Actions of Ethanol on Ligand-Gated Ion Channels

描述（由申请方提供）：本项目的广泛、长期目标是确定和表征乙醇对GABAA受体的作用机制。这项工作将在三个主要方向上取得进展。首先，我们将建立乙醇和神经活性类固醇之间的相互作用的性质α 1 β 2 γ 2 GABAA受体。在这些实验中，我们将确定的范围内的内源性类固醇，可以与受体相互作用，产生乙醇介导的增强，并进行投入实验，以深入了解乙醇行动的分子和动力学机制，通过使用神经活性类固醇的不同效果和定点诱变的GABAA受体亚基。其次，我们将研究乙醇的直接增强效应的机制（即，在没有辅因子的情况下）对来自α 4 β 2 δ受体的GABA激活电流的影响。第三，我们将建立乙醇对啮齿动物脑片中天然受体的增强作用。 本研究的具体目标是：（1）确定内源性神经活性类固醇和乙醇是否与重组α 1 β 2 γ 2 GABAA受体正相互作用以增强受体功能。(2)研究乙醇对α 1 β 2 γ 2 GABAA受体的分子和动力学调节机制。(3)确定乙醇对重组α 4 β 2 δ GABAA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（含α 1 β 2 γ 2和α 4 β-δ亚基）GABAA受体以及表达天然GABAA受体的丘脑切片上进行。受体激活及其调节类固醇和乙醇将检查从单通道和全细胞电流的记录。 这项研究的目的是检验酒精相关性醉酒的假设， 是由中枢神经系统中GABAA受体的激活介导的，并且受体与内源性神经活性类固醇的相互作用是GABAA受体系统的这种激活中的关键组分。实验结果将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.