Actions of Ethanol on Ligand-Gated Ion Channels

描述(由申请人提供)：该项目广泛的、长期的目标是建立和表征乙醇对GABAA受体的作用机制。这项工作将朝着三个主要方向推进。首先，我们将确定乙醇和神经活性类固醇在α1β2伽马2 GABAA受体上相互作用的性质。在这些实验中，我们将确定可以与受体相互作用产生乙醇介导的增强作用的内源性类固醇的范围，并通过使用不同作用的神经活性类固醇和GABAA受体亚基的定点突变来深入了解乙醇作用的分子和动力学机制。其次，我们将研究乙醇(即，在没有辅因子的情况下)对来自α4β2受体的GABA激活电流的直接增强作用的机制。第三，我们将确定乙醇对啮齿类动物脑片中天然受体的增强作用。 这项研究的具体目标是：(1)确定内源性神经活性类固醇和乙醇是否与重组α1β2Gamma2 GABAA受体正向相互作用以增强受体功能。(2)研究乙醇对α1β2γ2 GABAA受体调控的分子和动力学机制。(3)探讨乙醇对重组α4β2βGABAA受体的直接作用机制。(4)研究乙醇对脑片全细胞电流的影响。这项工作将在HEK 293细胞中瞬时表达的重组野生型和突变型α1β2Gamma2和α4β-增量亚单位)GABAA受体以及表达天然GABAA受体的丘脑切片上进行。我们将通过单通道和全细胞电流的记录来研究受体的激活及其被类固醇和乙醇的调节。 这项拟议的研究旨在检验酒精与醉酒有关的假设 是通过激活中枢神经系统中的GABAA受体介导的，而受体与内源性神经活性类固醇的相互作用是激活GABAA受体系统的关键组成部分。实验结果将被用于研究乙醇介导的GABAA受体功能调节的机制和结构要求。

DESCRIPTION (provided by applicant): The broad, long-term objective of this project is to establish and characterize the mechanism of action of ethanol on the GABAA receptor. The work will progress in three principal directions. First, we will establish the nature of interactions between ethanol and neuroactive steroids on the alpha1beta2gamma2 GABAA receptor. In these experiments, we will determine the range of endogenous steroids that can interact with the receptor to produce ethanol-mediated potentiation, and carry put experiments to gain insight into the molecular and kinetic mechanisms of ethanol action by using neuroactive steroids of varying effects and site-directed mutagenesis of the GABAA receptor subunits. Second, we will examine the mechanism of direct potentiating effect of ethanol (i.e., in the absence of a cofactor) on GABA activated currents from alpha4beta2delta receptors. And third, we will establish the potentiating effect of ethanol on native receptors in rodent brain slices. The specific goals of the proposed research are: (1) To establish whether endogenous neuroactive steroids and ethanol positively interact on the recombinant alpha1beta2gamma2 GABAA receptor to potentiate the receptor function. (2) To study the molecular and kinetic mechanisms of alpha1beta2gamma2 GABAA receptor modulation by ethanol. (3) To determine the mechanism of direct action of ethanol on recombinant alpha4beta2delta GABAA receptors. (4) To study the effect of ethanol on whole-cell currents in brain slices. The work will be carried out on recombinant wild type and mutant alpha1beta2gamma2 and alpha4beta-delta subunit-containing) GABAA receptors transiently expressed in HEK 293 cells, and on thalamic slices expressing native GABAA receptors. Receptor activation and its modulation by steroids and ethanol will be examined from the recordings of single-channel and whole-cell currents. The proposed research has been designed to test the hypothesis that ethanol-linked inebriation is mediated by the activation of the GABAA receptor in the central nervous system, and that receptor interaction with endogenous neuroactive steroids is a critical component in such activation of the GABAA receptor system. The results from the experiments will be used to examine the mechanism and structural requirements in the ethanol-mediated modulation of the GABAA receptor function.