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的治疗方法至关重要。在这个正在进行的研究项目的更新中，我们专注于了解酒精如何影响神经元表达的关键离子通道的功能。这个通道，N-甲基-D-天冬氨酸(NMDA)受体，在兴奋性谷氨酸能传递中发挥重要作用，并关键地参与构成学习、记忆和其他高级认知过程的复杂过程。在上一次资助期间进行的先前研究证实，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中可能定义酒精作用部位的关键残基。在这一应用中，我们提出了一系列特定的目标，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。目的1验证NMDA受体亚域中的特定氨基酸决定乙醇抑制受体功能的能力的假说。以此为目的的实验将使用重组表达和记录技术来测试关键跨膜区残基的定点突变如何改变酒精对通道功能的影响。目的2验证酒精抑制NMDA受体也可通过表达新的NR3亚基和通过磷酸化NR1和NR2亚基胞内区的关键残基来改变这一假说。这一目标的实验将利用表达和记录技术，并结合使用转基因动物。目的3将验证这样一种假设，即对酒精的行为反应可以通过突变的NMDA受体的表达来改变，该受体显示出改变的酒精敏感性。这一目标的实验将使用目前正在开发的一种新型NMDA基因敲入小鼠和病毒过度表达技术来改变离散脑区的NMDA亚单位表达。总体而言，这些研究的结果有望导致对NMDA受体在调节酒精的大脑活动中的作用有更全面的了解。 公共卫生相关性：影响个人对酒精醉人效应的敏感性的过程是未来酒精问题的重要预测因素。这项提案中将要进行的研究将确定影响大脑离子通道酒精敏感性的因素，该离子通道是调节大脑活动的关键因素。

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.