Mechanism of Arginine Transport in Cardiac Myocytes

描述(申请人提供)：精氨酸(Arg)是一氧化氮合酶(NOS)产生一氧化氮(NO)的唯一底物，一氧化氮(NO)是一种对许多生理和病理过程至关重要的信号分子。精氨酸不是由心肌细胞合成的，必须从血浆中输入。因此，负责Arg转运的膜结合载体蛋白(S)在Arg-NO系统的病理生理中可能起到与Arg本身同样重要的作用。我们在心肌细胞中的初步数据显示了大的Arg激活电流，其特性与低亲和力的阳离子氨基酸转运体CAT-2A一致。我们的数据还表明，心肌细胞中存在CAT-2AmRNA转录本，b)Arg依赖的瞬时电荷运动，这使得详细的动力学研究依赖于Fm依赖的Arg运输，c)Arg激活的NO释放，以及d)NO抑制Arg电流，提示该信号分子对Arg运输的急性调节。本项目的目的是鉴定和鉴定这种心脏精氨酸转运体，定量研究其动力学机制，并研究NO对其潜在的调节作用。为了实现这一目标，将结合分子生物学、生化、荧光和电生理技术对Arg转运和载体蛋白进行研究，以探讨心肌细胞Arg激活电流是由低亲和力CAT-2A转运蛋白产生的假设。拟议的实验将通过确定底物的特异性、表观亲和力、对抑制剂的敏感性以及Arg转运的Vm依赖性来表征这种转运蛋白的电生理和生化特征。实验还将利用我们的初步数据显示依赖于Arg的电荷运动来解决描述Arg运输的动力学反应方案。最后，实验将研究NO对Arg激活电流的抑制以及NO敏感的蛋白激酶介导的转运体的磷酸化，以解决NO尖锐地调节心肌细胞Arg运输的机制。总之，这些研究将提供阳离子氨基酸进入细胞运输过程中发生的分子事件的详细图景，以及调控机制如何改变运输功能。

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.