THE BRAIN ACETYLCHOLINE STORAGE SYSTEM

这个项目将描述生物化学和药理学方面的特点 大鼠脑突触小泡对乙酰胆碱的储存。存储空间 该系统由质子泵ATPase和单独的ACh组成 与变构受体结合的转运蛋白 抑制性药物维沙米考。之前描述的ACh转运体- 令人惊讶的是，维沙米考受体(AchT-VR)复合体的电射线 与突触小泡蛋白多糖密切相关。这个项目将 确定大鼠脑Acht-VR与电射线Acht-VR有多相似。 大鼠脑突触小泡将以自由形式准备(即， P3制剂)或在代谢活跃的突触体内。主动型 放射性标记ACh的转运和放射性标记维沙米考或a的结合 将研究与这些制剂更紧密结合的类似物 过滤分析。其他生化、生物有机和免疫化学技术 也将视情况使用。具体目的是：(1)研究 大鼠脑ACh主动转运的机制及特异性离子依赖 系统，(2)研究维沙米考类似物结合与转运的关系 抑制，(3)检测大鼠小泡内Acht-VR是否与 蛋白多糖，(4)用光亲和标记法鉴定大鼠脑Acht-VR 用氚高亲和力的ACh类似物，(5)建立一种测定 Acht-VR在大鼠脑突触体内结合高密度脂蛋白的研究 亲和力，维沙米考的放射性类似物，以及(6)使突触体 不同的药物操作，改变新陈代谢和 这可能会影响Acht-VR的性能。 该项目的长期目标是了解 哺乳动物突触小泡储存ACh的生物化学，无论是 受监管，以及储存和释放是否结合在一起。这 可能会导致一种药理学的发展，这种药理将增加ACh的储存 进入和释放出受损的胆碱能终末。由于许多疾病 毒性状态会影响胆碱能神经系统，这种能力 可能会有显著的临床益处。

This project will characterize biochemical and pharmacological aspects of acetylcholine (ACh) storage by rat brain synaptic vesicles. The storage system is composed of a proton-pumping ATPase and a separate ACh transporter that is associated with an allosteric receptor site for the inhibitory drug vesamicol. The previously characterized ACh transporter- vesamicol receptor (AChT-VR) complex of the electric ray, surprisingly, is strongly associated with synaptic vesicle proteoglycan. This project will determine how similar the rat brain AChT-VR is to the electric ray AChT-VR. Rat brain synaptic vesicles will be prepared either in free form (that is, a P3 preparation) or inside metabolically active synaptosomes. Active transport of radiolabeled ACh and binding of radio-labeled vesamicol or a more tightly binding analog to these preparations will be studied using filter assays. Other biochemical, bioorganic and immunochemical techniques also will be used as appropriate. The specific aims are to (1) study the mechanism and specific ion dependence of the rat brain ACh active transport system, (2) study the relationship of vesamicol analog binding to transport inhibition, (3) test whether the AChT-VR in rat vesicles is linked to proteoglycan, (4) identify the rat brain AChT-VR by photo-affinity labeling with a tritiated high affinity ACh analog, (5) develop an assay for the AchT-VR inside of rat brain synaptosomes based on binding of a high affinity, radioactive analog of vesamicol, and (6) subject the synaptosomes to different pharmacological manipulations that alter the metabolism and that might affect the properties of the AChT-VR. The long term goal of the project is to understand the complete biochemistry of ACh storage by synaptic vesicles in mammals, whether it is subject to regulation, and whether storage and release are coupled. This might lead to development of a pharmacology that would increase ACh storage in and release from compromised cholinergic terminals. Since many disease and toxic states impact on the cholinergic nervous system, this capability could be of significant clinical benefit.