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。这个提议中的实验来自我们在当前支持期间的几个有趣的发现。具体目标1跟进我们的观察，即孔环和相邻的跨膜螺旋之间的相互作用支配红藻氨酸受体对顺式不饱和脂肪酸（如二十二碳六烯酸（DHA））抑制的敏感性。在这个目标的实验将使用突变周期分析，以确定在通道中的残基相互作用，以控制渗透，门控和调制。具体目标2建立在我们的发现，即暴露于DHA似乎改变了开放状态下红藻氨酸受体跨膜螺旋的构象。取代半胱氨酸的反应性、金属离子结合和二硫键形成将用于确定脂肪酸引入的结构变化。具体目标3继续我们发现的小分子拮抗剂，其防止DHA增强NMDA受体，但对DHA抑制红藻氨酸受体的作用很小或没有作用。将来自NMDA和红藻氨酸受体的联合收割机结构域结合的嵌合亚基将用于研究不同的结构相互作用是否是通道活性增强和抑制的基础，并分析产生功能通道的结构要求。总的来说，这些实验将提供有关离子型谷氨酸受体操作的结构基础的新信息，以及有关离子通道如何受脂质双层组分相互作用影响的新信息。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 several interesting discoveries that we made during the current period of support. Specific Aim 1 follows up on our observation that interactions between the pore loop and adjacent transmembrane helices govern kainate receptor susceptibility to inhibition by cis-unsaturated fatty acids, such as docosahexaenoic acid (DHA). Experiments in this aim will use mutant cycle analysis to determine which residues in the channel interact with each other to control permeation, gating and modulation. Specific Aim 2 builds on our discovery that exposure to DHA appears to change the conformation of kainate receptor transmembrane helices in the open state. Reactivity of substituted cysteines, metal ion binding, and disulfide bond formation will be used to determine structural changes introduced by fatty acids. Specific Aim 3 follows up on our discovery of small molecule antagonists that prevent DHA from potentiating NMDA receptors but have little or no effect on DHA inhibition of kainate receptors. Chimeric subunits that combine domains from NMDA and kainate receptors will be used to investigate whether distinct structural interactions underlie the potentiation and inhibition of channel activity and t analyze the structural requirements for generation of functional channels. Collectively, these experiments will provide new information about the structural basis for ionotropic glutamate receptor operation and new information about how ion channels are affected by interactions with components of the lipid bilayer. 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.