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进入突触的运输 囊泡 (SV)，一种由 H+ 电化学梯度 (ΔμH+) 驱动的活动。与相对稳定的离子相比 质膜上的梯度、ΔμH+ 和其他离子（包括 Cl-）随着外吞作用和内吞作用而波动 SV。囊泡填充需要与这些变化的条件相协调，从而调节运输。 与大多数变送器的 SV 吸收主要依赖于 ΔμH+ 的化学成分相反 (ΔpH)，主要兴奋性递质谷氨酸的摄取主要取决于膜电位。 囊泡谷氨酸转运蛋白（VGLUT）也表现出不寻常的特性，包括变构调节 腔内 H+、细胞质和腔内 Cl- 以及相关的 Cl- 电导。我们假设这些 机制协调谷氨酸通量与突触外吞和内吞循环中的不同步骤 囊泡。该提案的长期目标是了解 VGLUT 的这些特性如何发挥作用 到兴奋性神经传递。该策略是确定这些机制如何调节 VGLUT 活动， 并利用这些信息来表征它们的生理作用。这个程序利用了我们之前的 确定这些调节机制的工作、我们开发的研究方法、最新的结构信息 和 VGLUT 敲除神经元，我们可以用它们来测试突变体的救援。 目标 1：阐明 pH 在囊泡谷氨酸转运中的机制和生理作用。这 腔内 H+ 对 VGLUT 变构激活的要求表明了一种防止强直外流的机制 谷氨酸盐穿过质膜，会降低量子信号。我们最近确定了一个 赋予囊泡谷氨酸运输的 pH 要求的残留物。我们现在将使用此信息来 确定 pH 如何调节谷氨酸转运以及这种调节如何影响兴奋性传递。 目标 2：确定 Cl- 如何变构调节囊泡谷氨酸转运。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腔内​​ pH 值的变构调节 SV 膜，表明参与谷氨酸转运的交替通路取决于 细胞质门和腔门之间的强度平衡。我们假设 Cl- 也会影响两个门， 直接或间接。因此，我们将确定细胞质相互作用网络和腔残基如何 有助于细胞质和腔内 Cl- 谷氨酸通量的变构调节。 目标 3： 目标 3：确定腔内 Cl- 如何影响谷氨酸储存和释放。去除细胞外质 Cl- 可以防止强刺激后突触抑制的恢复。确定 这是否反映了由 VGLUT 相关电导介导的管腔 Cl- 流出的要求， 我们将用缺乏电导的突变体来拯救 VGLUT1/2 双敲除，并监测对 谷氨酸释放和 SV pH。