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)在酒精行为中的作用 在大脑上。尽管已知NMDAR是酒精作用的主要中枢神经系统靶点，但NMDAR的确切作用 NMDAR及其取代基亚单位在调节酒精效应中的作用尚不完全清楚。 先前使用药物或分子生物学技术的研究，如“基因敲除”或“敲入” 动物受到药物不完全特异性和严重并发症等限制的阻碍。 与酒精敏感氨基酸位置突变相关的受体生理变化有关。工作 在影响离子通道门控和酒精的NMDAR GluN2a-C亚基的第三和第四膜相关(M)区域中的氨基酸位置 敏感度。我们的观察发现，在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可以类似地影响乙醇 敏感度)与酒精敏感度和门控可以分开调节的观点一致。我们的 最近的研究表明，酒精敏感部位的多重突变可以保持较低的酒精敏感性。 在改善门控特性的同时，在初步研究中，我们已经确定了配体结合区域(LBD)的突变，该突变可以进一步恢复酒精不敏感的自然生理特征 GluN2A亚单位。在这些研究中，我们将使用定点突变结合全细胞和 宏膜片浓度跳跃膜片钳记录验证NMDAR亚单位与 改变的酒精敏感性，但本质上是正常的生理可以通过引入 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。为了更好地定义 NMDAR亚单位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 生理上正常的亚基。此项目的目的是绕过 通过开发酒精不敏感的NMDAR GluN2亚基的现有方法的缺点 未改变的生理学，用作分子工具，并使这些亚基可用于 由神经科学和酒精研究团体进行的神经生理和行为研究。这个 从这些研究中获得的知识可以为更好地理解 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.