Ethanol Sensitivity of Native and Cloned NMDA Receptors

描述(申请人提供)：本项目的主要目标是阐明乙醇在N-甲基-D-天冬氨酸类离子通道上的分子位置和作用机制。NMDA受体是谷氨酸激活的配体门控离子通道的一个亚型，参与脑内兴奋性突触传递。它们调节多种形式的突触可塑性，这些突触可塑性是学习和记忆的基础，通过药物或遗传手段抑制它们的功能可以扰乱这些复杂的行为。急性地，乙醇抑制通过NMDA受体的离子流动，而长期暴露在乙醇中的神经元诱导这些受体功能状态的上调，表现为在戒断过程中中枢神经系统兴奋的迹象。确定调控乙醇抑制NMDA受体的因素是重要的，因为对乙醇的醉酒效应的急性敏感性似乎是未来酒精滥用的重要预测因素。之前在该项目期间开展的工作确定了几个可以调节NMDA受体对乙醇的抑制程度的因素。这些包括亚基组成和翻译后修饰，包括磷酸化和细胞骨架相互作用。此外，该项目在受体(F639)的NR1亚基中发现了一种氨基酸，当突变为丙氨酸时，显著降低了乙醇对含有任何NR2亚基的NMDA受体的抑制作用。在这一应用中，提出了三个具体的假设，以进一步定义调节NMDA受体的乙醇敏感性的分子机制。AIM 1将扩展我们的突变研究，以检查F639和同源NR2位点的额外突变以及其他TM结构域的残基。目的2研究NR1和NR2残基的C末端磷酸化对乙醇抑制NMDA受体的影响。AIM 3将在体外和体内表达AIMS 1和AIMS 2中确定的乙醇不敏感受体，以阐明乙醇的哪些作用是通过抑制NMDA受体介导的。

DESCRIPTION (provided by applicant): The major goals of this project are to elucidate the molecular sites and mechanisms of action of ethanol on the N-methyI-D-aspartate class of ion channels. NMDA receptors are a subtype of glutamate-activated ligand-gated ion channel and they are involved in excitatory synaptic transmission in the brain. They mediate several forms of synaptic plasticity that underlie learning and memory and inhibition of their function by pharmacological or genetic means disrupts these complex behaviors. Acutely, ethanol inhibits ion flux through NMDA receptors while chronic exposure of neurons to ethanol induces an up-regulation in the functional status of these receptors that is manifested by signs of CNS excitability during withdrawal. Identifying the factors that regulate the inhibition of NMDA receptors by ethanol is important as acute sensitivity to the intoxicating effects of ethanol appears to be an important predictor of future alcohol abuse. Previous work carried out during this project identified several factors that can regulate the magnitude of inhibition of NMDA receptors to ethanol. These included subunit composition and post-translational modifications including phosphorylation and cytoskeleton interactions. In addition, this project identified an amino acid in the NR1 subunit of the receptor (F639) that when mutated to alanine significantly reduced the inhibitory effects of ethanol on NMDA receptors containing any NR2 subunit. In this application, three specific hypotheses are proposed to further define the molecular mechanisms that regulate the ethanol sensitivity of NMDA receptors. Aim 1 will extend our mutagenesis studies to examine additional mutations at F639 and homologous NR2 sites as well as residues in other TM domains. Aim 2 will investigate the effects of C-terminal phosphorylation of NR1 and NR2 residues on ethanol inhibition of NMDA receptors. Aim 3 will express ethanol-insensitive receptors identified in Aims 1 and 2 in neurons in vitro and in vivo to address which effects of ethanol are mediated by inhibition of NMDA receptors.