Construction and Expression of Concatemeric alpha6beta2* Nicotinic Acetycholine R

描述（申请人提供）：烟碱乙酰胆碱受体（nAChRs）是配体门控离子通道，发挥许多重要的生理作用。nachr自然以不同亚型的家族形式存在。每个nAChR亚型都是一个五聚体，由它的亚基组成来定义，在哺乳动物中是16种不同基因的产物。每个nAChR亚型都具有独特的分布和一组特征。脑内nAChR亚型多样性具有重要的生理意义，为神经精神疾病的靶向药物治疗提供了机会。本项目特别感兴趣的是，含有a6， b2, b3， a4亚基（a6*-nAChR）的天然nAChR亚型在中脑，多巴胺能，愉悦和奖励中心表达。这意味着选择性靶向a6*-nAChR的药物可用于治疗尼古丁或其他药物依赖，和/或帕金森病。然而，这些a6*-nAChR亚型的分离表征一直难以捉摸，部分原因是它们在其他nAChR亚型的背景下自然表达，但也因为在异源表达过程中难以精确控制亚基组成、化学计量和排列，这可能是由于缺乏伴侣引导的过程。我们试图克服这些障碍，实现a6*-nAChR的表征和使用系链五聚体亚基构建的异源表达。我们将开始测试一个假设，即在缺乏神经元特异性伴侣蛋白的情况下，由系留亚基构建的nAChR会强制正确的亚基组装，从而保留天然nAChR亚型的特性。具体目的是首先在非洲爪蟾卵母细胞中，然后在哺乳动物细胞宿主中异种表达和表征由确定的系链亚基组成的a6*-nAChR。这些亚基将通过系统地将a6和b3亚基引入已被证明编码全功能nAChR的结构中来创建。标准测定将用于确认从信息到功能性a6*-nAChR的构建表达，并表征功能性a6*-nAChR。根据R21项目，成功异源表达共价连接的a6b2b3-和a6a4b2b3- nAChR构建体将为使用系链五聚体方法表达其他复杂nAChR亚型提供概念证明。没有其他方法可以确保亚基组成，化学计量和排列，定义一个给定的nAChR亚型。a6*-nAChR作为系链五聚体的纯、丰富表达将能够明确地确定亚基对亚型功能的贡献，并测试药物在这些靶标上的亚型选择性。与公共卫生相关的是，这些进展可以扩展到改善从成瘾到运动障碍等疾病的治疗。公共卫生相关性：含有a6亚基的尼古丁乙酰胆碱受体（nAChRs）与从成瘾到帕金森病等疾病有关。由于稀缺性和其他nAChR亚型的污染，这些受体已被证明难以使用天然来源进行研究。人工表达含有a6的nAChRs将提供一种有价值的替代方法，但获得这些复杂受体的适当表达一直极具挑战性。我们提出了一种使用连接受体亚基的方法，使含有a6的nachr具有与其自然表达的对应物相匹配的特性，作为通常单个亚基关联的替代方法。这将加强所需的亚基相互作用，促进在各种实验可适应的系统中的表达。反过来，这将大大简化对这些重要受体的研究，增强对其特性的了解，并有助于指导nAChR导向成瘾和帕金森病治疗的发展。

DESCRIPTION (provided by applicant): Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that play many important physiological roles. nAChRs naturally exist as a family of diverse subtypes. Each nAChR subtype is a pentamer and is defined by its composition of subunits, which in mammals are products of sixteen different genes. Each nAChR subtype has a distinctive distribution and set of characteristics. nAChR subtype diversity in the brain is physiologically important and provides opportunities for target-based drug treatment of neuropsychiatric conditions. Of particular interest in this project, native nAChR subtypes containing a6, b2, b3 a4 subunits (a6*-nAChR) are expressed in midbrain, dopaminergic, pleasure and reward centers. This means that drugs selectively targeting a6*-nAChR could be used to treat nicotine or other drug dependence, and/or Parkinson's disease. However, characterization of these a6*-nAChR subtypes in isolation has been elusive, in part because they are naturally expressed over a background of other nAChR subtypes, but also because of difficulties during heterologous expression in precisely controlling subunit composition, stoichiometry, and arrangement, perhaps due to the absence of chaperone-guided processes. We seek to overcome these obstacles toward a6*-nAChR characterization and heterologous expression using tethered pentameric subunit constructs. We will begin to test the hypothesis that nAChR construction from tethered subunits enforces correct subunit assembly in the absence of neuron-specific chaperone proteins, preserving properties of native nAChR subtypes. The specific aims are to heterologously express and characterize, first in Xenopus oocytes and then in a mammalian cell host, a6*-nAChR composed of defined, tethered subunits. These subunits will be created by systematically introducing a6 and b3 subunits into a construct proven to encode fully functional nAChR. Standard assays will be used to confirm construct expression from message to functional a6*-nAChR and to characterize functional a6*-nAChR. As befits an R21 project, successful heterologous expression of covalently-linked a6b2b3- and a6a4b2b3- nAChR constructs will provide proof of concept for expression of other, complex nAChR subtypes using the tethered pentamer approach. No other approach can ensure the subunit composition, stoichiometry and arrangement that define a given nAChR subtype. Pure, plentiful expression of a6*-nAChR as tethered pentamers would enable unambiguous determination of subunit contributions to subtype function, and tests of subtype selectivity of drugs at these targets. Of public health relevance, these advances could extend to improved treatment of diseases ranging from addictions to movement disorders. PUBLIC HEALTH RELEVANCE: Nicotinic acetylcholine receptors (nAChRs) containing the a6 subunit are implicated in diseases ranging from addictions to Parkinson's disease. These receptors have proved difficult to study using natural sources, due to a combination of scarcity and contamination with other nAChR subtypes. Artificial expression of a6-containing nAChRs would provide a valuable alternative, but obtaining appropriate expression of these complex receptors has been extremely challenging. We propose an approach using linked receptor subunits to make a6- containing nAChRs with properties matching those of their naturally-expressed counterparts, as an alternative to the usual association of individual subunits. This will enforce desired subunit interactions, facilitating expression in a variety of experimentally amenable systems. In turn, this will greatly simplify studies of these important receptors, enhancing knowledge of their properties and helping to guide development of nAChR- directed treatments for addictions and Parkinson's disease.