Department of Biomedical Sciences, Marquette University, Milwaukee, Wisconsin, USA.
Biobank, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Alcohol Clin Exp Res. 2022 Dec;46(12):2203-2213. doi: 10.1111/acer.14965. Epub 2022 Nov 9.
The N-methyl-D-aspartate receptor (NMDAR) is a major molecular target of alcohol action in the central nervous system, yet many aspects of alcohol's modulation of the activity of this ion channel remain unclear. We and others have shown that ethanol inhibition of NMDAR involves alterations in gating, especially a reduction in mean open time. However, a full description of ethanol's effects on NMDAR kinetics, including fitting them to a kinetic model, has not been reported.
To determine ethanol's effects on NMDAR kinetics, we used steady-state single-channel recording in outside-out patches from HEK-293 cells transfected with recombinant GluN1/GluN2A or GluN1/GluN2B NMDAR subunits. Very low glutamate concentrations were used to isolate individual activations of the receptor.
In both subunit types, ethanol, at approximate whole-cell IC values (156 mM, GluN2A; 150 mM, GluN2B), reduced open probability (p ) by approximately 50% and decreased mean open time without changing the frequency of opening. Open and shut time distributions exhibited two and five components, respectively; ethanol selectively decreased the time constant and relative proportion of the longer open time component. In the GluN2A subunit, ethanol increased the time constants of all but the longest shut time components, whereas in the GluN2B subunit, shut times were unchanged by ethanol. Fitting of bursts of openings (representing individual activations of the receptor) to the gating portion of a kinetic model revealed that ethanol altered two rates: the rate associated with activation of the GluN2A or GluN2B subunit, and the rate associated with the closing of the longer of the two open states.
These results demonstrate that ethanol selectively alters individual kinetic rates and thus appears to selectively affect distinct conformational transitions involved in NMDAR gating.
N-甲基-D-天冬氨酸受体（NMDAR）是中枢神经系统中酒精作用的主要分子靶点，但酒精对该离子通道活性的许多调节作用仍不清楚。我们和其他人已经表明，乙醇对 NMDAR 的抑制作用涉及门控的改变，特别是平均开放时间的减少。然而，乙醇对 NMDAR 动力学的影响，包括将其拟合到动力学模型中，尚未有报道。
为了确定乙醇对 NMDAR 动力学的影响，我们使用 HEK-293 细胞的外切斑中的稳态单通道记录，这些细胞转染了重组 GluN1/GluN2A 或 GluN1/GluN2B NMDAR 亚基。使用非常低的谷氨酸浓度来分离受体的单个激活。
在两种亚基类型中，乙醇在近似全细胞 IC 值（GluN2A 为 156 mM，GluN2B 为 150 mM）下，将开放概率（p ）降低约 50%，并减少平均开放时间，而不改变开放频率。开放和关闭时间分布分别呈现两个和五个分量；乙醇选择性地降低了时间常数和较长开放时间分量的相对比例。在 GluN2A 亚基中，乙醇增加了除最长关闭时间分量之外的所有时间常数，而在 GluN2B 亚基中，乙醇对关闭时间没有影响。对开放爆发（代表受体的单个激活）进行拟合到动力学模型的门控部分，结果表明乙醇改变了两个速率：与 GluN2A 或 GluN2B 亚基的激活相关的速率，以及与两个开放状态中较长的一个关闭相关的速率。
这些结果表明，乙醇选择性地改变了单个动力学速率，因此似乎选择性地影响了 NMDAR 门控中涉及的不同构象转变。
