ENDOGENOUS REGULATORS OF GLUTAMATE ACTIVATED CHANNELS

谷氨酸激活的离子通道的作用决定了 通过兴奋性突触在哺乳动物的大脑中传递信息。 因此，谷氨酸通道的正常功能和调节 (AMPA、Kainate和NMDA亚型)参与虚拟 所有的大脑功能。在过去的10-15年里，N-N的基础研究 甲基-D-天冬氨酸(NMDA)受体提供了最清晰的 基础研究与临床问题相关的理论基础。 这些研究为了解正常的大脑功能提供了新的见解 如突触的可塑性，记忆的形成，以及 苯环利定(PCP)等拟精神病药物对人体的影响 行为。谷氨酸受体的过度刺激可导致 神经细胞死亡在癫痫发作和卒中中可能起重要作用 作用于其他神经精神疾病。令人惊叹的复杂性 调节机制影响谷氨酸受体。例如, NMDA受体受变构机制调节，多个 激酶、磷酸酶和可溶性第二信使。尽管这样 考虑到兴奋性的核心作用，复杂性似乎是合适的 突触，是什么决定了这种突触的特异性的问题 交互作用还没有被探索。钙离子通过开放进入神经元 突触上的NMDA通道启动了几个这样的调节 机制，因此我们将重点放在海马区的调节上 NMDA受体通过细胞内钙离子传递。我们的结果表明 谷氨酸的区隔作用和局部相互作用 细胞中的受体、调节蛋白和细胞骨架元件 突触后密度(PSD)是解开这个谜题的关键。这些相互作用 可能影响突触NMDA通道的活性以及 海马区突触的形成和受体组成。我们 将在这项提案中检验这一一般假设的两个方面。第一, 我们将研究NMDA受体的结构域，该受体负责 钙调节(目标1-2)和脱敏(目标3)。初步 结果表明，钙调节是NR2a特异性的，并且 嵌合/缺失结构提示NR1和NR2a区域是 可能是通过一种球链机制参与其中。我们还将 研究NMDA受体和NR2B可能的诱导作用 特别是亚单位，在个体的功能和定位上 海马神经元上的突触(目标4)。这些研究将使 使用缺乏NR2B亚单位的转基因小鼠。建议数 研究将使用在293细胞中表达的重组NMDA受体 和非洲爪哇卵母细胞以及培养的天然受体 海马神经元。我们开发的新方法用于研究 突触NMDA受体和单个突触部位的功能 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.