Trafficking of a Neuronal Glutamate Transporter, EAAC1

描述（由申请人提供）：谷氨酸是哺乳动物CNS中主要的快速兴奋性神经递质，并参与发育和成年神经系统中几种形式的可塑性。谷氨酸受体的过度激活有助于在对CNS的急性损伤中观察到的脑损伤，包括缺血和头部创伤。谷氨酸的细胞外浓度由钠依赖性转运蛋白家族控制。我们和其他人已经发现，这些转运蛋白之一，称为EAAC 1的活性，可以通过激活蛋白激酶C（PKC）或血小板衍生生长因子受体迅速增加。这些影响与EAAC 1从亚细胞区室到细胞表面的重新分布有关，并且独立于新转运蛋白的合成。基于我们的初步研究，我们想追求以下四个具体目标：1）我们建议检查所需的信号分子的PDGF依赖的EAAC 1的再分布。2)我们最近发现，EAAC 1组成型之间的质膜和细胞内的隔间，半衰期约为5-7分钟。我们建议，以确定EAAC 1是否被内化的小窝蛋白依赖的内吞途径和开始，以确定用于回收EAAC 1的亚细胞隔间。3)我们已经发现EAAC 1与蛋白质C激酶（PICK 1）和PKC的α亚型相互作用形成复合物。我们建议确定这些相互作用所需的域，并确定这些相互作用如何影响EAAC 1贩运。4)调节EAAC 1运输和谷氨酸受体之一的机制似乎非常相似，增加EAAC 1的信号会降低GluR 2。我们建议使用各种方法来确定EAAC 1和离子型谷氨酸受体是否以这种明显互补的方式进行调节，使用其他方法来化学诱导长时程抑制（LTD）或长时程增强（LTP）。我们对这种调节很感兴趣，因为EAAC 1在突触传递高度可塑的区域和对兴奋性毒性损伤非常敏感的神经元中富集。EAAC 1位于突触后膜上，并且在突触周围围绕GluRs，在那里它理想地位于微调突触传递。我们怀疑，了解EAAC 1的调节可能对了解谷氨酸毒性和/或突触可塑性有影响。

DESCRIPTION (provided by applicant): Glutamate is the predominant rapid excitatory neurotransmitter in the mammalian CNS and is involved in several forms of plasticity in the developing and adult nervous system. Excessive activation of glutamate receptors contributes to brain damage observed in acute insults to the CNS, including ischemia and head trauma. The extracellular concentrations of glutamate are controlled by a family of sodium-dependent transporters. We and others have found that the activity of one of these transporters, called EAAC1, can be rapidly increased by either activation of protein kinase C (PKC) or the platelet-derived growth factor receptor. These affects are associated with a redistribution of EAAC1 from a subcellular compartment to the cell surface and are independent of synthesis of new transporters. Based on our preliminary studies, we would like to pursue the following four specific aims: 1) We propose to examine the signaling molecules that are required for the PDGF-dependent redistribution of EAAC1. 2) We have recently found that EAAC1 constitutively recycles between the plasma membrane and intracellular compartments with a half-life of approximately 5-7 min. We propose to determine if EAAC1 is internalized by a caveolin-dependent endocytosis pathway and begin to identify the subcellular compartments used for recycling of EAAC1. 3) We have found that EAAC1 forms complexes with protein interacting with C kinase (PICK1) and the alpha subtype of PKC. We propose to identify domains required for these interactions and to determine how these interactions affect EAAC1 trafficking. 4) The mechanisms that regulate EAAC1 trafficking and one of the glutamate receptors appear to be quite similar, and a signal that increases EAAC1 decreases GluR2. We propose to use a variety of approaches to determine if EAAC1 and ionotropic glutamate receptors are regulated in this apparently complementary fashion using other approaches to chemically induce either long term depression (LTD) or long term potentiation (LTP). We are intrigued by this regulation because EAAC1 is enriched in areas where synaptic transmission is highly plastic and in neurons that are exquisitely sensitive to excitotoxic insults. EAAC1 is localized on the post-synaptic membrane and is perisynaptic surrounding GluRs where it is ideally situated to fine-tune synaptic transmission. We suspect that understanding the regulation of EAAC1 could have implications for understanding glutamate toxicity and/or synaptic plasticity.