Structural Basis of Vesicular Neurotransmitter Transport

囊泡神经递质运输的结构基础

• 批准号: 10392888
• 负责人: ROBERT H EDWARDS
• 金额: $ 64.24万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10392888
• 关键词: Allosteric Regulation; Allosteric Site; Anions; Arginine; Binding; Biological Assay; Cell surface; Couples; Cryoelectron Microscopy; Crystallography; Cytoplasm; Disease; Drops; Electrophysiology (science); Electrostatics; Elements; Escherichia coli; Exhibits; Exocytosis; Family; Family member; Glutamate Transporter; Glutamates; Lobe; Lysosomes; Membrane Potentials; N-terminal; Neurotransmitters; Physiological; Physiology; Process; Property; Protein Analysis; Proteins; Recycling; Rest; Role; Sialic Acids; Site; Structure; Synapses; Synaptic Transmission; Synaptic Vesicles; Transmembrane Domain; Vesicle; driving force; extracellular; glutamatergic signaling; member; neurotransmission; neurotransmitter release; neurotransmitter transport; pH gradient; periplasm; programs; protein structure function; protonation; response; solute; stoichiometry; tool; uptake; vacuolar H+-ATPase

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.

突触囊泡对经典递质的摄取依赖于H+的电化学驱动力 （ΔµH+），并且通常涉及胞质递质与内腔H+的交换。然而，泡状的 谷氨酸转运几乎完全依赖于该梯度的电分量（Δ ε），而不是 pH梯度（ΔpH），并通过H+和Cl-进行不寻常的变构调节。囊泡 谷氨酸转运体（VGLUT）也表现出相关的Cl-电导， 这些属性仍然未知。此外，VGLUT属于溶质载体17（SLC 17）。 该家族包括依赖于ΔpH而不是Δ pH进行相反运输的其他成员 从VGLUT的方向。本提案的长期目标是了解 囊泡谷氨酸转运有助于突触传递。该战略使用结构， 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 囊泡谷氨酸转运的特性。我们已经确定了一个SLC 17家族成员E. coliD-半乳糖酸转运体DgoT，其在序列上与VGLUT密切相关。DgoT在N-末端叶内含有一个极性口袋，通过一个推定的H+通道连接到周质，这在DgoT中是明显的。 内向型结构。向外定向的结构包含封闭在半乳糖酸盐中的半乳糖酸盐， 底物识别位点。该结构预测，在细胞中，细胞周质H+向谷氨酸的传递， 跨膜结构域（TM）4释放TM 1中的相互作用精氨酸以结合底物。相比 与VGLUT相似，但与其他SLC 17蛋白一样，DgoT催化H+共转运。虽然关键的 由于这些残基对于VGLUT是保守的，因此它们在DgoT中发挥不同的功能。我们现在将 1)阐明DgoT中半乳糖酸与H+的偶联转运机制。 使用交换和结合以及净吸收的测定，我们将确定质子化的 DgoT有助于底物识别。2)确定囊泡谷氨酸转运的结构基础。 我们将结合晶体学和低温电子显微镜来确定 VGLUT。3)阐明负责VGLUT变构调节的机制。 我们将利用DgoT和哺乳动物细胞的结构以及可用的测定方法。 蛋白质，以了解VGLUT的变构调节H+和Cl-。我们还将使用 电生理学来评估由结构暗示的通道，并确定其与 谷氨酸通量