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

本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。虽然已知NMDAR是酒精作用的主要CNS靶点，但NMDAR的确切作用还不清楚。 NMDAR及其取代基亚基在介导酒精作用中的作用仍不完全清楚。 既往使用药物或分子生物学技术（如“敲除”或“敲入”）的研究 动物已经受到限制，包括不完全的药物特异性和严重并发症， 与酒精敏感性氨基酸位置突变相关的受体生理学改变。工作 来自该实验室的J.K.已经鉴定并表征了NMDAR GluN 2A-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。 灵敏度我们的观察结果是，在离子通道门控中观察到的变化并不直接与 乙醇敏感性（例如，离子通道门控测量的相反变化可以类似地影响乙醇 敏感性）与酒精敏感性和门控可以分开调节的想法一致。我们 最近的研究表明，在酒精敏感位置的多个突变可以保持低酒精敏感性 同时改善门控特性，在初步研究中，我们已经确定了配体结合结构域（LBD）中的突变，可以进一步恢复天然生理特性，使酒精不敏感 GluN 2A亚基。在这些研究中，我们将使用定点诱变结合全细胞和 用大膜片浓度跳跃膜片钳记录来检验NMDAR亚基与 改变的乙醇敏感性，但基本上正常的生理学可以通过引入多个 在调节酒精敏感性、离子通道门控和/或配体结合的位置处的突变。为了最好地定义 NMDAR亚基在中枢神经系统酒精作用中的作用，理想的分子工具将是酒精不敏感的 在生理学上正常的亚基。该项目的目的是规避 通过开发具有以下的醇不敏感性NMDAR GluN 2亚基的当前可用方法的缺点 未改变的生理学用作分子工具，并使这些亚基可用于 神经生理学和行为学研究的神经科学和酒精研究社区。的 从这些研究中获得的知识可以为更好地了解 NMDA受体在酒精的神经生理和行为效应以及酒精中毒中的作用。

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.