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，用于从胆碱能神经末梢诱发释放。它被一种叫做维索霉素的化合物变构抑制。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ACh储存（从而释放）的药理学工具，用于治疗胆碱能疾病。使用定点诱变，表达和突变特性的表征实验提出。VAChT中的残基必须质子化才能运输。第一个目的是测试是否最近的初步分配的天冬氨酸残基在推定的跨膜结构域Xi（TMD Xi）的这一作用是正确的。一个不同的天冬氨酸或谷氨酸残基，最近被证明是不是在TMD必须去质子化结合乙酰胆碱和vesamicol。第二个目的是鉴定它，第三个目的是测试和改进最近对ACh和vesamicol结合位点与推定的TMD XIII和X的分配。已知推定TMD X中的天冬氨酸残基参与向VAChT提供能量输入的质子易位反应。第四个目的是测试和完善拟议的作用（S），使用一种新的测定，监测跨膜重定向的乙酰胆碱结合位点的这种残基。第五个目的是测试VAChT是否包含12个TMD，如现在所假设的，通过将一个半胱氨酸残基插入序列的每个亲水区域，并使用化学标记技术确定残基暴露于膜的哪一侧。第六个目的是确定哪些亲水区域改变构象时，VAChT结合乙酰胆碱和vesamicol和运输乙酰胆碱通过使用标记技术的条件下，检测不同的标记率。实验将扩展到负责所需的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极端pH值下改变构象。研究结果将为VAChT的结构和动力学研究提供新的信息。

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.