Immunochemical Protocols for Nicotinic Receptors

描述（由申请人提供）：尼古丁是烟草的主要生理活性成分，负责建立和维持慢性烟草使用。尼古丁通过与多种烟碱乙酰胆碱受体（nAChR）相互作用发挥其作用，这些受体由同源亚基的五聚体组装体组成。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。由于它们在介导尼古丁效应中的核心作用，了解哪些烟碱亚型表达，它们的分布及其特征对于理解烟草使用的开始和维持至关重要。指导所提出的研究的中心假设是，建立的技术探测nAChR，而强大的，固有的限制，他们不能积极隔离和识别nAChR亚型的基础上，其组成亚基。免疫化学技术解决了这一限制，使他们有可能大大扩展我们的知识nAChR的组成和性质。提出了两个具体目标：1。开发神经元nAChR的免疫沉淀/免疫捕获方案。2.建立神经元nAChR的蛋白质印迹技术。我们已经组装了大量的抗体储备，针对哺乳动物神经元nAChR亚基表达的全方位。将评估和优化每种抗体储备液在免疫沉淀/免疫捕获和Western印迹技术中的性能。使用抗体有2个主要困难：它们可能无法识别靶标（由于亲和力差或不利的实验条件），或可能发生交叉反应。nAChR亚基缺失突变动物将用作阴性对照，以评估这两个问题的严重程度。由于无效突变体组织应表达与野生型组织中发现的抗原基本相同的抗原补体，除了靶向基因产物之外，我们预期无效突变体将在此能力方面表现出色。首先将追求目标1，因为所建立的每个亚基特异性方案可立即用于分离和分析天然nAChR的组成。这种方法已经产生了新的数据（见初步结果），预计这种方法将获得进一步的快速科学回报。[125]我们实验室开发的Epibatidine结合技术将用于监测nAChR亚型的分离，并确定捕获的药理学。目标2的工作将在第二年开始，因为这些实验很可能需要比目标1中所涵盖的更深入的优化，并且在任何情况下，免疫印迹技术将通过免疫沉淀纯化的nAChR来源的可用性来促进。

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.