Molecular Analysis of Presynaptic Choline Transporters

乙酰胆碱（Acetylcholine，ACh）是中枢和外周神经系统的主要神经递质。的限速 乙酰胆碱合成中的一个步骤被认为是突触前获得前体胆碱，通过高水平的 亲和性，Na+依赖性，半胆碱-3（HC-3）敏感性胆碱转运蛋白（CHT）。近期在以下方面的突破 无脊椎动物和哺乳动物CHT基因的分子阐明，CHT特异性 抗体，以及易处理的体外和转基因模型系统的创建已经建立了新的机会 确定神经元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，我们确定海隧是 在体内以及体外模型系统中，主要是囊状定位。CHT似乎居住在 ）n胆碱能突触囊泡的亚群，其表达囊泡乙酰胆碱转运体（VAChT）， 储存ACh。初步的研究证明了CHT在突触膜中的定位变化， 野生型小鼠对去极化的反应和达到正常胆碱水平的翻译后机制 尽管在CHT +/-小鼠中CHT蛋白减少50%，但在转运和HC-3结合方面仍存在显著差异。在这个新的应用程序中，我们 应用生物化学、成像和功能方法学，使用体外和体内模型系统来研究 携带CHT的囊泡池的性质，并阐明囊泡靶向的物理要求， 突触CHT运输。其次，我们探索了质膜穿梭作为活性的主要途径， 信号和行为诱导的胆碱摄取的变化，并实施酵母双杂交筛选新的 CHT互动者。最后，我们分析了完全和部分基因CHT消融小鼠的后果， 胆碱能生物化学、药理学、生理学和行为学。这些研究将阐明 CHT调节，阐明CHT如何支持胆碱能突触/行为可塑性，并提供新的CHT联系 大脑疾病。

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.