Structural Basis of Vesicular Neurotransmitter Transport

经典递质对突触小泡的摄取依赖于H+电化学驱动力 (ΔµH+)，通常涉及胞质递质与管腔H+的交换。然而，水泡状 谷氨酸的运输几乎完全依赖于这种梯度的电子成分(Δψ)，而不是 PH梯度(ΔpH)，并经历了H+和Cl-的不寻常的变构调节。水泡 谷氨酸转运体(VGLUT)也表现出与氯离子电导相关的生理作用 这些属性中的哪些仍不清楚。此外，VGLUT属于溶质载体17(SLC17) 包括依赖ΔpH而不是Δψ进行相反运输的其他成员 从VGLUT方向行驶。这项建议的长期目标是了解 囊泡谷氨酸运输有助于突触传递。该战略使用结构来 确定所有家庭成员共有的机制，并了解它们的适应如何赋予 囊泡谷氨酸运输的特性。我们已经确定了SLC17家族成员的第一个结构，E.coliD-半乳糖酸转运体DgoT，它在序列上与VGLUT密切相关。DgoT在N-末端叶内含有一个极袋，通过一个假定的H+隧道连接到周质， 内向型结构。一个向外的结构包含被遮挡在 底物识别部位。这些结构预测了周质H+向谷氨酸的输送。 跨膜结构域(TM)4释放TM1中相互作用的精氨酸与底物结合。与之形成鲜明对比的是 VGLUT但像其他SLC17蛋白一样，DgoT催化H+共转运。尽管关键的 残基对VGLUT是保守的，因此它们在DgoT中起着不同的作用。我们现在就会 1)阐明DgoT中半乳糖酸偶联到H+的转运机制。 使用交换和结合以及净摄取的分析，我们将确定质子化如何 DgoT有助于底物识别。2)确定囊泡谷氨酸转运的结构基础。 我们将使用结晶学和冷冻电子显微镜相结合的方法来确定 一辆VGLUT。3)阐明VGLUT的变构调节机制。 我们将利用DgoT和哺乳动物的结构和可用的分析方法 了解H+和Cl-对VGLUT的变构调节作用。我们还将使用 电生理学，以评估结构所暗示的通道，并确定其与 谷氨酸流量。

The synaptic vesicle uptake of classical transmitters depends on a H+ electrochemical driving force (ΔµH+), and generally involves the exchange of cytosolic transmitter for lumenal H+. However, vesicular glutamate transport relies almost entirely on the electrical component of this gradient (Δψ) rather than the pH gradient (ΔpH), and undergoes unusual, allosteric regulation by H+ and Cl-. The vesicular glutamate transporters (VGLUTs) also exhibit an associated Cl- conductance, and the physiological role of these properties remains unknown. Further, the VGLUTs belong to the solute carrier 17 (SLC17) family which includes other members that rely on ΔpH rather than Δψ for transport in the opposite direction from VGLUTs. The long-term objective of this proposal is to understand how the properties of vesicular glutamate transport contribute to synaptic transmission. The strategy uses structure to identify the mechanisms common to all family members and understand how their adaptation confers the specific properties of vesicular glutamate transport. We have determined the first structures of an SLC17 family member, E. coli D-galactonate transporter DgoT, which is closely related in sequence to the VGLUTs. DgoT contains a polar pocket within the N-terminal lobe connected to the periplasm through a putative H+ tunnel evident in the inwardly oriented structure. An outwardly oriented structure contains galactonate occluded in the substrate recognition site. The structures predict that delivery of periplasmic H+ to a glutamate in transmembrane domain (TM) 4 liberates an interacting arginine in TM1 to bind substrate. In contrast to the VGLUTs but like other SLC17 proteins, DgoT catalyzes H+ cotransport. Although the critical residues are conserved to the VGLUTs, they thus serve a different function in DgoT. We will now 1) Elucidate the mechanism that couples transport of galactonate to H+ in DgoT. Using assays for exchange and binding as well as net uptake, we will determine how protonation of DgoT contributes to substrate recognition. 2) Determine the structural basis for vesicular glutamate transport. We will use a combination of crystallography and cryo-electron microscopy to determine the structure of a VGLUT. 3) Elucidate the mechanisms responsible for allosteric regulation of the VGLUTs. We will leverage the structures as well as the available assays for both DgoT and the mammalian proteins to understand the allosteric regulation of VGLUTs by H+ and Cl-. We will also use electrophysiology to assess a channel suggested by the structure, and determine its relationship to glutamate flux.