Acetylcholine Receptor Biogenesis, Structure, Function

本项目的长期目标仍然是阐明烟碱的结构和功能， 乙酰胆碱受体（nAChRs）。识别功能重要的神经元nAChR的经典方法 中枢神经系统（CNS）中的亚型由于有限数量的选择性 药理学试剂。本项目将使用基于结构的信息， 新的研究工具，以调查nAChR亚单位在神经系统中的特定功能。 第一个目标集中在“敲入”小鼠Chrna 3 tm 1（Hwrt）的表征上，该小鼠通过以下方式产生： 同源重组介导的靶向基因置换。有针对性的DMA编码五种肌肉类型 α 1衍生的氨基酸取代赋予受体对纳摩尔α-银环蛇毒素的功能敏感性 （Bgtx），甚至在2个受体α 3亚基中只有1个是突变的那些情况下。杂合小鼠 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 表型上与杂合受体的随机表达一致， 来自Met小鼠的交感神经元可以被Bgtx阻断。生化和电生理方法 将用于全面评估该突变在杂合子小鼠回交中的功能后果 C57 BI/6/J的背景。突变α 3亚基在CNS中的表达将通过以下方法研究： 荧光、放射自显影和将Bgtx微量注射到富含α 3的离散脑区域中。在 第二个目标是制备Bgtx敏感的P2、β 3、β 4和α 5亚基，并对其进行电化学表征。 在卵母细胞和腺病毒转染的神经元中异源表达后，在生理学上可以产生这种效应。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，在所有可预防的死亡中， 西方世界它还能显著增强认知能力， 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 没有有效的治疗。因此，了解烟碱受体在中枢神经系统中的功能作用， 对人类健康有益的巨大潜力。此外，这项研究的结果可能会导致 开发治疗药物，重现尼古丁的某些有益作用。

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 DMAencoding 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.