Transport of Neurotransmitter Into Synaptic Vesicles

突触传递依赖于经典神经递质从细胞质中的运输，它们在那里被合成，并在从细胞外空间重新摄取后积累到突触小泡，然后突触小泡经历调节的胞吐作用。为了了解运输到突触小泡如何有助于神经递质的释放、突触后反应和最终的行为，我们重点研究了负责神经末梢这一基本功能的蛋白质。在以前的工作中，我们已经确定了两个蛋白质家族，负责将阳离子和中性递质运输到突触小泡中。然而，负责囊泡谷氨酸运输的蛋白质已被证明是难以捉摸的。我们最近发现，一种先前被认为通过质膜介导Na+依赖的无机磷吸收的蛋白质，实际上将谷氨酸运输到突触小泡中。除了H+驱动的谷氨酸转运外，该蛋白(VGLUT1)还表现出一种不寻常的氯离子电导，被谷氨酸抑制。为了评估它的通道性质和它的运输功能，我们将表征VGLUT1与氯离子和质子的相互作用。我们还将评估VGLUT1在质膜上运输谷氨酸(和磷酸盐)的潜力。由于VGLUT1的调控可能会影响突触囊泡的充盈，而且VGLUT1含有两个多脯氨酸结构域，我们将进一步研究它与其他蛋白质的相互作用。仅有一部分谷氨酸神经元表达VGLUT1也表明存在另一种异构体，最近的工作描述了一种密切相关的假定的磷酸转运蛋白。因此，我们将检查这种蛋白质的分布，并研究其运输功能。为了评估VGLUT1在体内的作用，我们将在小鼠中干扰该基因并检测其生理效应。这些实验将有助于了解谷氨酸在神经末梢的运输及其在递质释放、突触可塑性、兴奋性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.