Acetylcholine Receptor Biogenesis, Structure, Function

描述(申请人提供)：该项目的长期目标仍然是阐明烟碱型乙酰胆碱受体(NAChRs)的结构和功能。在中枢神经系统(CNS)中识别具有重要功能的神经元nAChR亚型的经典方法因选择性药理药物的数量有限而受挫。基于结构的信息将在这个项目中用于推动新研究工具的开发，以研究神经系统中nAChR亚单位的特定功能。第一个目标是研究通过同源重组介导的靶向基因替换而产生的“敲入”小鼠--chrna3tm1(HWRT)。编码5个肌肉A1型衍生氨基酸替换的靶向DNA赋予受体对纳米α-银环蛇毒素(BGTX)的功能敏感性，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。杂合子小鼠表达包含一个突变型和一个野生型A3亚基的杂交nAChRs，但其他表型正常。与杂合受体的随机表达一致，BGTX可以阻断蛋氨酸小鼠交感神经元-2/3的烟碱反应。生化和电生理学方法将被用来全面评估这种突变在杂合子小鼠中的功能后果，这些小鼠回交到C57BI/6/J背景中。突变的A3亚单位在中枢神经系统中的表达将通过荧光、放射自显影和向富含A3的离散脑区微量注射BGTX来研究。在第二个目标中，我们将制备BGTX敏感的P2，(33，04和a5)亚基，并在卵母细胞和腺病毒感染的神经元中进行异源表达后的电生理特性研究。这些结果将决定未来在这4个亚基中产生BGTX敏感敲入鼠的可行性。相关性：尼古丁是一种极易上瘾的毒品，占西方世界所有可预防的死亡人数的20%。它还显著提高认知能力，一些遗传性癫痫涉及尼古丁受体。胆碱能神经元的丧失与阿尔茨海默病有关，这是一种没有有效治疗方法的疾病。因此，了解尼古丁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意义。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。

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.