Mechanism of Arginine Transport in Cardiac Myocytes

心肌细胞中精氨酸的转运机制

• 批准号: 7108534
• 负责人: RUBEN DANIEL PELUFFO
• 金额: $ 34.17万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-15 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7108534
• 关键词: aminoacid transport; arginine; cardiac myocytes; electrophysiology; fluorescence microscopy; intracellular transport; laboratory rat; membrane transport proteins; nitric oxide; phosphorylation; polymerase chain reaction; protein kinase; stoichiometry; voltage /patch clamp

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.

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（NO）是许多生理和病理过程中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，在Arg- no系统的病理生理中，负责Arg运输的膜结合载体蛋白(s)可能与Arg本身一样发挥重要作用。我们在心肌细胞中的初步数据显示，大的精氨酸激活电流的性质与低亲和力的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转录物，b) Arg依赖的瞬时电荷运动，这允许对fm依赖的Arg运输进行详细的动力学研究，c) Arg激活的NO释放，d) NO抑制Arg电流，表明该信号分子对Arg运输的急性调节。本项目旨在鉴定和表征该心脏精氨酸转运体，定量求解其动力学机制，并研究NO对其的潜在调控作用。为了实现这一目标，我们将结合分子生物学、生物化学、荧光和电生理技术研究精氨酸转运和载体蛋白，以研究心肌细胞中精氨酸激活电流是由低亲和力的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Arg转运的Vm依赖性来表征这种转运体的电生理和生化特性。实验还将利用我们显示氩依赖电荷运动的初步数据来解决描述氩输运的动力学反应方案。最后，实验将研究NO对精氨酸激活电流的抑制以及NO敏感蛋白激酶介导的转运蛋白磷酸化，以解决NO在心肌细胞中急性调节精氨酸转运的机制。总之，这些研究将提供阳离子氨基酸转运到细胞过程中发生的分子事件的详细图像，以及调节机制如何改变转运功能。