Structural Basis of Vesicular Neurotransmitter Transport

囊泡神经递质运输的结构基础

• 批准号: 9920217
• 负责人: ROBERT H EDWARDS
• 金额: $ 65.21万
• 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/9920217
• 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.

经典发射器的突触囊泡吸收取决于H+电化学驱动力 （ΔμH+），通常涉及胞质发射器的腔内H+ 谷氨酸转运几乎完全依赖于该梯度的电气成分（Δψ），而不是 pH梯度（ΔPH），通过H+和Cl-进行异常的变构调节 谷氨酸转运蛋白（VGLUTS）也表现出相关的CL导率和硫代型的作用 TESE的性质仍然未知。 包括其他依赖其他成员而不是δ进行运输的家庭 vgluts的方向。 囊泡谷氨酸的运输有助于突触传播。 确定所有家庭成员共有的机制，并了解其适应如何赋予 囊泡谷氨酸转运的特定性质。这 向内定向的结构。 底物识别位置。 跨膜结构域（TM）4在TM1中释放了与底物相互作用的相互作用 vgluts，但与其他SLC17蛋白一样，DGOT催化H+ Cotransport。 残基是vgluts的，因此它们在DGOT中具有不同的功能 1）阐明了将半乳酸酯耦合到DGOT中H+的机制。 使用分析进行交换和绑定以及净吸收，我们将确定质子化的质子化 DGOT有助于底物识别。 我们将使用晶体学和冷冻电子显微镜的组合来确定 vglut。 我们将利用结构作为DGOT和哺乳动物的可用测定 通过H+和Cl-了解VGLUT的变构调节的蛋白质。 电生理学评估结构所建议的通道，并确定均值。 谷氨酸通量。