Acetylcholine Receptor Biogenesis, Structure, Function

描述（由申请人提供）：本项目的长期目标仍然是阐明烟碱乙酰胆碱受体（nAChR）的结构和功能。经典的方法来辨别功能显着的神经元nAChR亚型在中枢神经系统（CNS）已受挫的选择性药物的数量有限。基于结构的信息将用于该项目，以推动新的研究工具的开发，以研究神经系统中nAChR亚基的特异性功能。第一个目标重点关注通过同源重组介导的靶向基因替换创建的“敲入”小鼠Chrna 3 tm 1（Hwrt）的表征。编码5个肌肉型α 1衍生的氨基酸取代的靶向DNA赋予受体对纳摩尔α-银环蛇毒素（Bgtx）的功能敏感性，即使在2个受体α 3亚基中只有1个是突变体的那些情况下。杂合子小鼠表达含有一个突变体和一个野生型α 3亚基的杂合nAChR，但在其他方面是正常的表型。与杂合受体的随机表达一致，来自Met小鼠的交感神经元中约2/3的烟碱反应可被Bgtx阻断。将使用生物化学和电生理学方法全面评估该突变在回交到C57 BI/6/J背景的杂合小鼠中的功能后果。CNS中突变α 3亚基的表达将通过荧光、放射自显影和将Bgtx显微注射到富含α 3的离散脑区域中来研究。在第二个目的中，在卵母细胞和腺病毒转染的神经元中异源表达后，将制备Bgtx敏感性β 2、β 3、β 4和α 5亚基并对其进行电生理学表征。这些结果将确定在这4个亚基中产生Bgtx敏感性敲入小鼠的未来可行性。相关性：尼古丁是一种极易上瘾的药物，在西方世界，可预防的死亡率高达20%。它还显著增强认知能力，并且一些遗传形式的癫痫涉及烟碱受体。胆碱能神经元的丧失与阿尔茨海默病有关，阿尔茨海默病是一种没有有效治疗的疾病。因此，了解烟碱受体在中枢神经系统中的功能作用对人类健康具有重要的潜在益处。此外，这项研究的结果可能会导致治疗药物的开发，重现尼古丁的一些有益作用。

DESCRIPTION (provided by applicant): The long term goal in this project remains the elucidation of the structure and function of nicotinic acetylcholine receptors (nAChRs). Classical approaches to discern functionally significant neuronal nAChR subtypes in the central nervous system (CNS) have been frustrated by the limited number of selective pharmacological agents. Structure-based information will be used in this project to drive the development of new research tools to investigate nAChR subunit-specific functionality in the nervous system. The first aim focuses on the characterization of a "Knock-In" mouse, Chrna3tm1(Hwrt), created through homologous recombination-mediated targeted gene replacement. Targeted DNA encoding five muscle-type a1-derived amino acid substitutions confers functional sensitivity of receptors to nanomolar a-bungarotoxin (Bgtx) even in those cases where only 1 of the 2 receptor a3 subunits is mutant. Heterozygous mice express hybrid nAChRs containing one mutant and one wild-type a3 subunit, but are otherwise normal phenotypically. Consistent with a stochastic expression of hybrid receptors, -2/3 of the nicotinic response in sympathetic neurons from Met mice can be blocked by Bgtx. Biochemical and electrophysiological methods will be used to fully assess the functional consequences of this mutation in heterozygous mice backcrossed into the C57BI/6/J background. The expression of the mutant a3 subunit in the CNS will be investigated by fluorescence, autoradiography, and micro-injection of Bgtx into discrete brain regions rich in a3. In the second aim, Bgtx-sensitive P2, (33, 04 and a5 subunits will be prepared and characterized electro- physiologically following heterologous expression in oocytes and in adenovirus-transfected neurons. These results will determine the future feasibility of generating Bgtx-sensitive knock-in mice in these 4 subunits. Relevance: Nicotine is an extremely addictive drug responsible for up to 20% of all preventable mortality in the western world. It also significantly enhances cognitive performance, and some inherited forms of epilepsy involve nicotinic receptors. Loss of cholinergic neurons is implicated in Alzheimer's disease, a disorder with no effective treatment. Understanding the functional role of nicotinic receptors in the CNS therefore has significant potential to benefit human health. In addition, the results from this study could lead to the development of therapeutic drugs reproducing some of the beneficial effects of nicotine.