ALCOHOL ACTIONS--MOLECULAR TARGETS ON BRAIN PROTEINS

我们的假设是乙醇通过结合在蛋白质腔内改变了离子通道的功能，并且这些腔的一些结构特征对于相关和不相关的离子通道将是相似的。此外，我们认为乙醇的某些行为作用需要增强甘氨酸或GABAA受体的功能。我们的总体目标是(L)确定几个负责乙醇作用的大脑受体/通道的特定蛋白质区域，以及(2)测试其中两个受体在体内的重要性。第一个目标将通过体外测试嵌合和突变的受体/通道来实现，第二个目标将使用转基因小鼠在体外表达对乙醇不敏感的突变受体。首先，我们将确定GABAA和甘氨酸受体TM2-3区域的特定氨基酸在乙醇调节受体功能中的作用。这些研究将在非洲爪哇卵母细胞中进行。为了确定GABAA和甘氨酸受体的结果是否推广到另一个相关的配体门控离子通道，我们将表征重组神经元烟碱型乙酰胆碱受体对乙醇的反应。接下来，我们将通过阐明乙醇作用于G蛋白激活的内向整流钾(GIRK)通道的分子基础来扩展我们最近的研究，表明乙醇激活了G蛋白激活的内向整流钾(GIRK)通道。这将通过在GIRK2和IRK1通道之间构建嵌合受体，然后对单一氨基酸进行突变来实现。将比较甘氨酸、GABAA和GIRK通道对乙醇敏感性的结构决定因素(即氨基酸性质和位置)。最后，我们将通过构建具有乙醇抗性突变受体的转基因小鼠来确定甘氨酸和GABA受体对乙醇特定行为行为的体内意义。这项研究的长期、与健康有关的目标是确定酒精作用的分子位点，这些分子位点将成为药物治疗的有用靶点，以减少酒精作用，如强化、渴求和依赖。

Our hypotheses are that ethanol alters the function of ion channels by binding within protein cavities and that some structural features of these cavities will be similar for related and unrelated ion channels. In addition, we propose that some behavioral actions of ethanol require enhancement of glycine or GABAA receptor function. Our overall goals are (l) to determine the specific protein regions of several brain receptors/channels that are responsible for ethanol action, and (2) to test the importance of two of these receptors in vivo. The first goal will be accomplished by in vitro testing of chimeric and mutated receptors/channels, and the second will use transgenic mice to express mutated receptors that are insensitive to ethanol in vitro. First, we will define the role of specific amino acids in the TM2-3 region of GABAA and glycine receptors in ethanol modulation of receptor function. These studies will be carried out in Xenopus oocytes. To determine if results from GABAA and glycine receptors generalize to another, related, ligand-gated ion channel, we will characterize the response of recombinant neuronal nicotinic acetylcholine receptors to ethanol. Next, we will extend our recent studies showing ethanol activation of G-protein activated inwardly rectifying potassium (GIRK) channels by elucidating the molecular basis of ethanol action on this channel. This will be done by construction of chimeric receptors between GIRK2 and IRK1 channels, followed by mutation of single amino acids. Structural determinants (i.e., amino acid properties and locations) of ethanol sensitivity will be compared for glycine, GABAA, and GIRK channels. Lastly, we will determine the in vivo significance of the glycine and GABA receptors for specific behavioral actions of ethanol by constructing transgenic mice with mutant receptors that are ethanol-resistant. The long-term, health-related, goal of this research is to identify molecular sites of alcohol action that would be useful targets for pharmacotherapies that would reduce alcohol actions such as reinforcement, craving, and dependence.