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

基本信息

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
项目类别：
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

项目摘要

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）：酒精对突触前功能产生了普遍的影响，但是这些可观影响的机制在很大程度上尚不清楚。这些突触前机制可能对酒精依赖的发展产生了重大贡献。确定负责完成乙醇突触前影响的分子参与者对于发展成瘾和累犯的新目标是必要的。定义乙醇结合的靶标特别重要，因为这可能会带来最完整的效果。该应用的长期目标是展现乙醇作用的突触前机制。该建议的目的是描述乙醇与Munc13-1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。 MUNC13-1是一种保守的突触前活性区蛋白，对于大脑中的神经递质释放必不可少。初步数据表明，乙醇与Munc13-1的C1二酰基甘油结合结构域结合。 Dunc13的降低，果蝇UNC13同源物导致对乙醇具有抗性并具有耐受性和自我给药的缺陷。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 C1结构域的结合所致。该假设将以4个目标进行检验。在AIM 1中，将确定与Munc13-1 C1结构域结合的乙醇的原子结构。在AIM 2中，将在体外测量乙醇对UNC13囊泡融合的影响。在AIM 3中，具有降低乙醇亲和力的突变的野生型Munc13-1和Munc13-1将用于功能补充DUNC13单倍弹性的行为表型。此外，将使用突触 - 磷脂传感器来检查乙醇-MUNC13-1相互作用的效果，以在陶醉和清醒的苍蝇中图像突触囊泡释放。这些实验将决定乙醇与MUNC13-1的结合如何改变该蛋白在体内的活性。在AIM 4中，我们确定杂合MUNC13-1KO/+小鼠（类似于果蝇Dunc13突变体）是否具有乙醇敏感性和自我给药的缺陷。这种方法具有创新性，因为它适用于一个项目，其原子水平分辨率，体外生物化学和中的优势体内行为和生理学，以了解乙醇效应器相互作用的功能。该提案很重要，因为它可能会提供有关乙醇影响突触前功能的一般机制重要性的明确信息，以及该机制在酒精依赖所需的过程中如何至关重要。最终，这些结果将提供有关酒精在何处结合Munc13-1的精确结构信息，以及这种相互作用如何改变Munc13-1的活性，这种相互作用如何影响乙醇的敏感性和自我给药。结果将使可以用于与依赖性和累犯的药物开发中的小分子抑制剂的设计和验证。