Molecular Analysis of Presynaptic Choline Transporters

突触前胆碱转运蛋白的分子分析

• 批准号: 8073467
• 负责人: Randy D. Blakely
• 金额: $ 38.59万
• 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/8073467
• 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（Acetylcholine, ACh）是中枢和外周神经系统的主要神经递质。的病原