Presynaptic Choline Transporters in the Heart

项目2 心血管功能的自主控制依赖于迷走神经与迷走神经的高度协调相互作用 胆碱能神经支配和去甲肾上腺素能交感神经投射。在之前的项目期间，布莱克利 该项目评估了去甲肾上腺素通过基因调控改变去甲肾上腺素信号的作用 运输商(篮网)。在当前项目期间，重点转向了解对 小鼠和人的心血管表型由胆碱能信号的遗传改变所致。在……里面 特别是，人们的注意力集中在突触前，高亲和力的胆碱转运体(CHT)上 药理学和遗传学研究表明，通过突触前维持胆碱能信号是必需的， 胆碱能终末高亲和力胆碱摄取(HACU)。布莱克利实验室首次克隆了老鼠和人类 并已开发出CHT特异性抗体和CHT缺陷小鼠模型以供当前开发 项目。布莱克利的团队检验了CHT是胆碱能的高度调控成分这一假说 心脏中的信号，这是由于CHT蛋白的活性依赖于穿梭到质膜 和转运蛋白的催化激活。其次，他们研究了基因改变的CHT表达如何影响 副交感神经的产生和释放，最终影响心血管功能。在具体目标1中，他们 建立CHT在正常小鼠心脏中的定位和活性，包括亚细胞评价 ACh合成的分布、转运特性和活性依赖的调节，以及CHT的支持 释放..。研究包括对小鼠心脏CHT分布的LIGH和EM免疫细胞化学研究 以及对心房突触小体的功能和亚细胞分离研究。在第二个具体目标中，他们 CHT基因缺失对心脏HACU、ACh水平及ACh/NE受体的影响 表情/敏感度。研究包括CHT转运和生物素化研究以及高效液相和 放射性配基测量ACh/NE信号潜力，比较野生型和CHT杂合子小鼠。在……里面 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 清醒、未麻醉野生型和CHT杂合子小鼠心功能的远程记录 以及评估人类CHT基因多态的频率和对心血管的影响。

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.