Immunochemical Protocols for Nicotinic Receptors

说明(申请人提供)：尼古丁是烟草中的主要生理活性成分，负责建立和维持长期烟草使用。尼古丁通过与不同的烟碱型乙酰胆碱受体(NAChRs)相互作用发挥作用，nAChRs由同源亚基的五聚体组成。不同的亚基组合产生不同的nAChR亚型，具有不同的性质和分布。由于它们在调节尼古丁的作用中起着核心作用，了解哪些尼古丁亚型被表达、它们的分布和它们的特征对于理解烟草使用的开始和维持至关重要。指导这项研究的中心假设是，现有的探测nAChR的技术虽然功能强大，但由于无法根据其组成亚基积极分离和识别nAChR亚型，因此存在固有的局限性。免疫化学技术解决了这一局限性，使它们有可能极大地扩展我们对nAChR组成和性质的知识。提出了2个具体目标：1.建立神经元nAChRs的免疫沉淀/免疫捕获方案。2.建立神经元nAChRs的蛋白质印迹技术。我们已经收集了大量针对哺乳动物神经元nAChR亚单位表达的抗体。将评估和优化每个抗体库在免疫沉淀/免疫捕获和Western blotting技术中的性能。2主要困难与抗体的使用有关：它们可能无法识别目标(由于亲和力差或不利的实验条件)，或者可能发生交叉反应。NAChR亚单位缺失突变动物将被用作阴性对照，以评估这两个问题的严重性。由于零突变组织应该表达与野生型组织中发现的大致相同的抗原补充，除了靶基因产物外，我们预计零突变将在这方面表现出色。首先将实现目标1，因为所建立的每个特定亚基的方案可以立即用于分离和分析天然nAChRs的组成。这种方法已经产生了新的数据(见初步结果)，预计这种方法还会有更快的科学回报。[125L]我们实验室开发的Epibatidine结合技术将用于监测nAChR亚型的分离，并确定捕获的亚型的药理学。AIM 2的工作将于第二年开始，因为这些实验可能需要比AIM 1中涵盖的实验更密集的优化，而且无论如何，通过免疫沉淀纯化的nAChR来源的可用性将促进Western blotting技术。

DESCRIPTION (provided by applicant): Nicotine is the major physiologically active component of tobacco that is responsible for the establishment and maintenance of chronic tobacco use. Nicotine exerts its actions through interactions with a diverse population of nicotinic acetylcholine receptors (nAChRs), composed of pentameric assemblies of homologous subunits. Different subunit combinations produce different nAChR subtypes, with diverse properties and distributions. Because of their central role in mediating nicotine's effects, knowing which nicotinic subtypes are expressed, their distribution, and their characteristics is vitally important to understanding the initiation and maintenance of tobacco use. The central hypothesis directing the proposed studies is that established techniques for probing nAChRs, while powerful, are inherently limited by their inability to positively isolate and identify nAChR subtypes on the basis of their component subunits. Immunochemical techniques address this limitation, giving them the potential to greatly expand our knowledge of nAChR composition and properties. 2 specific aims are proposed: 1. Develop immunoprecipitation/immunocapture protocols for neuronal nAChRs. 2. Establish Western blotting techniques for neuronal nAChRs. We have assembled an extensive collection of antibody stocks, directed against the full range of mammalian neuronal nAChR subunits expressed. The performance of each antibody stock in immunoprecipitation/immunocapture and Western blotting techniques will be assessed and optimized. 2 major difficulties are associated with the use of antibodies: they may fail to recognize the target (due to poor affinity or unfavorable experimental conditions), or crossreactions may occur. nAChR subunit-null mutant animals will be used as negative controls to assess the severity of both of these problems. Since null-mutant tissue should express largely the same complement of antigens as found in wild-type tissue, with the exception of the targeted gene product, we anticipate that the null mutants will excel in this capacity. Aim 1 will be pursued first, since each subunit-specific protocol established may be used immediately to separate and analyze the composition of native nAChRs. This approach has already produced novel data (see Preliminary Results), and further quick scientific returns are anticipated for this approach. [125l]Epibatidine binding techniques developed in our laboratory will be used to monitor the separation of nAChR subtypes, and to determine the pharmacology of those captured. Work on Aim 2 will begin in the second year, since it is likely that these experiments will require more intensive optimization than those covered in Aim 1 and, in any case, Western blotting techniques will be facilitated by the availiblity of nAChR sources purified by immunoprecipitation.