Molecular Organization of the Organic cation-Proton Exchanger, MATE1

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。载体介导的这些“有机”物质的分泌 阳离子“(OCS)，特别是通过肾脏，对这些药物的药代动力学有深远的影响。 化合物，更重要的是，OC的分泌是许多临床上重要的药物相互作用的场所。 尽管肾脏和肝脏OC分泌的第一步(即进入)的分子基础已经很好地定义了，但 活性和限速步骤(即底物从细胞出口进入肾小管滤液或胆汁)知之甚少。 在膜小泡研究中确定的OC分泌退出步骤的生理标志 和完整的肾小管，是载体介导的OC/H+交换，以及一组新的转运体(多药 和毒素挤出器，Mate)是最近从人的肾脏和肝脏中克隆的(MATE1和Mate-2K)。 显示出这种“生理指纹”。强有力的证据支持越来越多的共识，即配偶是 肾脏和肝脏OC分泌的主要贡献者。尽管有大量的配偶运输者 (在人类中只有两个，但在原核生物、真菌和植物中有750+)，而且有可能 Mate转运蛋白是阳离子在肾脏和肝脏分泌活跃的主要驱动因素 药物，对这些蛋白质的分子特征几乎一无所知。在这项提案中，我们 描述假设驱动的实验，这些实验将确定Mate转运蛋白的底物结合区和 确定这些蛋白质的二级结构和螺旋结构。这些研究使用了一种组合 定点突变(半胱氨酸扫描和嵌合体)和蛋白质组学方法(光亲和标记 和质谱学)，以获得对配偶运输活动的分子基础的结构洞察。这些 研究对于建立准确预测并在理想情况下先发制人的模型是必不可少的。 阳离子药物在肾脏和肝脏中的相互作用。

The liver and kidney excrete from the body a wide array of positively charged organic molecules of physiological, pharmacological and toxicological significance. Carrier-mediated secretion of these "organic cations" (OCs), particularly by the kidney, has a profound influence on the pharmacokinetics of these compounds and, importantly, OC secretion is the site of many clinically significant drug-drug interactions. Although the molecular basis for the first (i.e., entry) step in renal and hepatic OC secretion is well defined, the active and rate-limiting step (i.e., substrate exit from cells into the tubular filtrate or bile) is poorly understood. The physiological hallmark of the exit step in OC secretion, as determined in studies with membrane vesicles and intact renal tubules, is carrier-mediated OC/H+ exchange, and a novel group of transporters (the Multidrug And Toxin Extruders, the MATEs) was recently cloned from human kidney and liver (MATE1 and MATE-2K) that displays this "physiological fingerprint." Strong evidence supports the growing consensus that MATEs are major contributors to renal and hepatic OC secretion. Despite the large number of MATE transporters that have been identified (only two in humans, but 750+ in prokaryotes, fungi, and plants), and the likelihood that MATE transporters represent the primary driving element in active renal and hepatic secretion of cationic drugs, virtually nothing is known about the molecular characteristics of these proteins. In this proposal, we describe hypothesis-driven experiments that will identify the substrate-binding region of MATE transporters and determine the secondary structure and helical organization of these proteins. The studies use a combination of site-directed mutagenesis (cysteine scanning and chimeras), and proteomic methods (photoaffinity labeling and mass spectrometry), to gain structural insight into the molecular basis of MATE transport activity. These studies will be essential for establishing models that accurately predict and, ideally, preempt unwanted interactions of cationic drugs in both the kidney and liver.