Structural Basis of Vesicular Neurotransmitter Transport

 描述（由申请人提供）：所有经典递质转运至突触囊泡取决于由液泡H+-ATP酶产生的外向H+电化学驱动力（H + µH+）。然而，囊泡谷氨酸转运不同于其他经典递质的囊泡转运，并且几乎完全依赖于该梯度的电组分（pH）而不是化学梯度（pH）。事实上，囊泡谷氨酸转运蛋白（VGLUT）是否介导H+交换仍不清楚。它们可以简单地催化易化扩散，甚至起到阴离子通道的作用。与此相反，密切相关的转运蛋白唾液酸催化的电中性共转运的H+与唾液酸，它仍然是未知的如何两个成员的SLC 17家族可以介导这种明显不同的活动。然而，唾液酸也被报道介导囊泡谷氨酸转运，这表明这两种不同的活动反映了一个共同的潜在机制。该计划的长期目标是了解SLC 17家族如何赋予质子驱动的扩散和H+共转运。该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，并且其衍射至3.7 nm的立方相。我们还重组成人工膜的重组蛋白，并表明，它催化的协同运输的有机阴离子与H+，类似唾液酸。我们现在建议：1）在原子分辨率下细化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）测试底物识别和H+运动中结构所涉及的特定残基的作用; 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转运蛋白，甚至可能是一种蛋白质如何以明显不同的方式与H+电化学驱动力偶联。与此同时，结构分析应阐明氯化物和我们最近发现的H+对VGLUT的变构调节机制，氯化物和H+对VGLUT的变构调节机制仍知之甚少。突变体的鉴定与改变的属性也为我们提供了工具，以测试这些属性的生理作用，在体外和体内的遗传操作。

 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.