Structure in vesicular acetylcholine transporter

囊泡乙酰胆碱转运体（VAChT）将乙酰胆碱（ACh）储存在突触囊泡中，用于诱发突触后神经元的兴奋。 从胆碱能神经末梢释放。它被一种叫做维索霉素的化合物变构抑制。的 该项目旨在了解VAChT的结构和运输机制，其长期目标是 开发一种药理学来控制乙酰胆碱的储存（从而释放），用于治疗胆碱能疾病。 使用定点诱变的实验、突变体特性的表达和表征是 提出了VAChT中的残基必须质子化才能运输。第一个目标是测试最近的 推测的跨膜结构域Xi（TMD Xi）中天冬氨酸残基对此作用的初步分配 是的。一个不同的天冬氨酸或谷氨酸残基，最近被证明不是在TMD必须是 去质子化以结合ACh和维索霉素。第二个目标是识别它。第三个目标是测试和提炼 ACh和vesamicol结合位点与推定的TMD XIII和X的最近分配。天冬氨酸 已知假定TMD X中的残基参与提供能量输入的质子移位反应 到VAChT。第四个目的是使用一种新的测定方法来测试和改进这种残留物的拟议作用， 监测ACh结合位点的跨膜重定向。第五个目标是测试VAChT是否 含有12个TMD，如现在所假设的，通过将一个半胱氨酸残基插入到该分子的每个亲水区域中， 序列并使用化学标记确定残留物暴露于膜的哪一侧 法第六个目的是确定当VAChT结合时，哪些亲水区域改变构象 ACh和vesamicol和运输ACh通过使用标记技术的条件下，检测不同的 标签率。该实验将扩展到负责所需功能的残基的突变体。 质子化和去质子化，以确定哪些亲水区域在pH极端时改变构象。 研究结果将为VAChT的结构和动力学研究提供新的信息。

Vesicular acetylcholine transporter (VAChT) stores acetylcholine (ACh) in synaptic vesicles for evoked ¿elease from cholinergic nerve terminals. It is allosterically inhibited by a compound called vesamicol. The project seeks understanding of VAChT structure and transport mechanism with the long term goal of developing a pharmacology to control ACh storage (and thus release) for treatment of cholinergic disorders. Experiments using site-directed mutagenesis, expression and characterization of mutant properties are proposed. A residue in VAChT must be protonated for transport. The first aim is to test whether the recent Dreliminary assignment of the aspartate residue in putative transmembrane domain XI (TMD XI) to this role s correct. A different aspartate or glutamate residue that recently was shown not to be in a TMD must be deprotonated to bind ACh and vesamicol. The second aim is to identify it. The third aim is to test and refine the recent assignments of ACh and vesamicol binding sites to putative TMDs XIII and X. The aspartate residue in putative TMD X is known to participate in proton translocation reactions that provide energy input to VAChT. The fourth aim is to test and refine the proposed role(s) of this residue using a novel assay that monitors transmembrane reorientation of the ACh binding site. The fifth aim is to test whether VAChT contains 12 TMDs, as is now assumed, by inserting one cysteine residue into each hydrophilic region of the sequence and determining which side of the membrane the residue is exposed to using a chemical labeling technique. The sixth aim is to identify which hydrophilic regions change conformation when VAChT binds ACh and vesamicol and transports ACh by using the labeling technique under conditions that detect different rates of labeling. The experiments will be extended to mutants of residues responsible for the required protonation and deprotonation to identify which hydrophilic regions change conformation at pH extremes. The results will provide substantial new information about VAChT structure and dynamics.