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.

本研究的长期目标是了解NMDA受体（NMDAR）在酒精作用中的作用