Molecular Analysis of Presynaptic Choline Transporters

突触前胆碱转运蛋白的分子分析

• 批准号: 8277373
• 负责人: Randy D. Blakely
• 金额: $ 38.61万
• 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: 2004-06-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8277373
• 关键词: Ablation; Acetylcholine; Affinity; Aging; Ally; Animal Model; Animals; Antibodies; Attention; Behavior; Behavioral; Binding; Biochemical; Biochemistry; Biological Models; Biotinylation; Brain Diseases; Caenorhabditis elegans; Carrier Proteins; Cell Culture Techniques; Cell membrane; Cell model; Choline; Cognitive; Complementary DNA; Dementia; Development; Elements; Fatigue; Financial compensation; Functional Imaging; Genes; Genetic; Hemicholinium 3; Human; Hybrids; In Vitro; Invertebrates; Lead; Learning; Lecithin; Life; Link; Mammalian Cell; Maps; Mediating; Membrane; Memory; Metabolic; Methodology; Molecular; Molecular Analysis; Monoclonal Antibodies; Mus; Nature; Nematoda; Neurons; Neurotransmitters; Pathway interactions; Peripheral; Peripheral Nervous System; Pharmacology; Physiological; Physiology; Preparation; Presynaptic Receptors; Primates; Proteins; Reagent; Regulation; Reporting; Respiration; Rodent; Role; Route; Second Messenger Systems; Signal Transduction; Sodium; Source; Stimulus; Surface; Synapses; Synaptic Membranes; Synaptic Transmission; Synaptic Vesicles; Synaptophysin; Tars; Techniques; Transgenic Model; Transgenic Organisms; Vesicle; Yeasts; acetylcholine transporter; choline transporter; cholinergic; cholinergic neuron; cytochemistry; density; extracellular; genetic manipulation; immunocytochemistry; in vitro Model; in vivo; in vivo Model; man; novel; polyclonal antibody; premature; presynaptic; prevent; receptor; response; second messenger; tool; trafficking; uptake; yeast two hybrid system

Acetylcholine (ACh) is a major neurotransmitter in the central and peripheral nervous system. The rate-limiting step in ACh synthesis is believed to be the presynaptic acquisition of the precursor choline, achieved by the high- affinity, Na+-dependent, hemicholinium-3 (HC-3)-sensitive choline transporter (CHT). Recent breakthroughs in the molecular elucidation of invertebrate and mammalian CHT genes, the development of CHT-specific antibodies, and the creation of tractable in vitro and transgenic model systems have established new opportunities to define neuronal CHT subcellular distribution, mechanisms of activity- and receptor-dependent CHT regulation, and the functional consequences of genetic manipulation of CHT. Recently, we have established that CHT is )redominantly vesicular in localization, both in vivo as well as in in vitro model systems. CHT appears to reside )n a subpopulation of cholinergic synaptic vesicles that express the vesicular ACh transporter (VAChT) and which store ACh. Preliminary studies document both a change in the localization of CHT in synaptic membranes in response to depolarization in wildtype mice and a posttranslational mechanism to achieve normal levels of choline transport and HC-3 binding despite a 50% reduction in CHT protein in CHT +/- mice. In this new application, we apply biochemical, imaging and functional methodologies using in vitro and in vivo model systems to investigate the nature of the vesicular pool harboring CHT and clarify the physical requirements for vesicular targeting and synaptic CHT trafficking. Secondly, we explore plasma membrane shuttling as a major route for activity, cell signaling and behaviorally induced changes in choline uptake and implement a yeast 2-hybrid screen for novel CHT interactors. Finally, we analyze the consequences of full and partial genetic CHT ablation in the mouse for cholinergic biochemistry, pharmacology, physiology and behavior. These studies will elucidate novel aspects of CHT regulation, clarify how CHT supports cholinergic synaptic/behavioral plasticity and provide new CHT links to brain disease.

乙酰胆碱（ACh）是中枢和周围神经系统的主要神经递质。速率限制 乙酰胆碱合成的步骤被认为是前体胆碱的突触前获取，通过高- 亲和力、Na+ 依赖性、hemicholinium-3 (HC-3) 敏感性胆碱转运蛋白 (CHT)。最近的突破 无脊椎动物和哺乳动物 CHT 基因的分子阐明、CHT 特异性的开发 抗体，以及易于处理的体外和转基因模型系统的创建创造了新的机遇 定义神经元 CHT 亚细胞分布、活性和受体依赖性 CHT 调节机制， 以及 CHT 基因操作的功能后果。最近，我们确定 CHT 是 ）在体内和体外模型系统中，定位主要为囊泡。 CHT 似乎存在 )n 表达囊泡乙酰胆碱转运蛋白 (VAChT) 的胆碱能突触囊泡亚群，并且 储存乙酰胆碱。初步研究记录了 CHT 在突触膜中的定位发生了变化 野生型小鼠对去极化的反应以及达到正常胆碱水平的翻译后机制 尽管 CHT +/- 小鼠中 CHT 蛋白减少了 50%，但转运和 HC-3 结合仍然存在。在这个新应用程序中，我们 使用体外和体内模型系统应用生化、成像和功能方法进行研究 含有 CHT 的囊泡池的性质，并阐明囊泡靶向的物理要求和 突触 CHT 贩运。其次，我们探索质膜穿梭作为活动的主要途径，细胞 信号传导和行为诱导的胆碱吸收变化，并实施酵母 2 杂交筛选新的 CHT 互动者。最后，我们分析了小鼠中完全和部分基因 CHT 消融的后果 胆碱能生物化学、药理学、生理学和行为。这些研究将阐明新的方面 CHT监管，阐明CHT如何支持胆碱能突触/行为可塑性并提供新的CHT链接 到脑部疾病。