MOLECULAR BIOLOGY OF ETHANOL ACTION ON RECEPTORS

乙醇对受体作用的分子生物学

• 批准号: 2893956
• 负责人: GEORGE R BREESE
• 金额: $ 9.76万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2893956
• 关键词: GABA receptor; NMDA receptors; cytoplasm; electrophysiology; ethanol; laboratory rat; membrane channels; messenger RNA; neurons; neuropharmacology; polymerase chain reaction; protein protein interaction; receptor sensitivity; tissue /cell culture; voltage /patch clamp

This Research Scientist Award is based upon current work being performed to understand the molecular basis of ethanol's specificity of action on ligand-gated ion channels. This proposal, tests the hypothesis that the selectivity of ethanol to affect responses to NMDA and GABA from some, but not all, neurons is due to the presence of specific NMDA and GABAA receptor subtypes. The model approach proposed to study the action of ethanol on NMDA and GABAA receptors is an update of progress made on R01s AA09122 and AA10025, grants defining the molecular mechanism(s) responsible for ethanol's specificity on specific receptor subtypes. In respect to ethanol's action on GABAA receptor subtypes, we have demonstrated zolpidem predicts one GABAA receptor subtype sensitive to ethanol. Likewise, we have shown a close relationship between ifenprodil and ethanol antagonism of NMDA responses. In this proposal, each cultured and dissociated neurons cell will be pharmacologically characterized with patch-clamp electrophysiology, the cytoplasm will be extracted from the cell, and mRNAs for GABAA and NMDA receptor subunits within each individual neuron will be identified with RT/PCR. In Specific Aim I, work will initially focus on the hypothesis that the alpha1beta2gamma2 subunit combination is one GABAA receptor subtype sensitive to ethanol and zolpidem. Due to present uncertainty, particular emphasis is being placed on which gamma2 variants in neurons with this subunit combination are sensitive to ethanol. A second portion of work on GABAA receptors relates to defining the molecular mechanism by which ethanol's action is dependent upon secondary neural inputs to a cell. Finally, we will determine if diazepam-insensitive receptors, containing alpha4 or alpha6 subunits, are sensitive to ethanol. Specific Aim II will identify the mRNAs for NMDA receptor subunits within individual neurons sensitive to ethanol antagonism of NMDA. Current results suggest that some, but not all, NMDA receptor subtypes sensitive to ifenprodil are sensitive to ethanol. Since ifenprodil binds to NMDA receptors purportedly containing the NMDAR-2b subunit, it can assumed that the receptor subtype sensitive to ethanol will contain this NMDA receptor subunit, as well as an appropriate NMDAR-1 variant. Studies are underway to confirm this hypothesis. The model proposed to define the molecular basis of ethanol's action to affect GABAA and NMDA receptor subtypes, when applied to all ligand- gated ion channels, will provide a means to identify neuroanatomical sites at which ethanol has the greatest probability of affecting brain function. Further, based upon structural analysis of ethanol's action on protein-protein inter-actions for the receptors, results may ultimately allow us to predict other protein-protein interactions influenced by ethanol.

这项研究科学家奖是基于目前正在进行的工作 为了了解乙醇作用的分子基础， 配体门控离子通道。 这一提议检验了一个假设，即 乙醇的选择性影响一些人对NMDA和GABA的反应， 但不是所有的神经元都是由于特定的NMDA和GABAA的存在 受体亚型 提出了一种模型方法来研究 乙醇对NMDA和GABAA受体的作用是对 R 01 AA 09122和AA 10025，赠款定义分子机制 负责乙醇对特定受体亚型的特异性。 关于乙醇对GABAA受体亚型的作用，我们有 证明唑吡坦预测一种GABAA受体亚型对 乙醇 同样，我们已经表明， 艾芬地尔和乙醇拮抗NMDA反应。 在这项提案中， 每个培养和分离的神经元细胞将被分离， 以膜片钳电生理学为特征， 从细胞中提取的GABAA和NMDA受体亚基的mRNA 将用RT/PCR鉴定每个单独神经元内的细胞。 在 具体目标一，工作将首先集中在假设， α 1 β 2 γ 2亚单位组合是一种GABAA受体亚型 对乙醇和唑吡坦敏感。 由于目前的不确定性， 特别强调的是神经元中的γ 2变体 对乙醇敏感。 第二 对GABAA受体的部分工作涉及定义分子 乙醇的作用依赖于次级神经元的机制 输入到细胞。 最后，我们将确定是否安定不敏感 含有α 4或α 6亚单位的受体对 乙醇 Specific Aim II将鉴定NMDA受体的mRNA 对乙醇拮抗作用敏感的单个神经元内的亚基 NMDA 目前的结果表明，一些，但不是全部，NMDA受体， 对艾芬地尔敏感的亚型对乙醇敏感。 以来 艾芬地尔与据称含有NMDAR-2b的NMDA受体结合 亚基，可以假定对乙醇敏感的受体亚型 将含有这种NMDA受体亚基，以及适当的 NMDAR-1变体。正在进行研究以证实这一假设。 的 提出了一个模型来定义乙醇作用的分子基础， 影响GABAA和NMDA受体亚型，当应用于所有配体- 门控离子通道，将提供一种方法， 乙醇最有可能影响大脑的部位 功能 此外，基于对乙醇作用的结构分析， 蛋白质-蛋白质相互作用的受体，结果可能 最终使我们能够预测其他蛋白质之间的相互作用 乙醇的影响。