Structural Basis of Vesicular Neurotransmitter Transport

 描述(申请人提供)：所有经典递质进入突触小泡的运输依赖于液泡H+-ATPase产生的向外定向的H+电化学驱动力(µH+)。然而，囊泡谷氨酸的运输不同于其他经典递质的囊泡运输，它几乎完全依赖于这种梯度的电子成分()，而不是化学梯度(pH)。事实上，囊泡型谷氨酸转运体(VGLUT)是否介导了H+交换仍不清楚。它们可能只是催化促进扩散，甚至起到阴离子通道的作用。相反，密切相关的转运体唾液酸催化H+与唾液酸的电中和共转运，目前尚不清楚SLC17家族的两个成员如何介导如此明显的不同活性。然而，唾液酸也被报道介导囊泡谷氨酸的运输，这表明这两种不同的活性反映了共同的潜在机制。该项目的长期目标是了解SLC17家族如何实现驱动的扩散和H+共转运。其策略是确定该家族中蛋白质的结构，并使用这些信息来指导机制的研究。筛选了一些与VGLUT相关的细菌蛋白质，我们已经鉴定出一种可以在许多不同条件下结晶的蛋白质，它在脂类立方相中衍射率为3.7？我们还将重组蛋白重组到人工膜中，并表明它催化了有机阴离子与H+的共转运，类似于唾液酸。我们现在建议1)在原子分辨率下提纯DgoT的结构；2)确定DgoT在不同功能状态下的结构，包括底物结合；3)测试结构所涉及的特定残基在底物识别和H+运动中的作用；以及4)确定后生动物VGLUT的结构。这些结果将有助于我们理解一类运输蛋白，甚至一种蛋白是如何以明显不同的方式与H+电化学驱动力耦合的。同时，结构分析应该阐明氯和我们最近发现的H+对VGLUT变构调节的机制，这一机制仍然知之甚少。具有改变性质的突变体的鉴定也为我们提供了通过体外和体内的遗传操作来测试这些性质的生理作用的工具。

 DESCRIPTION (provided by applicant): The transport of all classical transmitters into synaptic vesicles depends on an outwardly directed H+ electrochemical driving force (µH+) produced by the vacuolar H+-ATPase. However, vesicular glutamate transport differs from the vesicular transport of other classical transmitters, and relies almost entirely on the electrical component o this gradient () rather than the chemical gradient (pH). Indeed, it remains unclear whethe the vesicular glutamate transporters (VGLUTs) mediate H+ exchange at all. They may simply catalyze facilitated diffusion, or even function as anion channels. In contrast, the closely relate transporter sialin catalyzes the electroneutral cotransport of H+ with sialic acid, and it remains unknown how two members of the SLC17 family can mediate such apparently different activities. However, sialin has also been reported to mediate vesicular glutamate transport, suggesting that the two different activities reflect a common underlying mechanism. The long-term objective of this program is to understand how the SLC17 family confers both -driven diffusion and H+ cotransport. The strategy is to determine the structure of proteins in this family and use this information to guide studies of mechanism. Screening a number of bacterial proteins related to the VGLUTs, we have identified one that can be crystallized under a number of different conditions, and that diffracts to 3.7 Å in the lipidic cubic phase. We have also reconstituted the recombinant protein into artificial membranes and shown that it catalyzes the cotransport of an organic anion with H+, similar to sialin. We now propose to 1) refine the structure of DgoT at atomic resolution; 2) determine the structure of DgoT in different functional states, including substrate-bound; 3) test the role of specific residues implicated by the structur in substrate recognition and H+ movement; and 4) determine the structure of a metazoan VGLUT. The results will help us to understand how one class of transport proteins and perhaps even one protein can couple in apparently different ways to the H+ electrochemical driving force. At the same time, structural analysis should illuminate the mechanism for allosteric regulation of the VGLUTs by chloride, which remains poorly understood, and by H+, which we have recently discovered. The identification of mutants with altered properties also provides us with tools to test the physiological role of these properties by genetic manipulation in vitro and n vivo.