Acute Regulation of Norepinephrine Transporters

去甲肾上腺素转运蛋白的急性调节

• 批准号: 7091428
• 负责人: AURELIO GALLI
• 金额: $ 33.18万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7091428
• 关键词: amphetamines; biophysics; calcium flux; cell line; cell surface receptors; central nervous system stimulants; endocytosis; exocytosis; gene expression; immunocytochemistry; laboratory mouse; laboratory rat; membrane transport proteins; molecular biology; neurons; neurotransmitter transport; norepinephrine; protein localization; protein quantitation /detection; protein structure function; receptor sensitivity; syntaxin; tricyclic antidepressant

DESCRIPTION (provided by applicant): The long-term objective of this project is to elucidate mechanisms supporting acute modulation of the norepinephrine (NE) transporter (NET) as established by coordinated trafficking and intrinsic activity modulation. NE signaling at PNS and CNS noradrenergic synapses regulates a variety of physiological functions including attention, motivation, vasoconstriction and heart rate. The NET dictates the magnitude and duration of NE signaling. For decades, the main focus on NET has been as a target for antidepressant and psychostimulant (e.g. amphetamine (AMPH), cocaine, and methylphenidate (Ritalin)) action. Our recent findings indicate that the stability of NET protein-protein associations, including interactions with syntaxin (SYN) 1A and protein phosphatase 2A (PP2A), dictate NET cell surface distribution and NET intrinsic activities, including transport rates, NET-gated currents and efflux potential. This renewal application unites two laboratories at the forefront of neurotransmitter transporter research, linking methods in biochemistry and molecular biology with real-time imaging and biophysical approaches, and includes studies on neuronal cell lines expressing NET and NET mutants as well as an evaluation of native noradrenergic neurons. We focus our efforts on three distinct, but coordinated, aspects of modulated NET behavior: the exocytosis of NET proteins, the endocytosis of NET proteins and the modulation of NET associated ion/substrate flux properties. Our specific aims are to: 1) Determine whether activity-dependent changes in NET surface density reflect increased insertion of cytoplasmic transporters or reduced transporter endocytosis (or both) and define the sites supporting SYN 1A/PP2Ac associations and their functional consequences with respect to NET localization and surface insertion; 2) Elucidate structural and signaling determinants of NET endocytic trafficking as triggered by GPCRs and AMPH; and 3) Determine relationships between NET/associated proteins/Ca2+ and AMPH-triggered NE efflux. Our overall goal then is to develop a more sophisticated understanding of the nature of acute NET regulation and to determine how the formation/dissolution of NET protein complexes promotes the coordination of NE clearance with NE release and clarifies intrinsic mechanisms by which psychostimulants impact NET mediated ion and NE flux.

描述（由申请人提供）：该项目的长期目标是阐明支持去甲肾上腺素（NE）转运蛋白（NE）急性调节的机制，该机制是通过协调的运输和内在活性调制确定的。 PNS和CNS去甲肾上腺素能突触的NE信号传导调节了各种生理功能，包括注意力，动机，血管收缩和心率。净决定了NE信号传导的幅度和持续时间。几十年来，对NET的主要重点一直是抗抑郁药和精神刺激剂（例如，苯丙胺（Amph），可卡因和甲基苯甲酸酯（Ritalin））作用的靶标。我们最近的发现表明，净蛋白质蛋白关联的稳定性，包括与语法（SYN）1A和蛋白质磷酸酶2a（PP2A）的相互作用，决定了净细胞表面分布和净固有活性，包括运输速率，净门控电流和外排势。该更新应用将两个实验室在神经递质转运蛋白研究的最前沿，将生物化学中的方法和分子生物学的方法与实时成像和生物物理方法联系起来，并包括对表达净和净突变体的神经元细胞系的研究，以及对天然甲腺癌神经元的评估。我们将精力集中在调制净行为的三个不同但协调一致的方面：净蛋白的胞吐作用，净蛋白的内吞作用以及净相关离子/底物通量特性的调节。我们的具体目的是：1）确定净表面密度的活性依赖性变化是否反映了细胞质转运蛋白的插入或减少转运蛋白的内吞作用（或两者），并定义支持Syn 1A/PP2AC关联的位点及其在网定位和表面插入方面的功能后果； 2）阐明由GPCR和AMPH触发的净内吞运输的结构和信号传导决定因素； 3）确定净/相关蛋白/Ca2+和AMPH触发的NE外排之间的关系。然后，我们的总体目标是对急性净调控性质有更复杂的理解，并确定净蛋白质复合物的形成/溶解如何促进NE清除率与NE释放的协调，并阐明精神刺激物影响净介导的net介导的离子和NE磁通的内在机制。