Presynaptic Choline Transporters in the Heart

心脏中的突触前胆碱转运蛋白

• 批准号: 7252844
• 负责人: Randy D. Blakely
• 金额: $ 29.7万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7252844
• 关键词: 3' Untranslated Regions; Acetylcholine; Affinity; Alleles; Antibodies; Attention; Axon; Binding; Biotinylation; Cardiac; Cardiovascular Physiology; Cardiovascular system; Carrier Proteins; Catecholamines; Cell Fractionation; Cell membrane; Chimeric Proteins; Choline; Cholinergic Agents; Cholinergic Receptors; Collaborations; Condition; Cytoplasmic Tail; Disease; Disruption; Elevation; Equilibrium; Evaluation; Exercise; Frequencies; Gene Dosage; Genes; Genetic; Genetic Polymorphism; Genetic Variation; Heart; Heart Atrium; Heart Rate; Hemicholinium 3; High Pressure Liquid Chromatography; Human; In Vitro; Knockout Mice; Light; Link; Localized; Measures; Membrane Transport Proteins; Modeling; Molecular; Mus; Mutation; Nature; Neuromuscular Junction; Phenotype; Physiological; Plasma; Production; Property; Rate; Reagent; Receptor Signaling; Regulation; Relative (related person); Rest; Signal Pathway; Signal Transduction; Source; Synaptosomes; Techniques; Variant; Vesicle; awake; cardiovascular disorder risk; choline transporter; cholinergic; heart innervation; in vivo; man; mouse model; nerve supply; noradrenergic; novel; postnatal; presynaptic; protein aminoacid sequence; radioligand; receptor expression; response; trait; uptake

Project 2 Autonomic control of cardiovascular function depends on a highly coordinated interaction between vagal cholinergic innervation and noradrenergic sympathetic projections. In the prior project period, the Blakely project evaluated the contribution of altered noradrenergic signaling via genetic modulation of norepineprhine transporters (NETs). In the current project period, focus shifts to understanding contributions to cardiovascular phenotypes in mouse and man contributed by genetic alterations in cholinergic signaling. In particular, attention is focused on the presynaptic, high-affinity choline transporter (CHT) that pharmacological and genetic studies indicate is required to sustain cholinergic signaling through presynaptic, high-affinity choline uptake (HACU) in cholinergic terminals. The Blakely lab first cloned mouse and human CHT and has developed CHT-specific antibodies and CHT-deficient mouse models to exploit in the current project. Blakely's group examines the hypothesis that CHT is a highly regulated component off cholinergic signaling in the heart, owing to both activity-dependent shuttling of CHT proteins to the plasma membrane and transporter catalytic activation. Secondly, they examine how genetically altered CHT expression impacts parasympathetic ACh production and release and ultimately cardiovascular function. In Specific Aim 1, they establish the localization and activity of CHT in the normal mouse heart, including evaluation of subcellular distribution, transport properties and activity dependent-regulation, and CHT support for ACh synthesis and release.. Studies include light and EM immunocytochemical studies of CHT distribution in the mouse heart as well as functional and subcellular fractionation studies on atrial synaptosomes. In Specific Aim II, they determine the impact of genetic loss of CHT on cardiac HACU, ACh levels and ACh/NE receptor expression/sensitivity. Studies include CHT transport and biotinylation studies as well as HPLC and radioligand measures of ACh/NE signaling potential, comparing wildtype to CHT heterozygous mice. In Specific Aim 3, they elucidate the physiological impact of genetic variation/disruption in CHT. Studies include telemeterized recordings of cardiac function in awake, unanesthetized wildtype and CHT heterozygous mice as well as evaluations of the frequency and cardiovascular impact of human CHT polymorphisms.

计划2 心血管功能的自主控制依赖于迷走神经和迷走神经之间高度协调的相互作用。 胆碱能神经支配和去甲肾上腺素能交感神经投射。在项目前期，Blakely 该项目评估了通过去甲肾上腺素的遗传调节改变去甲肾上腺素能信号的贡献 转运蛋白（NET）。在当前项目期间，重点转移到理解对 小鼠和人的心血管表型由胆碱能信号传导的遗传改变贡献。在 特别是，注意力集中在突触前，高亲和力胆碱转运蛋白（CHT）， 药理学和遗传学研究表明需要通过突触前维持胆碱能信号传导， 胆碱能末梢的高亲和力胆碱摄取（HACU）。布莱克利实验室首次克隆小鼠和人类 CHT，并已开发出CHT特异性抗体和CHT缺陷小鼠模型，以利用在目前的 项目Blakely的小组检验了CHT是胆碱能神经系统中高度调节的成分的假设。 由于CHT蛋白质向质膜的活动依赖性穿梭， 和转运蛋白催化活化。其次，他们研究了基因改变的CHT表达如何影响 副交感神经ACh的产生和释放以及最终的心血管功能。在Aim Specific 1中，他们 建立CHT在正常小鼠心脏中的定位和活性，包括亚细胞 分布，运输特性和活性依赖性调节，以及CHT对ACh合成的支持， 释放..研究包括光和EM免疫细胞化学研究CHT分布在小鼠心脏 以及心房突触体的功能和亚细胞分级研究。在第二个具体目标中， 确定CHT基因缺失对心脏HACU、ACh水平和ACh/NE受体的影响 表达/敏感性。研究包括CHT转运和生物素化研究以及HPLC和 放射性配体测量ACh/NE信号传导潜力，比较野生型与CHT杂合小鼠。在 具体目标3，他们阐明了CHT中遗传变异/破坏的生理影响。研究包括 遥测记录清醒、未麻醉野生型和CHT杂合子小鼠的心脏功能 以及评估人类CHT多态性的频率和心血管影响。