GLUTAMATE-GATED CHANNELS IN CENTRAL & PERIPHERAL NEURONS

描述（由申请人提供）：我工作的长期目标是通过研究NMDA、AMPA和红藻氨酸受体的操作来更好地理解突触传递，这些受体形成由神经递质谷氨酸门控的离子通道。另一个主要目标是揭示这些受体的特性，这些特性可能允许临床干预以防止兴奋性毒性细胞死亡或提供镇痛。这项提议中的实验源于我们在当前支持期间的两个有趣发现。具体目标1遵循我们的观察，即通过顺式不饱和脂肪酸（如二十二碳六烯酸（DHA））阻断红藻氨酸受体取决于通道孔中Q/R位点处的RNA编辑。具有编辑的（R）形式的亚基的通道被强烈阻断，但在Q/R位点包含具有（Q）的亚基显著减弱抑制。孔环区段中的定点诱变将用于测试重组通道的这种调节的结构基础。具体目标2建立在我们的发现，突变附近的细胞外端的M3螺旋调节脂肪酸块。嵌合亚基和点突变将用于确定目标1中检测的孔环片段外结构域中脂肪酸抑制的结构要求。具体目标3将分析内源性脂质衍生介质释放引起的通道调节。与正常脑电活动相关的刺激，以及与脂肪酸释放相关的各种脑病理学，将用于触发脂质动员。这些实验将利用抵抗脂肪酸调节的亚基以及我们已经发现的阻断NMDA受体的脂肪酸增强的小分子拮抗剂。总的来说，这些实验将揭示神经元谷氨酸受体的运作和调节。构成这些受体的亚基彼此同源，并且被认为共享相同的膜拓扑结构。亚基之间的结构差异是NMDA、AMPA和红藻氨酸受体对突触通讯的独特贡献的基础。我们的实验集中在结构特征，允许特定的亚基组成的受体的差异调节。许多病理条件，包括脑外伤，癫痫和缺血，引起顺式不饱和脂肪酸的大量释放。这些化合物直接调节许多不同的膜蛋白，包括许多离子通道亚型。该项目分析了DHA对谷氨酸受体调节的分子基础，DHA在神经系统中含量很高，已知对正常的大脑功能至关重要。

DESCRIPTION (provided by applicant): The long-term objective of my work is to provide a better understanding of synaptic transmission by studying the operation of NMDA, AMPA and kainate receptors, which form ion channels gated by the neurotransmitter glutamate. Another major goal is to uncover properties of these receptors that may allow for clinical intervention to prevent excitotoxic cell death or to provide analgesia. The experiments in this proposal arise from two interesting discoveries that we made during the current period of support. Specific Aim 1 follows up on our observation that kainate receptor block by cis-unsaturated fatty acids, such as docosahexanoic acid (DHA), depends on RNA editing at the Q/R site in the channel pore. Channels with subunits in the edited (R) form are strongly blocked, but inclusion of subunits with a (Q) at the Q/R site dramatically weakens inhibition. Site-directed mutagenesis in the pore loop segment will be used to test the structural basis for this regulation of recombinant channels. Specific Aim 2 builds on our discovery that mutations near the extracellular end of the M3 helix regulate fatty acid block. Chimeric subunits and point mutations will be used to determine the structural requirements for fatty acid inhibition in domains outside of the pore loop segment tested in Aim 1. Specific Aim 3 will analyze channel modulation resulting from release of endogenous lipid-derived mediators. Stimuli relevant to normal brain electrical activity, and to a variety of brain pathologies associated with fatty acid release, will be used to trigger lipid mobilization. The experiments will make use of subunits that resist fatty acid modulation as well as small molecule antagonists that we have discovered which block fatty acid potentiation of NMDA receptors. Collectively, these experiments will shed light on the operation and regulation of neuronal glutamate receptors. Subunits that make up these receptors are all homologous to each other and are thought to share the same membrane topology. Structural differences among the subunits underlie the unique contributions made by NMDA, AMPA and kainate receptors to synaptic communication. Our experiments focus on structural features that allow for differential regulation of receptors with specific subunit composition. A number of pathologic conditions, including brain trauma, epilepsy, and ischemia, elicit massive release of cis-unsaturated fatty acids. These compounds directly regulate many different membrane proteins including a number of ion channel subtypes. This project analyzes the molecular basis of glutamate receptor modulation by DHA, which is present at high levels in the nervous system and is known to be essential for normal brain function.