Synaptic Glutamate Receptor Trafficking.

描述（由申请人提供）：中枢神经系统中的兴奋性突触释放谷氨酸，其作用于两种类型的离子性受体：AMPA受体（AMPARs）和NMDA受体（NMDARs）。有证据表明，与NMDARs相比，ampar具有高度的移动性，其活性可以迅速改变突触上受体的数量。为了开始了解突触AMPARs调控的分子基础，我们重点研究了共济失调和癫痫小鼠占星者。该小鼠的小脑颗粒细胞缺乏功能性ampar，尽管nmdar是正常的，兴奋性突触释放正常量的谷氨酸。在目前的Conte资助期间，我们对stargazin （v-2）的作用进行了一系列研究，这是一种突变基因，在stargazer小鼠中。我们发现stargazin是AMPARs的一个辅助亚基，不仅控制它们向细胞表面和突触的运输，而且控制它们的生物物理性质。我们已经确定了另外三种蛋白（v-3, y-4, v-8），它们在整个中枢神经系统中表达，可以挽救小脑颗粒细胞中的AMPAR缺陷。我们将这些蛋白称为跨膜AMPAR调节蛋白（TARPs）。我们已经成功地删除了老鼠体内每个tarp的基因。这些突变小鼠将成为本RO1拨款中许多拟议实验的基础，这是Conte拨款所开展工作的延续。有4个具体目标。(1)确定v-3在小鼠脑中的作用，(2)确定y-4在小鼠脑中的作用，(3)确定TARPs之间的功能差异，(4)确定TAPRs在中枢神经系统中是否具有与ampar无关的作用。TARPs在AMPAR运输、突触传递和可塑性中的作用将主要在海马中进行研究。初步研究表明，y-8在海马AMPAR转运中起重要作用，但AMPAR的大量传递仍然存在。我们还将比较不同的TARPs修改ampar失活和突触传递动力学的能力。我们有证据表明，每个tarp所起的作用（即表面传递、突触靶向和受体门控）都是不同的。我们发现Y-2/Y-3A/-4，三重KO是致命的，新生幼崽完全不能动。我们将确定脊髓丧失功能的原因。这些研究可能揭示了TARPs在神经系统中的新作用。鉴于受体转运在突触可塑性中发挥的关键作用，以及在学习和记忆的某些方面的暗示，预计这些研究的结果将具有直接的临床影响。事实上，临床上有希望的AMPAkines发挥其作用，部分是通过控制AMPAR门控动力学，类似于TARPs。此外，TARPs修饰AMPAkines的药理学性质，因此代表了药物设计的新靶点。

DESCRIPTION (provided by applicant): Excitatory synapses in the CNS release glutamate, which acts on two types of ionotropic receptors: AMPA receptors (AMPARs) and NMDA receptors (NMDARs). Evidence indicates that AMPARs, in contrast to NMDARs, are highly mobile and that activity can rapidly change the number of receptors at the synapse. To begin to understand the molecular basis underlying the regulation of synaptic AMPARs, we have focused on the ataxic and epileptic mouse stargazer. Cerebellar granule cells in this mouse lack functional AMPARs, although NMDARs are normal and excitatory synapses release normal amounts of glutamate. During the current Conte grant we have carried out a series of studies on the role of stargazin (v-2), the mutated gene in the stargazer mouse. We have found that stargazin is an auxiliary subunit of AMPARs, not only controlling their trafficking to the cell surface and to the synapse, but also controlling their biophysical properties. We have identified a total of three additional proteins (v-3, y-4, v-8) that are expressed throughout the CNS and can rescue the AMPAR defect in cerebellar granule cells. We refer to these proteins as transmembrane AMPAR regulatory proteins (TARPs). We have succeeded in deleting the gene for each of the TARPs in mice. These mutant mice will form the basis for many of the proposed experiments in this RO1 grant, which is a continuation of the work carried out on the Conte Grant. There are 4 Specific Aims. (1) Determine the role of v-3 in the mouse brain, (2) determine the role of y-4 in the mouse brain, (3) determine the functional differences among TARPs, and (4) determine whether TAPRs may have AMPAR-independent roles in the CNS. The role of TARPs in AMPAR trafficking, synaptic transmission and plasticity will be studied primarily in the hippocampus. Preliminary studies indicate that y-8 plays an important role in AMPAR trafficking in the hippocampus, but substantial AMPAR transmission remains. We will also compare the ability of various TARPs to modify the deactivation of AMPARs and the kinetics of synaptic transmission. We have evidence that each of the roles TARPs play (i.e., surface delivery, synaptic targeting and receptor gating) all vary for each of the TARPs. We have found that the Y-2/Y-3A/-4, triple KO is lethal and the newborn pups are completely immobile. We will determine why the spinal cord is nonfunctional. These studies may uncover novel roles for TARPs in the nervous system. Given the critical role that receptor trafficking plays in synaptic plasticity and, by implication in certain aspects of learning and memory, it is anticipated that findings from these studies will have direct clinical impact. Indeed, clinically promising AMPAkines exert their effect, in part, by controlling the kinetics of AMPAR gating similar to TARPs. In addition, TARPs modify the pharmacological properties of AMPAkines and, thus, represent a novel target for drug design.