PHYSIOLOGICAL REGULATION OF THE CHOLINE CO-TRANSPORTER

本项目的目标是更好地了解 支持胆碱能突触功能的机制。 实验将被设计和进行，以确定是否高 亲和胆碱共转运蛋白在两个不同的人群中发现， 马蹄蟹的胆碱能末梢据估计，一 一个群体存在于终膜中，另一个群体存在于 末端有胞质小泡。将努力查明， 分离，并表征这两个群体的胆碱共- 运输机该项目的成功完成将具有重大意义。 研究膜下分子机制的意义 胆碱转运蛋白的运输。具体目标如下： 阐明： 1.为了证明半胆碱-3（HC-3）结合与 选择性地与胆碱能末梢结合，HC-3结合是局部的， 在突触前膜的ChAT识别终端;（启动） 2.为了证明升高的K+刺激胆碱摄取的增加， 是通过胆碱能神经元中HC-3结合的可定量增加来实现的。 终端;（已完成） 3.为了证明乙酰胆碱囊泡和胆碱共转运囊泡 是不同的群体;分离可分离的突触群体 和突触样囊泡-一个富含VAChT和ACh; 其他富含HC-3结合位点;（已启动） 4.为了证明Ch协同转运蛋白的生理特性， 在分离的突触样囊泡群体（部分）中， HC-3结合;和 5.证明PKC在胆碱能神经元和终末的定位。 为了确定是否一个特定的PKC亚型或多种亚型， 定位于胆碱能末梢。（已开始）

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)