Acetylcholine Receptor Biogenesis, Structure, Function

这个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)。识别具有重要功能的神经元nAChR的经典方法 中枢神经系统(CNS)中的亚型一直受到有限数量的选择性 药理制剂。本项目将使用基于结构的信息来推动 新的研究工具，以研究神经系统中nAChR亚单位的特定功能。 第一个目标是描述一只“敲入”小鼠的特征，通过 同源重组介导的靶向基因置换。编码五种肌型的靶向DNA A1衍生氨基酸取代使受体对纳米级银环蛇毒素具有功能性敏感性 (BGTX)，即使在2个受体A3亚基中只有1个是突变的情况下也是如此。杂合子小鼠 含有一个突变体和一个野生型A3亚基的表达杂交nAChRs，但其他情况下是正常的 典型的。与杂交受体的随机表达一致，-2/3的尼古丁反应 BGTX可阻断Met小鼠的交感神经元。生化和电生理方法 将被用来全面评估这种突变在回交杂合小鼠中的功能后果 进入C57BI/6/J背景。突变的A3亚基在中枢神经系统中的表达将通过 荧光、放射自显影和将BGTX微量注射到富含A3的离散脑区。在 第二个目标，BGTX敏感的P2，(33，04和a5亚基将被制备和电学表征) 生理学跟踪异源表达在卵母细胞和腺病毒感染的神经元中。这些 结果将决定未来在这4个亚基中产生BGTX敏感敲入小鼠的可行性。 相关性：尼古丁是一种极易上瘾的药物，占所有可预防死亡的20%。 西方世界。它还显著提高认知能力，以及某些遗传性癫痫 涉及尼古丁受体。胆碱能神经元的丢失与阿尔茨海默病有关，阿尔茨海默病是一种具有 没有有效的治疗方法。因此，了解尼古丁受体在中枢神经系统中的功能作用 造福人类健康的巨大潜力。此外，这项研究的结果可能会导致 重现尼古丁的一些有益作用的治疗药物的开发。

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.