PHYSIOLOGICAL REGULATION OF THE CHOLINE CO-TRANSPORTER

这个项目的目的是为了更好地了解 支持胆碱能突触功能的机制。 将设计和进行实验，以确定高 亲和力胆碱共转运体在两个不同的人群中被发现 马蹄蟹的胆碱能终末。预计会有一位 群体存在于末端膜上，第二个群体存在于 末端有细胞质小泡。将努力确定， 分离并鉴定这两种胆碱类化合物-- 传送者。该项目的成功完成将具有重要的意义 膜支撑分子机制研究的意义 贩卖胆碱联合转运体。以下是具体目标 阐述： 1.证明半胱氨酸-3(HC-3)结合与 选择性地与胆碱能终末结合，并且HC-3结合是定位的 在Chat识别的终末的突触前膜中；(启动) 2.证明K+升高刺激胆碱摄取增加 是通过胆碱能受体中HC-3结合的可量化增加来实现的 终端；(已完成) 3.证明ACh囊泡和胆碱共转运体囊泡 是不同的种群；分离可分离的突触种群 和突触样囊泡--一个富含血管和乙酰胆碱的囊泡； 其他富含HC-3结合位点的；(启动) 4.阐明CH共转运蛋白的生理特性 在分离的突触样囊泡群(部分)中 HC-3结合；以及 5.研究PKC在胆碱能神经元和终末的定位。 确定PKC的一个特定亚型还是多个亚型 定位于胆碱能终末。(已启动)

The objective of this project is to gain a better understanding of the mechanisms which support the function of the cholinergic synapse. Experiments will be designed and carried out to determine if the high affinity choline co-transporter is found in two distinct populations in cholinergic terminals in the horseshoe crab. It is anticipated that one population exists in the terminal membrane and a second exists in cytoplasmic vesicles in the terminal. Efforts will be made to identify, separate, and characterize these two populations of choline co- transporters. Successful completion of this project will have significant implications for studying the molecular mechanisms undergirding membrane trafficking of the choline co-transporter. The following specific aims are set forth: 1. To demonstrate that hemicholinium -3(HC-3) binding is associated selectively with cholinergic terminals and that HC-3 binding is localized in the presynaptic membrane of ChAT identified terminals; (initiated) 2. To demonstrate that elevated K+ stimulated increase in choline uptake is accomplished by a quantifiable increase in HC-3 binding in cholinergic terminals; (completed) 3. To demonstrate that ACh vesicles and choline co-transporter vesicles are different populations; to isolate separable populations of synaptic and synaptic-like vesicles - One enriched with the VAChT and ACh; the other enriched with HC-3 binding sites; (initiated) 4. To demonstrated physiological characteristics of the Ch co-transporter in the isolated synaptic-like vesicle population (fraction)enriched with HC-3 binding; and 5. To demonstrate PKC localization in cholinergic neurons and terminals. To determine if one specific isoform or multiple isoforms of PKC are localized in cholinergic terminals. (initiated)