Single molecule determination of nAChR structural assembly for therapeutic targeting

 描述（由申请人提供）：烟草制品的消费导致几种健康风险的升高。吸烟者患心血管疾病、呼吸系统疾病和几种癌症的可能性更高。在美国，大约20%的死亡与烟草产品有关。尽管有明显的危害，但据估计，美国有4000多万成年人吸烟。减少吸烟人数的主要障碍之一是烟草的一种成分尼古丁具有高度成瘾性。我们提出了一种新的方法来阐明尼古丁成瘾的机制，并确定尼古丁戒断治疗的目标nAChR组件和表型。在R21阶段，我们将专注于开发一种新的方法来监测尼古丁受体在单分子水平上的组装和运输。烟碱 受体不能在细胞环境外纯化，使得它们不能用需要纯化和重建蛋白质的普通单分子技术来纯化。我们将利用在细胞衍生囊泡中分离的受体。从不同细胞器中分离囊泡将使我们能够确定尼古丁和治疗配体影响nAChRs的组装和运输。鉴定现有疗法靶向的特定结构组装将使我们能够鉴定戒烟疗法的特定靶点。在R33阶段，我们将通过从烟碱受体GFP敲除小鼠的大脑不同区域分离囊泡来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 活体动物产生的烟碱受体的分子研究。我们就能把 将不同的受体亚型组装到特定的大脑区域。这些研究将首先 时间允许我们对脑衍生的nAChR进行单分子荧光研究。了解现有的治疗方法如何改变特定脑区的结构组装，将有助于了解靶向哪些nAChR以及它们应该诱导的结构组装。这将提供深入了解尼古丁成瘾的机制和戒烟治疗最佳诱导的受体表型。

 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.