Role of Munc13-1 as a presynaptic effector of ethanol action

Munc13-1 作为乙醇作用突触前效应器的作用

• 批准号: 9223617
• 负责人: Joydip Das
• 金额: $ 33.14万
• 依托单位: UNIVERSITY OF HOUSTON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-20 至 2020-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9223617
• 关键词: Affect; Affinity; Alcohol abuse; Alcohol consumption; Alcohol dependence; Alcoholic Intoxication; Alcoholism; Alcohols; Behavior; Behavioral; Binding; Binding Proteins; Binding Sites; Biochemical; Biochemistry; Biological Models; Blood - brain barrier anatomy; Brain; Cell membrane; Complement; Crystallization; DAG/PE-Binding Domain; Data; Defect; Dependence; Development; Diglycerides; Drosophila genus; Drosophila melanogaster; Drug Targeting; Ethanol; Family; Genetic Models; Goals; Homologous Gene; Image; In Vitro; Individual; Intoxication; Knockout Mice; Measures; Membrane; Membrane Lipids; Molecular; Molecular Target; Mus; Mutation; Nervous System Physiology; Nervous system structure; Neurons; PHluorin; Participant; Pharmaceutical Preparations; Pharmacotherapy; Phenotype; Phosphotransferases; Physiology; Play; Process; Property; Protein Kinase; Proteins; Rattus; Resistance; Resolution; Role; Self Administration; Signal Pathway; Site; Societies; Stimulus; Structure; Synapses; Synaptic Transmission; Synaptic Vesicles; Testing; Validation; Vesicle; Work; X-Ray Crystallography; addiction; alcohol behavior; alcohol effect; alcohol response; alcohol sensitivity; alcohol-related death; atomic interactions; base; behavioral response; combat; cost; design; drug development; drug discovery; effective intervention; experimental study; fighting; fly; in vivo; innovation; mutant; neurotransmitter release; new therapeutic target; novel; novel therapeutics; presynaptic; protein complex; public health relevance; recidivism; relating to nervous system; sensor; small molecule inhibitor; sobriety; vesicular release

DESCRIPTION (provided by applicant): Alcohol has pervasive impacts on presynaptic functions, yet the mechanisms underlying these considerable impacts are largely unknown. It is likely that these presynaptic mechanisms contribute significantly to the development of alcohol dependence. Identifying the molecular participants responsible for consummating ethanol's presynaptic impact is necessary to develop new targets to fight addiction and recidivism. It is particularly important to define the target of ethanol binding as this may bring about the most complete effect. The long-term goal of this application is to unfold the presynaptic mechanisms for ethanol action. The objective of this proposal is to describe the interaction between ethanol and Munc13-1 and define the effects of this interaction in presynaptic physiology and behavior. Munc13-1 is a conserved presynaptic active zone protein essential for neurotransmitter release in the brain. Preliminary data demonstrate that ethanol binds to the C1 diacylglycerol-binding domain of Munc13-1. A reduction in Dunc13, the Drosophila Unc13 homolog results in flies that are resistant to ethanol and have defects in tolerance and self-administration. 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 C1 domain. This hypothesis will be tested in 4 aims. In Aim 1, the atomic structure of ethanol bound to the C1 domain of Munc13-1 will be determined. In Aim 2, the effect of ethanol on Unc13 vesicle fusion will be measured in vitro. In Aim 3, wild type Munc13-1 and Munc13-1 with mutations that reduced ethanol affinity will be used to functionally complement the Dunc13 haploinsufficient behavioral phenotypes. Moreover, the effect of the ethanol-Munc13-1 interaction will be examined using the synapto- pHluorin sensor to image synaptic vesicle release in intoxicated and sober flies. These experiments will determine how ethanol binding to Munc13-1 alters the activity of this protein in vivo. In Aim 4, we determine if heterozygous Munc13-1KO/+ mice, similar to the Drosophila Dunc13 mutants, have defects in ethanol sensitivity and self-administration. The approach is innovative as it applies in a single project the strengths of atomic level resolution, in vitro biochemistry, and in vivo behavior and physiology, to understand the function of an ethanol-effector interaction. This proposal is significant as it will likely provide unequivocal information on the importance of a general mechanism by which ethanol impacts presynaptic function, and how this mechanism is critical in the process required for alcohol dependence. Ultimately, these results will provide precise structural information on where alcohol binds Munc13-1, and how this interaction alters Munc13-1 activity, how this interaction impacts ethanol sensitivity and self-administration. The results will enable the design and validation of small molecule inhibitors that could be used in the development of drugs to fight dependence and recidivism.

描述（由申请人提供）：酒精对突触前功能有广泛的影响，但这些重大影响的机制在很大程度上是未知的。很可能这些突触前机制对酒精依赖的发展有重要作用。确定负责完成乙醇的突触前影响的分子参与者是必要的，以开发新的目标，以打击成瘾和累犯。确定乙醇结合的靶点尤其重要，因为这可能带来最完整的效果。这项应用的长期目标是揭示乙醇作用的突触前机制。本提案的目的是描述乙醇和Munc 13 -1之间的相互作用，并定义这种相互作用在突触前生理和行为中的影响。Munc 13 -1是一种保守的突触前活性区蛋白，对脑中神经递质的释放至关重要。初步数据表明，乙醇结合C1二酰基甘油结合域的Munc 13 -1。Dunc 13（果蝇Unc 13同源物）的减少导致果蝇对乙醇产生抗性，并在耐受性和自我给药方面存在缺陷。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Munc 13 C1结构域的结合。这一假设将在4个目标中进行检验。在目标1中，将确定与Munc 13 -1的C1结构域结合的乙醇的原子结构。在目的2中，将在体外测量乙醇对Unc 13囊泡融合的影响。在目标3中，野生型Munc 13 -1和具有降低乙醇亲和力的突变的Munc 13 -1将用于功能性地补充Dunc 13单倍不足行为表型。此外，乙醇-Munc 13 -1相互作用的影响将使用突触- pHluorin传感器来检查，以在中毒和清醒的果蝇中对突触囊泡释放进行成像。这些实验将确定乙醇与Munc 13 -1的结合如何改变该蛋白在体内的活性。在目标4中，我们确定是否杂合子Munc 13 - 1 KO/+小鼠，类似于果蝇Dunc 13突变体，具有乙醇敏感性和自我给药的缺陷。该方法是创新的，因为它适用于一个单一的项目，原子水平的分辨率，体外生物化学， 体内行为和生理学，以了解乙醇效应相互作用的功能。这一提议意义重大，因为它可能会提供明确的信息，说明乙醇影响突触前功能的一般机制的重要性，以及这种机制在酒精依赖所需的过程中如何至关重要。最终，这些结果将提供关于酒精结合Munc 13 -1的精确结构信息，以及这种相互作用如何改变Munc 13 -1活性，这种相互作用如何影响乙醇敏感性和自我给药。这些结果将使小分子抑制剂的设计和验证成为可能，这些抑制剂可用于开发药物以对抗依赖和累犯。