Single molecule determination of nAChR structural assembly for therapeutic targeting

 描述(申请人提供)：烟草产品的消费导致几种健康风险的增加。吸烟者患心血管疾病、呼吸系统疾病和多种癌症的可能性更高。因此，美国大约20%的死亡与烟草产品有关。尽管存在明显的危害，但据估计，美国有超过4000万成年人是烟民。减少吸烟者数量的主要障碍之一是，尼古丁是烟草的一种成分，具有很强的成瘾性。我们提出了一种新的方法来阐明尼古丁成瘾的机制，并确定尼古丁戒断疗法的靶向nAChR组件和表型。在R21阶段，我们将重点开发一种新的方法，在单分子水平上监测尼古丁受体的组装和运输。尼古丁 受体不能在细胞环境之外进行纯化，这使得普通的单分子技术无法获得它们，这些技术需要蛋白质的纯化和重组。我们将利用分离在细胞来源的囊泡中的受体。分离不同细胞器中的囊泡将使我们能够确定尼古丁和治疗性配体影响nAChRs的组装和运输。识别现有疗法靶向的特定结构组件将使我们能够识别戒断疗法的特定靶点。在R33阶段，我们将通过分离尼古丁受体GFP敲击小鼠大脑不同区域的小泡，将研究扩展到在活体动物中产生的受体。这将是第一首单曲 活体动物产生的尼古丁受体的分子研究。我们将能够将 将不同的受体亚型组装到特定的大脑区域。这些研究将是第一次 时间允许我们对大脑来源的nAChRs进行单分子荧光研究。了解现有疗法是如何改变特定大脑区域的结构组装的，将提供对哪些nAChR为靶点以及它们应该诱导的结构组装的理解。这将为深入了解尼古丁成瘾的机制和戒烟疗法最佳诱导的受体表型提供依据。

 DESCRIPTION (provided by applicant): The consumption of tobacco products leads to the elevation of several health risks. Smokers have a higher likelihood of developing cardiovascular disease, respiratory disease, and several types of cancer. As a result, approximately 20% of all deaths in the United States are associated with tobacco products. Despite the clear hazards, it is estimated that more than 40 million adults in the United States are smokers. One of the primary barriers to decreasing the number of smokers is that nicotine, a component of tobacco, is highly addictive. We propose a novel approach to elucidate the mechanism of nicotine addiction and to identify target nAChR assemblies and phenotypes for nicotine cessation therapeutics. In the R21 phase, we will focus on the development of a novel approach to monitor the assembly and trafficking of nicotinic receptors at the single molecule level. Nicotinic receptors cannot be purified outside of the cellular environment making them inaccessible to common single molecule techniques that require the purification and reconstitution of proteins. We will utilize receptors isolated in cell derived vesicles. Isolation of vesicles from different organelles will allow us to determine nicotine and therapeutic ligands affect the assembly and trafficking of nAChRs. Identification of the specific structural assemblies targeted by existing therapeutics will allow us to identify specific targets for cessation therapeutics. In the R33 phase, we will extend studies to receptors generated in live animals by isolating vesicles from different regions of the brain of nicotinic receptor GFP knock-mice. These will be the first single molecule studies of nicotinic receptors generated in live animals. We will be able to correlate the assembly of different receptor subtypes to specific brain regions. These studies will for the first time allow us to perform single molecule fluorescence studies on brain derived nAChRs. Understanding how existing therapeutics alter structural assembly in specific brain regions will provide an understanding of which nAChRs to target and the structural assembly they should induce. This will provide insight into the mechanism of nicotine addiction and on the receptor phenotypes optimally induced by cessation therapeutics.