Development of NMDA receptor subunits with alcohol insensitivity but unaltered physiology as molecular tools

开发对酒精不敏感但生理学未改变的 NMDA 受体亚基作为分子工具

• 批准号: 10303458
• 负责人: ROBERT WILLIAM PEOPLES
• 金额: $ 7.65万
• 依托单位: MARQUETTE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-25 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10303458
• 关键词: Acute; Affect; Affinity; Alcoholic Intoxication; Alcoholism; Alcohols; Amino Acids; Area; Behavior; Behavioral; Biological; Brain; C-terminal; Cells; Characteristics; Communities; Development; Ethanol; Exhibits; Glutamates; Goals; Human; Ion Channel; Ion Channel Gating; Kinetics; Knock-out; Knockout Mice; Knowledge; Laboratories; Ligand Binding; Ligand Binding Domain; Link; Measures; Mediating; Membrane; Methods; Molecular; Mus; Mutation; N-Methyl-D-Aspartate Receptors; N-terminal; NMDA receptor A1; NMDA receptor antagonist; Neonatal; Neurons; Neurosciences Research; Pharmaceutical Preparations; Pharmacology; Physiological; Physiology; Positioning Attribute; Proteins; Reporting; Research Design; Role; Site; Site-Directed Mutagenesis; Specificity; Structure; Synapses; Techniques; Testing; Work; alcohol consequences; alcohol effect; alcohol research; alcohol sensitivity; base; behavioral study; extracellular; improved; knock-down; knockin animal; mutant; neurophysiology; overexpression; patch clamp; receptor; receptor function; receptor sensitivity; simulation; tool

The long term goal of this study is to understand the role of NMDA receptors (NMDAR) in the actions of alcohol on the brain. Although NMDAR are known to be major CNS targets of alcohol action, the precise roles of NMDAR and their substituent subunits in mediating the effects of alcohol are still incompletely understood. Prior studies using pharmacological agents or molecular biological techniques such as “knockout” or “knock-in” animals have been hindered by limitations including incomplete drug specificity and severe complications due to alterations in receptor physiology associated with mutations at alcohol-sensitive amino acid positions. Work from this laboratory has identified and characterized amino acid positions in the third and fourth membrane-associated (M) domains of the NMDAR GluN2A-C subunits that influence both ion channel gating and alcohol sensitivity. Our observations that changes observed in ion channel gating are not directly linked to changes in ethanol sensitivity (e.g., opposite changes in ion channel gating measures can similarly affect ethanol sensitivity) are consistent with the idea that alcohol sensitivity and gating may be regulated separately. Our recent work has shown that multiple mutations at alcohol-sensitive positions can retain low alcohol sensitivity while improving gating characteristics, and in preliminary studies, we have identified a mutation in the ligand-binding domain (LBD) that can further restore native physiological characteristics to an alcohol-insensitive GluN2A subunit. In these studies we will use site-directed mutagenesis combined with whole-cell and macropatch concentration-jump patch-clamp recording to test the hypothesis that NMDAR subunits with altered ethanol sensitivity but essentially normal physiology can be developed by introducing multiple mutations at positions regulating alcohol sensitivity, ion channel gating, and/or ligand binding. To best define the role of an NMDAR subunit in CNS alcohol actions, the ideal molecular tool would be an alcohol-insensitive subunit that is otherwise normal with respect to its physiology. The purpose of this project is to circumvent the shortcomings of currently-available methods by developing alcohol-insensitive NMDAR GluN2 subunits with unaltered physiology for use as molecular tools, and to make these subunits available for use in neurophysiological and behavioral studies by the neuroscience and alcohol research communities. The knowledge gained from these studies could provide a basis for a better understanding of the precise role of the NMDA receptor in the neurophysiological and behavioral effects of alcohol as well as in alcoholism.

这项研究的长期目标是了解NMDA受体（NMDAR）在酒精作用中的作用 在大脑上。尽管已知NMDAR是酒精作用的主要中枢神经系统目标，但 NMDAR及其在介导酒精作用中的亚tit量亚基仍然尚不完全了解。 先前使用药物或分子生物学技术（例如“敲除”或“敲入”的研究 动物受到限制的阻碍，包括不完整的药物特异性和严重的并发症 与酒精敏感氨基酸位置突变相关的受体生理学的改变。工作 从该实验室确定并表征了在第三和第四膜相关（M）域中的NMDAR GLUN2A-C亚基的氨基酸位置 灵敏度。我们认为在离子通道门控中观察到的变化的观察与变化无直接相关 乙醇敏感性（例如，离子通道门控措施的相反变化可能会影响乙醇 灵敏度）与酒精敏感性和门控可以分别调节的想法一致。我们的 最近的工作表明，对酒精敏感位置处的多个突变可以保持低酒精敏感性 在改善门控特征和初步研究中，我们已经确定了配体结合域（LBD）的突变，该突变可以进一步恢复对酒精不敏感的天然生理特征 Glun2a亚基。在这些研究中，我们将使用定位的诱变与全细胞和 宏观浓度 - 跳跃贴片钳记录，以测试NMDAR亚基的假设 乙醇敏感性的改变，但基本正常生理可以通过引入多种 在减轻酒精敏感性，离子通道门控和/或配体结合的位置的突变。最好定义 NMDAR亚基在CNS酒精作用中的作用，理想的分子工具将是一种不敏感的 相对于其生理学是正常的亚基。该项目的目的是规避 通过开发对酒精不敏感的NMDAR GLUN2亚基的缺点 将其用作分子工具的未改变的生理学，并使这些亚基可用于 神经科学和酒精研究社区的神经生理学和行为研究。 从这些研究中获得的知识可以为更好地理解确切作用提供基础 NMDA受体在酒精以及酒精中毒的神经生理和行为影响中。