Tools for inside-out pharmacology: nicotinic agents

描述（由申请人提供）：长期暴露于尼古丁是一种横切现象，既会导致尼古丁依赖，也会无意中产生治疗效果，如预防帕金森病。该项目测试了一个新的建议，即慢性尼古丁暴露的影响取决于细胞内的尼古丁，而不是通过质膜受体的传统信号转导。众所周知，尼古丁是被动进入细胞的，最近的研究表明尼古丁也会进入细胞器，如内质网（ER）。在内质网中，尼古丁可能在药理学上陪伴新生的尼古丁乙酰胆碱受体（nAChRs），“配对”亚基作为五聚体受体聚集。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：减少未折叠的蛋白质反应，从内质网“护送”其他蛋白质，或“诱变”蛋白质到异常途径。提出的机制是“由内而外”的，因为它始于内质网而不是质膜。尼古丁持续的由内而外的影响发生在浓度远低于其短暂激活质膜nAChR通道的情况下。该项目将发明新技术来测量和控制“由内而外”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，我们将开发区隔化的尼古丁配体。我们还将采用其他最先进的技术：COPII囊泡萌芽重建和FRET。子方法A发明了测量尼古丁作用区隔的工具。子方法B发明了将药理学局限于内质网的工具，既适用于尼古丁，也适用于临床重要的alpha4beta2 nachr选择性配体伐尼克兰。子方法C测试nachr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，可能会揭示成瘾和神经变性的新治疗靶点。

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.