Tools for inside-out pharmacology: nicotinic agents

描述(申请人提供)：长期接触尼古丁是一种交叉现象，导致尼古丁依赖以及无意中的治疗效果，如预防帕金森氏症。该项目测试了这一新的建议，即慢性尼古丁暴露的影响依赖于细胞内的尼古丁，而不是通过质膜受体的传统信号转导。众所周知，尼古丁被动地进入细胞，最近的研究表明，尼古丁也进入内质网(ER)等细胞器。在内质网中，尼古丁可能在药理上伴随新生的烟碱型乙酰胆碱受体(NAChRs)，随着五聚体受体的组装而“撮合”亚单位。其他研究表明，细胞内尼古丁-nAChR相互作用的其他潜在后遗症：降低未折叠的蛋白质反应，从内质网“护送”其他蛋白质，或“绑架”蛋白质到异常途径。所提出的机制是“由内向外”，因为它始于内质网而不是质膜。尼古丁持续的由内向外效应在远低于其对质膜nAChR通道的瞬时激活的浓度下进行。该项目将发明新的技术来测量和控制“由内而外”的尼古丁药理学的初始步骤。我们将开发用于测量药物结合的NanoSIMS，我们将开发分隔化的烟碱配体。我们还将采用其他最先进的技术：重建COPII囊泡发芽和FRET。次级方法A发明了测量尼古丁活动区隔的工具。次级方法B发明了将药理学限制在内质网的工具，既针对尼古丁，也针对临床上重要的α4β2 nAChR选择性配体varenicline。子方法C测试nAChRs与先前实验发现的“候选基因”之间的相互作用。由内而外的药理学是一个变革性的概念，它可能揭示成瘾和神经退化的新治疗靶点。

DESCRIPTION (provided by applicant): Chronic exposure to nicotine is a crosscutting phenomenon, leading to nicotine dependence as well as to inadvertent therapeutic effects such as protection against Parkinson's disease. The project tests the novel suggestion that effects of chronic nicotine exposure depend on intracellular nicotine-not on conventional signal transduction via receptors at the plasma membrane. It is known that nicotine passively enters cells and recent studies suggest that nicotine also enters organelles such as endoplasmic reticulum (ER). In ER, nicotine may pharmacologically chaperone nascent nicotinic acetylcholine receptors (nAChRs), "matchmaking" subunits as pentameric receptors assemble. Other studies suggest additional potential sequelae of intracellular nicotine-nAChR interactions: decreasing unfolded protein responses, "escorting" other proteins from ER, or "abducting" proteins to abnormal pathways. The proposed mechanism is "inside-out", because it begins in the ER rather than on the plasma membrane. Nicotine's sustained inside-out effects proceed at concentrations much lower than its transient activation of plasma membrane nAChR channels. The project will invent new techniques to measure and control the initial steps in "inside-out" nicotinic pharmacology. We will develop NanoSIMS for measuring drug binding, and we will develop compartmentalized nicotinic ligands. We will also employ other state-of-the-art techniques: reconstitution of COPII vesicle budding, and FRET. Sub-Approach A invents tools measuring the compartmentalization of nicotine action. Sub-Approach B invents tools for confining pharmacology to the ER, both for nicotine and for the clinically important alpha4beta2 nAChR-selective ligand, varenicline. Sub-Approach C tests for interactions between nAChRs and "candidate genes" discovered by previous experiments. Inside-out pharmacology is a transformative concept that may reveal new therapeutic targets for addiction and neurodegeneration.