Structure in vesicular acetylcholine transporter

描述（由申请人提供）：囊泡乙酰胆碱转运蛋白（VAChT）将乙酰胆碱（ACh）储存在突触囊泡中，以诱导胆碱能神经末梢释放。它被一种叫做维萨米醇的化合物变构抑制。该项目旨在了解VAChT的结构和运输机制，长期目标是开发一种控制乙酰胆碱能储存（从而释放）的药理学工具，以治疗胆碱能疾病。提出了利用位点定向诱变、表达和表征突变体特性的实验。VAChT中的残基必须被质子化才能进行运输。第一个目的是测试最近在假定的跨膜结构域XI （TMD XI）中对天冬氨酸残基的初步分配是否正确。一种不同的天冬氨酸或谷氨酸残基最近被证明不在TMD中，必须去质子化以结合乙酰胆碱和维萨霉素。第二个目标是识别它。第三个目的是测试和完善乙酰胆碱和维氨醇结合位点最近在推测的TMD XIII和X上的定位。已知推测的TMD X中的天冬氨酸残基参与质子易位反应，为VAChT提供能量输入。第四个目标是使用一种监测乙酰胆碱结合位点的跨膜重定向的新方法来测试和完善该残基的拟议作用。第五个目标是测试VAChT是否像现在假设的那样含有12个tmd，方法是在序列的每个亲水性区域插入一个半胱氨酸残基，并使用化学标记技术确定残基暴露在膜的哪一侧。第六个目的是在检测不同标记率的条件下，通过标记技术，确定当VAChT结合ACh和vesamicol并运输ACh时，哪些亲水区域改变了构象。实验将扩展到负责所需质子化和去质子化的残基突变体，以确定哪些亲水性区域在极端pH值下改变构象。研究结果将提供有关VAChT结构和动力学的大量新信息。

DESCRIPTION (provided by applicant): Vesicular acetylcholine transporter (VAChT) stores acetylcholine (ACh) in synaptic vesicles for evoked release 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 pharmacological tool with which 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 preliminary assignment of the aspartate residue in putative transmembrane domain XI (TMD XI) to this role is 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.