Structure in vesicular acetylcholine transporter

囊泡性乙酰胆碱转运体(Vacht)在突触小泡中储存乙酰胆碱(ACh)以供诱发 从胆碱能神经末梢释放。它被一种叫做维沙米考的化合物变构抑制。这个 该项目寻求对游艇结构和运输机制的了解，长期目标是 开发一种药理学来控制ACh的储存(从而释放)以治疗胆碱能障碍。 利用定点突变、表达和表征突变特性的实验 建议。Vacht中的残留物必须质子化才能运输。第一个目标是测试最近的 推测跨膜结构域XI(TMD XI)中天冬氨酸残基对这一作用的初步指定 S说得对。最近被证明不在TMD中的另一种天冬氨酸或谷氨酸残基必须是 去质子化以结合ACh和维沙米考。第二个目标是识别它。第三个目标是测试和改进 推测的TMDSXIII和X.天冬氨酸的ACh和维沙米考结合位点的最新指认 已知TMD X中的残基参与了提供能量输入的质子转移反应 去瓦希特。第四个目标是使用一种新的分析方法来测试和提炼这种残留物的拟议作用(S) 监测ACh结合部位的跨膜重定向。第五个目标是测试Vacht是否 包含12个TMD，如现在假设的那样，通过在每个亲水区域插入一个半胱氨酸残基 测序并使用化学标记来确定残基暴露在膜的哪一侧 技术。第六个目标是确定哪些亲水性区域在Vacht结合时改变了构象 利用标记技术在检测到不同的条件下传输ACh 贴标率。实验将扩大到对所需残基负责的突变体 质子化和去质子化，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。

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.