Transport of Neurotransmitter Into Synaptic Vesicles

突触传递依赖于经典神经递质从细胞质的转运，在细胞质中它们被合成并在从细胞外空间再摄取后积聚到突触囊泡中，然后突触囊泡经历受调节的胞吐作用。 为了了解进入突触囊泡的转运如何促进神经递质释放、突触后反应和最终行为，我们将重点放在负责神经末梢这一基本功能的蛋白质上。 在以前的工作中，我们已经确定了两个家庭的蛋白质负责运输阳离子和中性递质进入突触囊泡。 然而，负责囊泡谷氨酸转运的蛋白质已被证明是难以捉摸的。我们最近发现，以前建议的蛋白质介导的Na+依赖性的无机磷酸盐的跨质膜摄取，实际上运输谷氨酸进入突触囊泡。 除了H+驱动的谷氨酸转运，蛋白质（VGLUT1）表现出不寻常的氯离子电导抑制谷氨酸。 为了评估其通道样特性及其运输功能，我们将表征VGLUT 1与氯离子和质子的相互作用。 我们还将评估VGLUT 1在质膜上转运谷氨酸（和磷酸盐）的潜力。由于VGLUT1的调节可能会影响突触囊泡填充和VGLUT1包含两个多聚脯氨酸结构域，我们将进一步表征其与其他蛋白质的相互作用。 VGLUT1的表达，只有一个子集的谷氨酸神经元也表明存在另一种亚型，最近的工作描述了一个密切相关的假定磷酸盐转运蛋白。 因此，我们将检查这种蛋白质的分布并研究其转运功能。 为了评估VGLUT1在体内的作用，我们将在小鼠中破坏该基因并检查其生理效应。 这些实验将有助于了解谷氨酸在神经末梢的转运及其在递质释放、突触可塑性、兴奋毒性和行为中的作用。

Synaptic transmission depends on the transport of classical neurotransmitters from the cytoplasm, where they are synthesized and accumulate after reuptake from the extracellular space, into synaptic vesicles, which then undergo regulated exocytosis. To understand how transport into synaptic vesicles contributes to neurotransmitter release, the postsynaptic response and ultimately behavior, we have focused on the proteins responsible for this basic function of the nerve terminal. In previous work, we have identified two families of proteins responsible for the transport of cationic and neutral transmitters into synaptic vesicles. However, the proteins responsible for vesicular glutamate transport have proven elusive. We have recently found that a protein previously suggested to mediate the Na+-dependent uptake of inorganic phosphate across the plasma membrane, in fact transports glutamate into synaptic vesicles. In addition to H+-driven glutamate transport, the protein (VGLUT1) exhibits an unusual chloride conductance inhibited by glutamate. To assess its channel-like properties and its transport function, we will characterize the interactions of VGLUT1 with chloride and protons. We will also assess the potential for VGLUT1 to transport glutamate (and phosphate) at the plasma membrane. Since the regulation of VGLUT1 may influence synaptic vesicle filling and VGLUT1 contains two polyproline domains, we will further characterize its interaction with other proteins. The expression of VGLUT1 by only a subset of glutamate neurons also suggests the existence of another isoform, and recent work describes a closely related putative phosphate transporter. We will thus examine the distribution of this protein and study its transport function. To assess the role of VGLUT1 in vivo, we will disrupt the gene in mice and examine the physiological effects. These experiments will help to understand the transport of glutamate at the nerve terminal and its role in transmitter release, synaptic plasticity, excitotoxicity and behavior.