BRAIN ACETYLCHOLINE STORAGE SYSTEM

该项目将描述生物化学和药理学方面的特点， 乙酰胆碱（ACh）储存的大鼠脑突触囊泡。 存储 系统由质子泵ATP酶和单独的ACh组成 转运蛋白，其与用于所述受体的变构受体位点相关。 抑制药物vesamicol。 先前描述的乙酰胆碱转运蛋白- 令人惊讶的是， 与突触囊泡蛋白聚糖密切相关。 该项目将 确定大鼠脑AChT-VR与电射线AChT-VR的相似程度。 大鼠脑突触囊泡将以游离形式（即， P3制剂）或代谢活性突触体内。 活性 放射性标记ACh的转运和放射性标记vesamicol或a 与这些制剂更紧密结合的类似物将使用 过滤测定。 其他生物化学、生物有机和免疫化学技术 也将酌情使用。 具体目标是：（1）研究 大鼠脑ACh主动转运的机制及离子依赖性 研究了vesamicol类似物的结合与转运的关系 抑制，（3）测试大鼠囊泡中的AChT-VR是否与 光亲和标记法鉴定大鼠脑AChT-VR 与氚标记的高亲和力乙酰胆碱类似物，（5）开发一种测定方法， AchT-VR在大鼠脑突触体内的结合基于高浓度的 vesamicol的亲和性、放射性类似物，和（6）使突触体 不同的药理学操作，改变新陈代谢， 可能会影响乙酰胆碱受体VR的特性 该项目的长期目标是了解完整的 哺乳动物突触囊泡储存乙酰胆碱的生物化学，无论是 以及储存和释放是否耦合。 这 可能会导致一种药理学的发展， 并从受损的胆碱能末梢释放。 由于许多疾病 和毒性状态对胆碱能神经系统的影响， 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.