ENDOGENOUS REGULATORS OF GLUTAMATE ACTIVATED CHANNELS

谷氨酸激活的离子通道的作用决定了 通过哺乳动物大脑中的兴奋性突触传递信息。 因此，谷氨酸通道的正常功能和调节 (of AMPA、红藻氨酸盐和NMDA亚型）实际上参与了 所有的大脑功能。 在过去的10-15年里，N- 甲基-D-天冬氨酸（NMDA）受体提供了一个最清晰的 基础研究与临床问题相关的基本原理。 这些研究为正常的大脑功能提供了新的见解 例如突触可塑性，记忆的形成， 拟精神病药物如苯环己哌啶（PCP）对人类的影响 行为 谷氨酸受体的过度刺激会导致 神经元细胞死亡在癫痫发作和中风，并可能发挥重要作用， 其他神经精神疾病。 一个惊人的复杂性， 调节机制影响谷氨酸受体。 比如说， NMDA受体受变构机制调节，多种 激酶、磷酸酶和可溶性第二信使。 虽然这样 考虑到兴奋性神经元的核心作用， 突触，是什么决定了这种特异性的问题 互动是未被探索的。 钙离子通过开放通道流入神经元 突触上的NMDA通道启动了几种调节性的 机制，因此，我们专注于调节海马 NMDA受体通过细胞内钙。 我们的结果表明 谷氨酸间的区室化和局部相互作用 受体、调节蛋白和细胞骨架元件 突触后密度（PSD）是这个难题的关键。 这些相互作用 可能会影响突触NMDA通道的活性， 海马突触的形成和受体组成。 我们 将在本提案中检验这一一般假设的两个方面。 第一、 我们将研究NMDA受体的区域， 钙调节（Aim 1-2）和脱敏（Aim 3）。 初步 结果表明，钙调节是NR 2A特异性的， 嵌合/缺失构建体表明NR 1和NR 2A的区域是 也许是通过一个球链机制。 我们还将 检查NMDA受体和NR 2B的可能诱导作用， 特别是亚基，在个体的功能和定位中， 海马神经元上的突触（目的4）。 这些研究将使 使用缺乏NR 2B亚基的转基因小鼠。 拟议 研究将使用在293细胞中表达的重组NMDA受体 和非洲爪蟾卵母细胞以及天然受体在培养 海马神经元 我们开发的新方法用于研究 突触NMDA受体和单个突触位点的功能将 被利用

The action of glutamate-activated ion channels determines the flow of information via excitatory synapses throughout the mammalian brain. As a result, the normal function and regulation of glutamate channels (of the AMPA, kainate and NMDA subtypes) are involved in virtually all brain functions. In the past 10-15 years, fundamental studies of N- methyl-D-aspartate (NMDA) receptors provide one of the clearest rationales for the relevance of basic research to clinical problems. These studies have provided new insights into normal brain functions such as synaptic plasticity, the formation of memories, and the action of psychomimetic drugs such as phencyclidine (PCP) on human behavior. Excessive stimulation of glutamate receptors can cause neuronal cell death in seizures and stroke, and may play an important role other neuropsychiatric illnesses. An amazing complexity of regulatory mechanisms influence glutamate receptors. For example, NMDA receptors are regulated by allosteric mechanisms, multiple kinases, phosphatases and soluble second messengers. Although such complexity may seem fitting given the central role of excitatory synapses, the question of what determines the specificity of such interactions is unexplored. Calcium influx into neurons through open NMDA channels at synapses initiates several of these regulatory mechanisms, thus we have focused on the regulation of hippocampal NMDA receptors by intracellular calcium. Our results suggest that compartmentalization and local interactions between glutamate receptors, regulatory proteins and cytoskeletal elements in the postsynaptic density (PSD) are keys to this puzzle. These interactions are likely to affect the activity of synaptic NMDA channels as well as the formation and receptor composition of hippocampal synapses. We will test two aspects of this general hypothesis in this proposal. First, we will examine the domains of the NMDA receptor responsible for calcium regulation (Aim 1-2) and desensitization (Aim 3). Preliminary results demonstrate that calcium regulation is NR2A specific and chimeric/deletion constructs suggest regions of NR1 and NR2A that are involved, perhaps by a ball-and-chain mechanism. We will also examine the possible inductive role of NMDA receptors, and the NR2B subunit in particular, in the function and localization of individual synapses on hippocampal neurons (Aim 4). These studies will make use of transgenic mice lacking the NR2B subunit. The proposed studies will use recombinant NMDA receptors expressed in 293 cells and Xenopus oocytes as well as native receptors in cultured hippocampal neurons. Novel methods we developed for studies of synaptic NMDA receptors and function of individual synaptic sites will be used.