ALCOHOL ACTIONS--MOLECULAR TARGETS ON BRAIN PROTEINS

我们的假设是，乙醇改变了离子通道的功能，通过结合在蛋白质空腔内，这些空腔的一些结构特征将是相似的相关和不相关的离子通道。此外，我们提出乙醇的某些行为作用需要甘氨酸或GABAA受体功能的增强。我们的总体目标是（1）确定负责乙醇作用的几种脑受体/通道的特定蛋白质区域，以及（2）测试这些受体中的两种在体内的重要性。第一个目标将通过嵌合和突变受体/通道的体外测试来实现，第二个目标将使用转基因小鼠来表达对乙醇不敏感的突变受体。首先，我们将确定特定的氨基酸在GABAA和甘氨酸受体的TM 2 -3区域中的作用，在乙醇调节受体功能。这些研究将在非洲爪蟾卵母细胞中进行。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乙酰胆碱受体对乙醇的反应。接下来，我们将扩展我们最近的研究表明乙醇激活G蛋白激活内向整流钾（GIRK）通道，阐明乙醇对这个通道的作用的分子基础。这将通过在GIRK 2和IRK 1通道之间构建嵌合受体，然后突变单个氨基酸来完成。结构决定因素（即，氨基酸性质和位置）对甘氨酸、GABAA和GIRK通道的乙醇敏感性进行比较。最后，我们将通过构建具有耐乙醇突变受体的转基因小鼠来确定乙醇特定行为作用的甘氨酸和GABA受体的体内意义。这项研究的长期、健康相关的目标是确定酒精作用的分子位点，这些位点将成为药物治疗的有用靶点，从而减少酒精作用，如强化、渴望和依赖。

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.