Characterization of a novel presynaptic target for ethanol action

乙醇作用的新型突触前靶点的表征

• 批准号: 8328677
• 负责人: Joydip Das
• 金额: $ 17.81万
• 依托单位: UNIVERSITY OF HOUSTON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-05 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8328677
• 关键词: 1,2-diacylglycerol; Affect; Alcohol abuse; Alcohol dependence; Alcoholism; Alcohols; Anesthetics; Animal Model; Animals; Behavioral; Binding; Binding Sites; Biochemical; Biochemistry; Biological Assay; Biological Models; Blood - brain barrier anatomy; Brain; Caenorhabditis elegans; Cell membrane; DAG/PE-Binding Domain; Data; Dependence; Development; Diglycerides; Distant; Drosophila genus; Drosophila melanogaster; Drug Design; Economic Burden; Ethanol; Family; Genetic Models; Goals; Homologous Protein; Image; In Vitro; Individual; Invertebrates; Lead; Mass Spectrum Analysis; Measures; Membrane; Molecular; Molecular Target; Monitor; Motor Activity; Nervous System Physiology; Nervous system structure; Neuraxis; Neurons; PHluorin; Pharmaceutical Preparations; Pharmacotherapy; Phosphotransferases; Physiological; Play; Population; Process; Protein Family; Protein Kinase; Protein Kinase C; Proteins; Role; Sedation procedure; Signal Pathway; Site; Societies; Stimulus; Synapses; Synaptic Transmission; Synaptic Vesicles; Techniques; Testing; Therapeutic Effect; Time; United States; Vesicle; Work; alcohol effect; alcohol response; alcohol-related death; base; combat; cost; drug discovery; effective intervention; fighting; fly; in vivo; innovation; neurogenetics; neurotransmitter release; novel; novel therapeutics; preference; presynaptic; receptor; recidivism; relating to nervous system; sedative; sensor; simulation; sobriety; weapons

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.

描述（由申请人提供）：酗酒和酒精中毒会影响美国4.5％的人口，造成约1840亿美元的经济负担。开发新的药物治疗的能力，以帮助将血统与酒精依赖性和累犯作斗争，这需要了解神经系统上酒精作用的机制。定义乙醇结合的靶标尤其重要，因为这些靶标可能会带来最完整的治疗作用。尽管众所周知，酒精对突触前功能具有明显的深远影响，但这种巨大影响的基本上是未知的。该建议的长期目标是定义酒精对突触前功能作用的分子机制。该探索性应用的目的是检查Munc13.1蛋白与乙醇之间的生理和行为相互作用。 MUNC13.1是突触前活性区蛋白，对于脑中神经递质释放必不可少的。在秀丽隐杆线虫中，同源的UNC13蛋白是对挥发性麻醉剂的敏感性。 Munc13蛋白质家族的C1或二酰基甘油结合结构域在结构上与蛋白激酶C（PKC）的结构相似，该蛋白激酶C（PKC）调节酒精的行为效应并具有酒精结合位点（S）。初步数据表明，乙醇也将在Munc13.1中与该C1结构域结合。要测试的中心假设是乙醇对神经系统功能的显着影响是由于乙醇与Munc13.1 C1结构域的结合所致。该提案的第一个目的将研究以下假设：乙醇与Munc13.1的C1结构域结合修饰了这种突触前蛋白的活性。这将通过光标记和质谱法来实现，以鉴定酒精结合残基，并阐明这种结合对膜易位测定中MUNC13.1活性的影响。第二个目的是确定DUNC13活性的降低如何改变果蝇中对乙醇的行为和生理反应。选择该无脊椎动物模型系统的能力快速和经济改变DUNC13水平并监测功能后果。乙醇-DUNC13相互作用的这些后果将通过测量野生型乙醇偏好，刺激和镇静作用以及功能动物的DUNC13降低来揭示。此外，将使用Synapto-Phluorin传感器检查相互作用的效果，以从醉酒和清醒蝇中的GABA能神经元的特定子集中对囊泡释放。这些GABA能神经元是乙醇对果蝇的刺激和镇静作用的关键调节剂。该方法是创新的，因为它首次运用了超敏生物化学和强大的神经遗传技术的优势，以剖析乙醇受体相互作用的功能。该建议很重要，因为预计将发现乙醇对突触前功能的影响的新型主要机制。最终，这种理解可能导致新药旨在破坏乙醇 - unc13.1相互作用，从而为抗清醒提供了宝贵的武器。