Ethanol Sensitivity of Native and Cloned NMDA Receptors

描述（由申请人提供）：了解酒精对大脑功能的影响对于开发更好的酒精相关问题治疗方法至关重要。在这个正在进行的研究项目的更新中，我们重点了解酒精如何影响神经元表达的关键离子通道的功能。该通道，即 N-甲基-D-天冬氨酸 (NMDA) 受体，在兴奋性谷氨酸能传递中发挥着重要作用，并且关键参与构成学习、记忆和其他高级认知过程的复杂过程。之前的资助期间进行的研究表明，酒精对 NMDA 受体的抑制可以通过受体上的离散结构域进行调节，并确定了蛋白质跨膜结构域内的关键残基，这些残基可能定义了酒精的作用位点。在本申请中，我们提出了一系列具体目标，旨在确定这些结构域如何控制受体对酒精的敏感性以及体内乙醇不敏感受体的表达如何影响酒精诱导的行为。目标 1 将检验以下假设：NMDA 受体子域内的特定氨基酸决定乙醇抑制受体功能的能力。该目的的实验将使用重组表达和记录技术来测试关键跨膜结构域中残基的定点诱变如何改变酒精对通道功能的影响。目标 2 将检验以下假设：新型 NR3 亚基的表达以及 NR1 和 NR2 亚基胞内结构域上关键残基的磷酸化也可改变 NMDA 受体的酒精抑制作用。此目的的实验将利用表达和记录技术以及转基因动物的使用。目标 3 将检验这样一个假设：对酒精的行为反应可以通过表达突变 NMDA 受体来改变，这些受体表现出改变的乙醇敏感性。这一目标的实验将使用目前正在开发的新型 NMDA 敲入小鼠和病毒过度表达技术来改变离散大脑区域的 NMDA 亚基表达。总体而言，这些研究的结果有望使人们更全面地了解 NMDA 受体在介导酒精对大脑的作用中的作用。 公共卫生相关性：影响个人对酒精中毒效应敏感性的过程是未来酒精问题的重要预测因素。该提案中将进行的研究将确定影响大脑离子通道酒精敏感性的因素，该离子通道对调节大脑活动至关重要。

DESCRIPTION (provided by applicant): Understanding the effects of alcohol on brain function is critical for developing better treatments for alcohol-related problems. In this renewal of an ongoing research project, we focus on understanding how alcohol affects the function of a key ion channel expressed by neurons. This channel, the N- methyl-D-aspartate (NMDA) receptor, plays a major role in excitatory glutamatergic transmission and is critically involved in complex processes that underlie learning, memory and other higher cognitive processes. Previous studies carried out during the previous funding period established that alcohol's inhibition of the NMDA receptor could be modulated by discrete domains on the receptor and identified key residues within transmembrane domains of the protein that may define an alcohol site of action. In this application, we propose a series of specific aims designed to establish how these domains control the receptor's sensitivity to alcohol and how expression of ethanol-insensitive receptors in vivo affects alcohol-induced behaviors. Aim 1 will test the hypothesis that specific amino acids within sub-domains of the NMDA receptor determine the ability of ethanol to inhibit receptor function. Experiments in this aim will use recombinant expression and recording techniques to test how site-directed mutagenesis of residues in key transmembrane domains alters the effects of alcohol on channel function. Aim 2 will test the hypothesis that alcohol inhibition of NMDA receptors is also modified by expression of the novel NR3 subunit and through phosphorylation of key residues on the intracellular domain of the NR1 and NR2 subunits. Experiments in this aim will utilize expression and recording techniques coupled with the use of genetically modified animals. Aim 3 will test the hypothesis that the behavioral responses to alcohol can be modified by expression of mutant NMDA receptors that show altered ethanol sensitivity. Experiments in this aim will use a novel NMDA knock-in mouse that is currently under development and viral over-expression techniques to alter NMDA subunit expression in discrete brain areas. Overall, results from these studies are expected to lead to a more complete understanding of the role of NMDA receptors in mediating alcohol's action of the brain. PUBLIC HEALTH RELEVANCE: Processes that affect an individual's sensitivity to the intoxicating effects of alcohol are an important predictor of future alcohol problems. Research to be carried out in this proposal will determine the factors that influence the alcohol sensitivity of a brain ion channel that is critically involved in regulating brain activity.