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+电化学驱动力