GLUTAMATE-GATED CHANNELS IN CENTRAL & PERIPHERAL NEURONS

描述（申请人提供）：我工作的长期目标是通过研究NMDA， AMPA和kainate受体的运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，以防止兴奋毒性细胞死亡或提供镇痛。本提案中的实验源于我们在当前支持期间所做的几个有趣的发现。特异性目的1是在我们观察到孔环和相邻的跨膜螺旋之间的相互作用控制红酸盐受体对顺式不饱和脂肪酸（如二十二碳六烯酸（DHA））抑制的敏感性的基础上进行的。本目的实验将使用突变周期分析来确定通道中哪些残基相互作用以控制渗透，门控和调制。特异性目标2建立在我们的发现，暴露于DHA似乎改变了开放状态下的盐酸盐受体跨膜螺旋的构象。取代半胱氨酸、金属离子结合和二硫键形成的反应性将用于确定脂肪酸引起的结构变化。特异性目标3是在我们发现的小分子拮抗剂的基础上进行的，这些拮抗剂可以阻止DHA增强NMDA受体，但对DHA抑制海因酸盐受体的作用很小或没有作用。结合NMDA和盐酸盐受体结构域的嵌合亚基将用于研究不同的结构相互作用是否在通道活性增强和抑制的基础上，并分析生成功能通道的结构要求。总的来说，这些实验将提供关于嗜离子性谷氨酸受体运作的结构基础的新信息，以及关于离子通道如何受到与脂质双分子层组分相互作用的影响的新信息。许多病理条件，包括脑外伤、癫痫和缺血，引起大量的顺式不饱和脂肪酸的释放。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。本项目分析了DHA调节谷氨酸受体的分子基础，DHA在神经系统中存在高水平，已知对正常脑功能至关重要。

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.