Characterization of a novel presynaptic target for ethanol action

描述（由申请人提供）：酒精滥用和酒精中毒影响美国4.5%的人口，造成约1840亿美元/年的经济负担。开发新的药物疗法以帮助对抗酒精依赖和累犯的能力需要了解酒精对神经系统作用的机制。确定乙醇结合的靶点尤其重要，因为这些靶点可能带来最完整的治疗效果。虽然已知酒精对突触前功能有明显而深远的影响，但这种巨大影响的机制基本上是未知的。这项建议的长期目标是确定酒精对突触前功能发挥作用的分子机制。该探索性应用的目的是研究Munc13.1蛋白和乙醇之间的生理和行为相互作用。Munc13.1是一种突触前活性区蛋白，对脑内神经递质的释放至关重要。在秀丽隐杆线虫中，同源Unc 13蛋白负责对挥发性麻醉剂的敏感性。Munc 13蛋白家族的C1或二酰基甘油结合结构域在结构上类似于调节酒精行为效应的蛋白激酶C（PKC）的结构域，并且具有酒精结合位点。初步数据表明，乙醇也将结合Munc13.1中的C1结构域。待检验的中心假设是乙醇对神经系统功能的显著影响是由于乙醇与Munc13.1 C1结构域的结合。该提案的第一个目的将检查的假设，即乙醇结合到Munc13.1的C1结构域修改这个突触前蛋白的活性。这将通过光标记和质谱法来鉴定醇结合残基，并通过阐明这种结合对膜易位测定中Munc13.1活性的影响来实现。第二个目的是确定Dunc 13活性的降低如何改变果蝇对乙醇的行为和生理反应。选择该无脊椎动物模型系统是因为其能够快速经济地改变Dunc 13水平并监测功能后果。乙醇-Dunc 13相互作用的这些后果将通过测量野生型和Dunc 13功能降低动物中的乙醇偏好、刺激和镇静来揭示。此外，将使用突触-pHluorin传感器来检查相互作用的效果，以在中毒和清醒的果蝇中从特定的GABA能神经元子集释放囊泡。这些GABA能神经元是果蝇中乙醇的刺激和镇静作用的关键调节剂。这种方法是创新的，因为它首次应用了超灵敏生物化学和强大的神经遗传学技术的优势来剖析乙醇-受体相互作用的功能。这一建议是有意义的，因为它有望揭示一种新的主要机制，乙醇对突触前功能的影响。最终，这种理解可能会导致新的药物设计破坏乙醇unc13.1相互作用提供了一个有价值的武器，在争取清醒。

DESCRIPTION (provided by applicant): Alcohol abuse and alcoholism affect 4.5% of the United States population causing an economic burden of approximately 184 billion dollars/year. The ability to develop new pharmacotherapies to help fight the descent into alcohol dependence and recidivism requires an understanding of mechanisms of alcohol actions on the nervous system. It is particularly important to define the targets of ethanol binding as these may bring about the most complete therapeutic effect. Although alcohol is known to have distinct and profound effects on presynaptic function, the mechanisms underlying this large impact are essentially unknown. The long-term goal of this proposal is to define the molecular mechanism by which alcohol exerts its action on presynaptic function. The objective of this exploratory application is to examine the physiological and behavioral interactions between Munc13.1 protein and ethanol. Munc13.1 is a presynaptic active zone protein essential for neurotransmitter release in brain. In Caenorhabditis elegans, the homologous Unc13 protein is responsible for the sensitivity to volatile anesthetics. The C1 or diacylglycerol binding domain of the Munc13 family of proteins is structurally similar to that of protein kinase C (PKC) which regulates behavioral effects of alcohol and has alcohol binding site(s). Preliminary data demonstrate that ethanol will also bind to this C1 domain in Munc13.1. The central hypothesis to be tested is that a significant effect of ethanol on nervous system function is due to the binding of ethanol to the Munc13.1 C1 domain. The first aim of this proposal will examine the hypothesis that ethanol binding to the C1 domain of the Munc13.1 modifies the activity of this presynaptic protein. This will be accomplished by photolabeling and mass spectrometry to identify alcohol binding residues, and by elucidating the effects of this binding on Munc13.1 activity in membrane translocation assays. The second aim is to determine how a reduction in Dunc13 activity changes the behavioral and physiological responses to ethanol in Drosophila melanogaster. This invertebrate model system was chosen for its ability to rapidly and economically alter Dunc13 levels and to monitor the functional consequences. These consequences of the ethanol-Dunc13 interaction will be revealed by measuring ethanol preference, stimulation, and sedation in wild type and Dunc13 reduction of function animals. Moreover, the effect of the interaction will be examined using the synapto-pHluorin sensor to image vesicle release from a specific subset of GABAergic neurons in intoxicated and sober flies. These GABAergic neurons are critical modulators of the stimulatory and sedative effects of ethanol in Drosophila. The approach is innovative as it applies the strengths of ultrasensitive biochemistry and powerful neurogenetic techniques for the first time to dissect the function of an ethanol-receptor interaction. This proposal is significant as it is expected to uncover a novel primary mechanism for an effect of ethanol on presynaptic function. Ultimately, this understanding may lead to new drugs designed to disrupt the ethanol-unc13.1 interaction providing a valuable weapon in the fight for sobriety.