Mechanism of Arginine Transport in Cardiac Myocytes

描述（由申请人提供）：精氨酸（Arg）是一氧化氮合酶（NOS）活性产生一氧化氮（NO）的唯一底物，一氧化氮是一种对许多生理和病理过程至关重要的信号分子。精氨酸不能由心肌细胞合成，必须从血浆中输入。因此，膜结合的载体蛋白（S）负责精氨酸运输可能发挥的作用一样重要的精氨酸本身的Arg-NO系统的病理生理。我们在心肌细胞中的初步数据显示大的Arg激活电流，其性质与低亲和力阳离子氨基酸转运蛋白CAT-2A一致。我们的数据还显示：a）心肌细胞中存在CAT-2A mRNA转录本，B）Arg依赖性瞬时电荷运动，这允许对FM依赖性Arg转运进行详细的动力学研究，c）Arg激活的NO释放，以及d）Arg电流的NO抑制，表明该信号分子对Arg转运的急性调节。本项目的目标是鉴定和表征这种心脏精氨酸转运蛋白，定量解决其动力学机制，并研究NO对它的潜在调控作用。为实现这一目标，将结合分子生物学、生化、荧光、和电生理学技术来研究心肌细胞中Arg激活电流由低亲和力CAT-2A转运蛋白产生的假设。拟议的实验将通过确定底物特异性，表观亲和力，对抑制剂的敏感性和精氨酸转运的Vm依赖性来表征这种转运蛋白的电生理学和生物化学特性。实验还将利用我们的初步数据显示精氨酸依赖的电荷运动来解决动力学反应方案，描述精氨酸运输。最后，实验将研究精氨酸激活电流的抑制NO以及NO敏感的蛋白激酶介导的磷酸化的转运蛋白，以解决的机制，NO急性调节心肌细胞中的精氨酸转运。总而言之，这些研究将提供一个详细的图片的分子事件发生在阳离子氨基酸运输到细胞，以及如何调节机制可能会改变运输功能。

DESCRIPTION (provided by applicant): Arginine (Arg) is the sole substrate for nitric oxide synthase (NOS) activity to produce nitric oxide (NO), a signaling molecule which is crucial for many physiologic and pathologic processes. Arg is not synthesized by cardiac myocytes and must be imported from the plasma. Thus, the membrane-bound carrier protein(s) responsible for Arg transport may play a role as important as Arg itself in the pathophysiology of the Arg-NO system. Our preliminary data in cardiac myocytes show large Arg-activated currents whose properties are consistent with the low-affinity cationic amino acid transporter CAT-2A. Our data also show a) the presence of CAT-2A mRNA transcripts in cardiac myocytes, b) Arg-dependent transient charge movements, which allow detailed kinetic studies on FM-dependent Arg transport, c) Arg-activated NO release, and d) NO inhibition of Arg currents, suggesting acute regulation of Arg transport by this signaling molecule. The goal of this project is to identify and characterize this cardiac arginine transporter, quantitatively solve its kinetic mechanism, and study its potential regulation by NO. To achieve this goal, Arg transport and the carrier protein will be studied with a combination of molecular biological, biochemical, fluorescence, and electrophysiological techniques to investigate the hypothesis that Arg-activated currents in cardiac myocytes are produced by the low-affinity CAT-2A transporter. Proposed experiments will characterize this transporter electrophysiologically and biochemically by determining substrate specificities, apparent affinities, sensitivity to inhibitors, and the Vm dependence of Arg transport. Experiments will also take advantage of our preliminary data showing Arg-dependent charge movements to solve the kinetic reaction scheme that describes Arg transport. Finally, experiments will study inhibition of Arg-activated currents by NO as well as NO-sensitive protein kinase-mediated phosphorylation of the transporter to solve the mechanism by which NO acutely regulates Arg transport in cardiac myocytes. Altogether, these studies will provide a detailed picture of the molecular events that take place during cationic amino acid transport into cells and how regulatory mechanisms may alter transport function.