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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

基本信息

批准号：
10494115
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
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-25 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10494115
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 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

项目摘要

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 NMDAR 被认为是酒精作用的主要中枢神经系统靶标，但其确切作用 NMDAR 及其取代亚基在介导酒精作用中的作用尚不完全清楚。先前使用药物或分子生物学技术（例如“敲除”或“敲入”）的研究动物的研究受到了一些限制，包括药物特异性不完全和由于药物引起的严重并发症与酒精敏感氨基酸位置突变相关的受体生理学改变。工作该实验室的研究人员鉴定并表征了 NMDAR GluN2A-C 亚基的第三和第四膜相关 (M) 结构域中影响离子通道门控和酒精的氨基酸位置敏感性。我们观察到离子通道门控中观察到的变化与乙醇敏感性（例如，离子通道门控措施的相反变化同样会影响乙醇敏感性）与酒精敏感性和门控可以单独调节的想法是一致的。我们的最近的工作表明，酒精敏感位置的多重突变可以保持低酒精敏感性在改善门控特性的同时，在初步研究中，我们发现了配体结合域（LBD）的突变，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，我们将使用定点诱变与全细胞和宏补丁浓度跳跃膜片钳记录来检验 NMDAR 亚基与改变乙醇敏感性，但基本上正常的生理机能可以通过引入多种调节酒精敏感性、离子通道门控和/或配体结合的位置的突变。最好地定义 NMDAR 亚基在中枢神经系统酒精作用中的作用，理想的分子工具是酒精不敏感的生理学方面正常的亚基。该项目的目的是为了规避通过开发酒精不敏感的 NMDAR GluN2 亚基来弥补目前可用方法的缺点未改变的生理学用作分子工具，并使这些亚基可用于神经科学和酒精研究界的神经生理学和行为研究。这从这些研究中获得的知识可以为更好地理解 NMDA 受体对酒精以及酒精中毒的神经生理和行为影响。