Molecular Organization of Renal Organic Cation Transport

肝脏和肾脏从体内排出大量带正电荷的有机分子， 生理、药理和毒理学意义。跨上皮分泌的第一步 肾脏和肝脏中组织的有机阳离子（OC）涉及介导的OC从血液中摄取到 穿过基底外侧膜。这一过程是OC分泌的进入步骤，由 转运蛋白SLC22A家族的成员：OCT 2（在肾脏中）和OCT 1（在肝脏中）。OCT是 临床上重要的药物相互作用的位点，以及这些转运蛋白的遗传多态性， 显示影响所选药物的功效和药代动力学。制定方案， 合理的药物设计需要了解这些蛋白质的结构。过程中 在该持续研究计划的当前资助周期中，我们开发了OCT 2结构的同源模型， 基于来自运输的主要易化剂超家族的几种相关转运蛋白的晶体结构 proteins.该模型，沿着其他SLC22 A转运蛋白的模型，提供了新的见解， 转运蛋白结构和功能之间的关系。然而，对这些准确性的信心 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。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直系同源物的结构和功能的检查， OCT 2. (1)使用定点突变和取代的半胱氨酸可及性方法（SCAM），我们将 确定跨膜螺旋和相邻环片段之间的过渡点， 这种蛋白质的拓扑结构。(2)额外的SCAM分析和交叉链接研究将确定可访问性 蛋白质的拟议裂缝区域中的残基，与结合裂缝相关的螺旋的组织， 并将测试转运蛋白的构象变化是否改变裂缝的结合表面。(3)蛋白组 方法（光亲和标记和质谱）将确定OC底物的裂缝区域 这家公司进退两难. (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，以完善OCT 2模型，评估其质量和稳定性，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。这些研究对于开发能够准确预测 并且理想地预先阻止阳离子药物在肾脏和肝脏中的不希望的相互作用。

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.