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.

突触传播的量化性质取决于神经递质转运到突触 Vesict（SVS），与相对稳定的离子相比，由H+电化学梯度驱动的活动 整个质膜，ΔH+和其他离子离子离子在内 囊泡填充需要与变化的条件和运输调节的协调。 与大多数发射器的SV吸收相反，该发射器依赖于∆H+的化学成分 （∆PH），主要兴奋性发射机谷氨酸的吸收主要取决于膜电位。 囊泡谷氨酸透明膜（VGLUTS）也表现出异常特性，包括通过 Lumenal H+，胞质和Lumenal CL及相关CL导率。 机理协调谷氨酸通量与突触的外移和内环中的不同步骤 vesict。 兴奋性神经传递。 并使用此帐户来表征他们的生理角色。 识别规则性机制，我们开发的测定方法的工作，最新的结构信息 和VGLUT敲除神经元，我们可以使用突变体进行测试休息。 AIM 1：阐明囊泡谷氨酸转运的机制和生理角色 腔内H+对VGLUT的变构激活的要求表明，机械师Tonic Effflux 跨质膜的谷氨酸将降解量子信号。 赋予水泡谷氨酸运输的pH值的残留物。 确定pH定期如何定期谷氨酸转运以及如何进行调节会影响精力传播。 AIM 2：确定Cl-Asterpory如何调节囊泡谷氨酸的运输。 广泛的细胞质相互作用网络影响了腔pH的变构调节的另一侧。 SV膜，表明在谷氨酸运输部门涉及的交替访问 细胞质和腔内门之间的强度平衡。 因此，我们将直接或间接地确定细胞质相互作用网络 有助于细胞质和腔内Cl-对谷氨酸通量的变构调节。 AIM 3：AIM 3：确定Lumenal Cl-对谷氨酸的储存和释放 Cl-Prevents从通常遵循强劲强劲强的突触抑郁症中恢复。 这是否反映了由VGLUT相关电导率介导的流体CL介导的外排的要求， 我们将在缺乏电导的情况下挽救VGLUT1/2双敲击，并监视对 谷氨酸释放和SV pH。