Structure in vesicular acetylcholine transporter

描述(申请人提供)：囊泡性乙酰胆碱转运体(VACHT)将乙酰胆碱(ACh)储存在突触小泡中，以从胆碱能神经末梢诱发释放。它被一种叫做维沙米考的化合物变构抑制。该项目旨在了解Vacht的结构和转运机制，长期目标是开发一种药理工具，用来控制ACh的储存(从而释放)，用于治疗胆碱能障碍。提出了使用定点突变、表达和表征突变特性的实验。Vacht中的残留物必须质子化才能运输。第一个目的是测试最近对假定跨膜结构域XI(TMD XI)中天冬氨酸残基的初步指定是否正确。另一种天冬氨酸或谷氨酸残基最近被证明不在TMD中，必须去质子化才能结合ACh和维沙米考。第二个目标是识别它。第三个目的是测试和完善最近对推测的TMD XIII和X的ACh和维沙米考结合位点的指定。已知TMD X中的天冬氨酸残基参与了为Vacht提供能量输入的质子转位反应。第四个目标是使用一种监测ACh结合位点跨膜重定向的新方法来测试和提炼这种残基的拟议作用(S)。第五个目的是通过在序列的每个亲水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，来测试Vacht是否像现在假设的那样包含12个TMD。第六个目的是利用标记技术，在检测不同标记率的条件下，确定Vacht结合ACh和维沙米考并运输ACh时，哪些亲水区改变了构象。实验将扩展到负责所需质子化和去质子化的残基的突变体，以确定哪些亲水性区域在pH极端时改变构象。研究结果将为游艇的结构和动力学提供大量新的信息。

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.