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

描述（由申请人提供）：酒精对突触前功能有普遍的影响，但这些影响背后的机制在很大程度上是未知的。这些突触前机制很可能对酒精依赖的发展起着重要作用。确定负责完善乙醇突触前影响的分子参与者对于开发对抗成瘾和再犯的新靶点是必要的。确定乙醇结合的靶点尤为重要，因为这可能带来最彻底的效果。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，对大脑中神经递质释放至关重要。初步数据表明，乙醇与Munc13-1的C1二酰基甘油结合域结合。果蝇un13同源基因Dunc13的减少会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。要验证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 C1结构域的结合。这一假设将在4个目标中得到验证。在Aim 1中，将确定与Munc13-1的C1结构域结合的乙醇的原子结构。在Aim 2中，我们将在体外测量乙醇对Unc13囊泡融合的影响。在Aim 3中，野生型Munc13-1和具有降低乙醇亲和力突变的Munc13-1将用于功能性补充Dunc13单倍不足的行为表型。此外，乙醇- munc13 -1相互作用的影响将通过突触-氟尿嘧啶传感器来检测醉酒和清醒果蝇的突触囊泡释放。这些实验将确定乙醇与Munc13-1结合如何在体内改变这种蛋白质的活性。在Aim 4中，我们确定杂合子Munc13-1KO/+小鼠是否与果蝇Dunc13突变体相似，在乙醇敏感性和自我给药方面存在缺陷。该方法具有创新性，因为它在单个项目中应用了原子水平分辨率、体外生物化学和体内生物化学的优势

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.