Molecular Organization of the Organic cation-Proton Exchanger, MATE1

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。载体介导的这些“有机”分泌 尤其是通过肾脏的“阳离子”（OC）对这些药物的药代动力学具有深远的影响。 重要的是，OC分泌是许多临床上显著的药物-药物相互作用的位点。 尽管第一种（即，在肾和肝OC分泌的进入）步骤是明确的， 主动和限速步骤（即，底物从细胞排出进入管状滤液或胆汁）。 OC分泌出口步骤的生理标志，如膜囊泡研究所确定 和完整的肾小管，是载体介导的OC/H+交换，和一组新的转运蛋白（多药 和毒素挤出机，MATE）最近从人类肾脏和肝脏（MATE 1和MATE-2K）克隆 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MATE是 肾和肝OC分泌的主要贡献者。尽管有大量的MATE转运蛋白， 已经被确定（人类中只有两个，但在原核生物，真菌和植物中有750多个）， MATE转运蛋白代表活性肾和肝分泌阳离子转运蛋白的主要驱动元件。 药物，实际上对这些蛋白质的分子特征一无所知。在本提案中，我们 描述了假设驱动的实验，这些实验将鉴定MATE转运蛋白的底物结合区域， 决定了这些蛋白质的二级结构和螺旋结构。这些研究结合了 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），以获得对MATE转运活性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至关重要。 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.