Glutamate Transport into Synaptic Vesicles

谷氨酸转运至突触小泡

• 批准号: 10568125
• 负责人: ROBERT H EDWARDS
• 金额: $ 49.3万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-15 至 2027-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10568125
• 关键词: Affect; Allosteric Regulation; Biochemical; Biological Assay; Cell membrane; Chemicals; Cytoplasm; Dependence; Endocytosis; Equilibrium; Event; Excision; Exhibits; Face; Family member; Glutamate Transporter; Glutamates; Ions; Knock-out; Location; Mediating; Membrane; Membrane Potentials; Molecular; Molecular Conformation; Monitor; Nature; Neurons; Neurotransmitters; Oocytes; Physiological; Physiology; Property; Recovery; Recycling; Regulation; Role; Side; Signal Transduction; Synapses; Synaptic Transmission; Synaptic Vesicles; System; Testing; Transmembrane Transport; Vesicle; Work; extracellular; insight; mutant; neural; neurotransmission; neurotransmitter transport; pH gradient; prevent; programs; synaptic depression; transmission process; uptake; vesicle transport

The quantal nature of synaptic transmission depends on the transport of neurotransmitter into synaptic vesicles (SVs), an activity driven by a H+ electrochemical gradient (∆µH+). In contrast to relatively stable ionic gradients across the plasma membrane, ∆µH+ and other ions including Cl- fluctuate with the exo- and endocytosis of SVs. Vesicle filling requires coordination with these changing conditions and hence regulation of transport. In contrast to the SV uptake of most transmitters that relies primarily on the chemical component of ∆µH+ (∆pH), uptake of the principal excitatory transmitter glutamate depends predominantly on membrane potential. The vesicular glutamate transporters (VGLUTs) also exhibit unusual properties, including allosteric regulation by lumenal H+, cytosolic and lumenal Cl- and an associated Cl- conductance. We hypothesize that these mechanisms coordinate glutamate flux with different steps in the exo- and endocytic recycling of synaptic vesicles. The long-term objective of this proposal is to understand how these properties of the VGLUTs contribute to excitatory neurotransmission. The strategy is to determine how these mechanisms regulate VGLUT activity, and use this information to characterize their physiological role. This program takes advantage of our previous work identifying these regulatory mechanisms, assays we developed to study them, recent structural information and VGLUT knockout neurons that we can use to test rescue by mutants. Aim 1: Elucidate the mechanism and physiological role of pH in vesicular glutamate transport. The requirement for allosteric activation of the VGLUTs by lumenal H+ suggests a mechanism to prevent tonic efflux of glutamate across the plasma membrane that would degrade the quantal signal. We recently identified a single residue that confers the pH requirement of vesicular glutamate transport. We will now use this information to determine how pH regulates glutamate transport and how this regulation influences excitatory transmission. Aim 2: Determine how Cl- allosterically regulates vesicular glutamate transport. We recently found that an extensive cytoplasmic interaction network influences the allosteric regulation by lumenal pH on the other side of the SV membrane, suggesting that the alternating access involved in glutamate transport depends on the balance in strength between cytoplasmic and lumenal gates. We hypothesize that Cl- also affects the two gates, either directly or indirectly. We will thus determine how the cytoplasmic interaction network and lumenal residues contribute to allosteric regulation of glutamate flux by cytoplasmic and lumenal Cl-. Aim 3: Aim 3: Determine how lumenal Cl- affects glutamate storage and release. Removal of extracellular Cl- prevents recovery from the synaptic depression that normally follows strong stimulation. To determine whether this reflects a requirement for the efflux of lumenal Cl- mediated by a VGLUT-associated conductance, we will rescue VGLUT1/2 double knockouts with mutants lacking the conductance, and monitor the effects on glutamate release and SV pH.

突触传递的量子性质取决于神经递质向突触的运输。 囊泡(Sv)，由H+电化学梯度(∆µH+)驱动的活动。与相对稳定的离子相反 质膜、∆µH+和包括Cl-在内的其他离子的梯度随外吞和内吞而波动 SVS.囊泡充盈需要与这些不断变化的条件相协调，从而调节运输。 与大多数发射机的SV摄取主要依赖于∆？H+的化学成分相反 (∆pH)，主要兴奋性递质谷氨酸的摄取主要取决于膜电位。 囊泡型谷氨酸转运体(VGLUT)也表现出不寻常的性质，包括通过 腔内H+、胞液和腔内氯-以及相关的氯电导。我们假设这些 突触内外循环中不同步骤调节谷氨酸流量的机制 水泡。这项建议的长期目标是了解VGLUT的这些属性如何贡献 到兴奋性神经传递。策略是确定这些机制如何调节VGLUT活动， 并利用这些信息来描述它们的生理作用。这个程序利用了我们以前的 识别这些调控机制的工作，我们为研究它们而开发的分析，最新的结构信息 和VGLUT基因敲除神经元，我们可以用来测试突变体的拯救。 目的1：阐明pH在囊泡谷氨酸转运中的机制和生理作用。这个 腔H+对VGLUT变构激活的要求提示了一种防止紧张性外流的机制 穿过质膜的谷氨酸会降低量子信号。我们最近发现了一支 给予囊泡谷氨酸运输pH要求的残留物。我们现在将使用此信息来 确定pH如何调节谷氨酸运输，以及这种调节如何影响兴奋性传递。 目的2：确定氯离子如何变构调节囊泡谷氨酸运输。我们最近发现，一个 广泛的细胞质相互作用网络影响另一侧管腔pH对变构的调节 SV膜，提示谷氨酸转运所涉及的交替通路依赖于 胞质门和管腔门之间的强度平衡。我们假设Cl-也影响这两个门， 无论是直接还是间接。因此，我们将确定细胞质相互作用网络和管腔残留物 胞质和管腔氯离子参与谷氨酸流量的变构调节。 目的3：目的3：确定氯离子对谷氨酸储存和释放的影响。清除细胞外 CL-阻止正常情况下强刺激后的突触抑制的恢复。要确定 这是否反映了对由VGLUT相关电导介导的管腔氯离子外流的要求， 我们将挽救VGLUT1/2双敲除缺失电导的突变体，并监测其对 谷氨酸释放和SV pH。