Prototoxin Effects on Nicotinic Receptor Function

摘要/摘要 在美国，吸烟，尤其是吸烟，是可预防死亡的主要原因 以及整个世界。吸烟行为是由尼古丁成瘾驱动的，尼古丁通过 烟碱型乙酰胆碱受体(NAChR)。更好地了解nAChR的功能和表达方式 因此，监管至关重要。原毒素是一个广泛的蛋白质家族，在生理上起作用 多种nAChR亚型的重要调节因子。然而，原毒素nAChR亚型的基础 选择性、它们的相互作用部位、它们改变nAChR功能的机制以及它们在 尼古丁依赖的途径在很大程度上还不确定。此应用程序解决了我们的 知识。我们的初步数据表明，原毒素lynx1以变构的方式调节多种亚型的 α3β4*-，α4β2*-和α5*-nAChR，它们都被反复地与人类吸烟行为联系在一起。我们还有 观察到lynx1在nAChR亚型上的宏观和单通道功能差异效应，这是 提供理想读数，用于定义发生Lynx1/α3β4*-nAChR相互作用的位置。这些 研究结果引出了我们的基本假设：变构原毒素效应产生于(通常很好) 保守的)与非激动剂结合的nAChRα(-)亚单位界面的相互作用，以及不同的结果 产生于每个原毒素/nAChR界面的相互作用的细节。新的初步数据也表明 在躯体尼古丁戒断过程中具有明确作用的吻侧IPN(IPR)GABA神经元共表达α3β4*- 和α4β2*-nAChR，以及α5亚单位和Lynx1mRNA的高水平表达。因此，它们代表着一种 优秀的，依赖相关的，天然的研究系统，用来研究nAChR群体(S)α5亚基 关联，以及lynx1如何调节IPN中这些相同的nAChR群体。因此，我们的理想状态是 用于比较在相同定义的nAChR人群中的lynx1调制的功能结果 原生神经元和体外模型，增强了对这些系统研究结果的验证和解释。 我们将通过结合来自多学科实验的精确实验数据来寻求这个机会 采用复杂的分子动力学建模方法。这一紧密结合的研究计划将使我们能够 首次建立一个通用框架来理解原毒素调节剂是如何产生的 天然神经元和体外培养神经元中多种nAChR亚型和异构体的功能结果 表达系统。它还将确保保持科学严谨性，迅速完善我们的实验设计， 并产生关键的生物学和机械学见解。此外，区域限制的原毒素表达可能 允许将nAChR功能的调制限制在特定的大脑区域或细胞类型。原毒素/nAChR 因此，相互作用可能代表着有希望的新药物靶点。通过探索自然和遗址 原毒素/nAChR相互作用，这项研究承诺消除科学和技术进步的关键障碍 与尼古丁成瘾有关的临床工作，以及受尼古丁受体功能影响的其他情况。

SUMMARY / ABSTRACT Tobacco use, and most-prominently cigarette smoking, is the leading cause of preventable death in the USA and across the world. Smoking behavior is driven by addiction to nicotine, which exerts its effects through nicotinic acetylcholine receptors (nAChR). Better understanding how nAChR function and expression is regulated is thus crucial. Prototoxins are an extensive family of proteins which serve as physiologically important regulators of multiple nAChR subtypes. However, the basis of prototoxins' nAChR subtype selectivities, their interaction sites, the mechanisms by which they alter nAChR function, and their roles within nicotine dependence pathways are largely undetermined. This application addresses these critical gaps in our knowledge. Our Preliminary Data indicate that the prototoxin lynx1 allosterically regulates multiple isoforms of α3β4*-, α4β2*- and α5*-nAChR, which have repeatedly been linked to human smoking behavior. We have also observed differential macroscopic and single-channel functional effects of lynx1 across nAChR isoforms, which provide ideal readouts for use in defining the sites at which lynx1/α3β4*-nAChR interactions occur. These findings led us to our underlying hypothesis: that allosteric prototoxin effects arise from (generally well- conserved) interactions with non-agonist-binding nAChR α(-)- subunit interfaces, and that differential outcomes arise from the details of interactions at each prototoxin/nAChR interface. New Preliminary data also indicate that rostral-IPN (IPR) GABA neurons, with a well-defined role in somatic nicotine withdrawal, coexpress α3β4*- and α4β2*-nAChR, together with high levels of both α5 subunit and lynx1 mRNA. They therefore represent an excellent, dependence-related, native system for studying with which nAChR population(s) α5 subunits associate, and how lynx1 modulates these same nAChR populations in the IPN. We therefore are ideally placed to compare functional outcomes of lynx1 modulation across the same defined nAChR populations in native neurons and in vitro models, enhancing validation and interpretation of findings across these systems. We will pursue this opportunity by combining precise experimental data from a multidisciplinary experimental approach with sophisticated molecular dynamics modeling. This closely integrated research plan will allow us to establish for the first time a generalized framework to understand how prototoxin modulators produce functional outcomes across multiple nAChR subtypes and isoforms, in both native neurons and in vitro expression systems. It will also ensure maintenance of scientific rigor, rapidly refine our experimental designs, and produce key biological and mechanistic insights. In addition, regionally-restricted prototoxin expression may permit modulation of nAChR function to be restricted to particular brain regions or cell types. Prototoxin/nAChR interactions may therefore represent promising new drug targets. By probing the nature and sites of prototoxin/nAChR interactions, this study promises to remove critical barriers to progress in scientific and clinical work related to nicotine addiction, as well as other conditions affected by nicotinic receptor function.