验来验证和完善这个模型，并探测这种蛋白质的结构变化与运输过程本身。这些研究的结果将有助于预测，理想情况下，预先阻止肾脏和肝脏中不必要的药物相互作用，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.