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Mechanism of Arginine Transport in Cardiac Myocytes

心肌细胞中精氨酸的转运机制

基本信息

批准号：
7666934
负责人：
RUBEN DANIEL PELUFFO
金额：
$ 33.17万
依托单位：
UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-08-15 至 2011-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7666934
关键词：
Acute Address Affinity Amino Acids Arginine Basic Amino Acid Transport Systems Binding Biochemical Biological CAT-2A Transporter Cardiac Cardiac Myocytes Carrier Proteins Cell Membrane Proteins Cell membrane Cells Charge Chronic Citrulline Cyclic GMP-Dependent Protein Kinases Data Dependence Event Feedback Fluorescence Functional disorder Goals Heart Heart failure Ions Kinetics Measurement Mediating Membrane Messenger RNA Molecular Movement Nitric Oxide Nitric Oxide Pathway Nitric Oxide Synthase Pathologic Processes Pathway interactions Phosphorylation Physiological Plasma Play Production Property Protein Kinase Proteins Rattus Reaction Regulation Reporting Research Personnel Role Scheme Side Signaling Molecule Site Substrate Specificity System Techniques Testing Transcript chronotropic electric field extracellular inhibitor/antagonist research study stoichiometry uptake voltage voltage clamp

项目摘要

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，一氧化氮是一种信号分子，对许多生理和病理过程至关重要。 Arg 不由心肌细胞合成，必须从血浆中输入。因此，负责 Arg 转运的膜结合载体蛋白在 Arg-NO 系统的病理生理学中可能发挥与 Arg 本身一样重要的作用。我们在心肌细胞中的初步数据显示大的精氨酸激活电流，其特性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) 精氨酸依赖性瞬时电荷运动，这允许对 FM 依赖性精氨酸转运进行详细的动力学研究，c) 精氨酸激活的 NO 释放，以及 d) NO 对精氨酸电流的抑制，表明该信号分子对精氨酸转运的急性调节。该项目的目标是鉴定和表征这种心脏精氨酸转运蛋白，定量解析其动力学机制，并研究 NO 对其的潜在调节。为了实现这一目标，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、对抑制剂的敏感性以及 Arg 转运的 Vm 依赖性，从电生理学和生物化学角度表征该转运蛋白。实验还将利用我们显示依赖于精氨酸的电荷运动的初步数据来解决描述精氨酸传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敏感蛋白激酶介导的转运蛋白磷酸化，以解决NO急性调节心肌细胞中Arg转运的机制。总而言之，这些研究将提供阳离子氨基酸转运到细胞期间发生的分子事件的详细情况，以及调节机制如何改变转运功能。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Cationic amino acid transporters and their modulation by nitric oxide in cardiac muscle cells.

心肌细胞中的阳离子氨基酸转运蛋白及其一氧化氮的调节。

DOI：
10.1007/s12551-021-00870-1
发表时间：
2021
期刊：
Biophysical reviews
影响因子：
0
作者：
Peluffo,RDaniel
通讯作者：
Peluffo,RDaniel

Molecular Determinants for Nitric Oxide Regulation of the Murine Cationic Amino Acid Transporter CAT-2A.

鼠阳离子氨基酸转运蛋白 CAT-2A 的一氧化氮调节的分子决定因素。

DOI：
10.1021/acs.biochem.0c00729
发表时间：
2020
期刊：
Biochemistry
影响因子：
2.9
作者：
Zheng,Ruifang;daRosa,Gabriela;Dans,PabloD;Peluffo,RDaniel
通讯作者：
Peluffo,RDaniel

L-Arginine currents in rat cardiac ventricular myocytes.

大鼠心室肌细胞中的 L-精氨酸电流。

DOI：
10.1113/jphysiol.2006.125054
发表时间：
2007
期刊：
The Journal of physiology
影响因子：
0
作者：
Peluffo,RDaniel
通讯作者：
Peluffo,RDaniel

L-lysine uptake in giant vesicles from cardiac ventricular sarcolemma: two components of cationic amino acid transport.

心室肌膜巨型囊泡中 L-赖氨酸的摄取：阳离子氨基酸转运的两个组成部分。

DOI：
10.1042/bsr20080159
发表时间：
2009
期刊：
Bioscience reports
影响因子：
4
作者：
Lu,Xiaodong;Zheng,Ruifang;Gonzalez,Jorge;Gaspers,Lawrence;Kuzhikandathil,Eldo;Peluffo,RDaniel
通讯作者：
Peluffo,RDaniel

D-enantiomers take a close look at the functioning of a cardiac cationic L-amino acid transporter.

D-对映体仔细观察心脏阳离子 L-氨基酸转运蛋白的功能。