Prototoxin Effects on Nicotinic Receptor Function

总结/摘要 在美国，烟草使用，尤其是吸烟，是可预防死亡的主要原因 and across横过the world世界.吸烟行为是由尼古丁成瘾驱动的，尼古丁通过以下方式发挥作用： 烟碱乙酰胆碱受体（nAChR）。更好地了解nAChR的功能和表达是如何 因此，监管至关重要。原毒素是一个广泛的蛋白质家族， 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，它们的相互作用位点，它们改变nAChR功能的机制，以及它们在 尼古丁依赖途径在很大程度上是不确定的。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lynx 1变构调节多种异构体， α3β4*-、α4β2*-和α5*-nAChR，它们一再与人类吸烟行为有关。我们还 观察到lynx 1在nAChR亚型中的差异宏观和单通道功能效应， 为确定lynx 1/α3β4*-nAChR相互作用发生的位点提供了理想的读数。这些 这些发现使我们得出了我们的基本假设：变构原毒素效应产生于（通常良好地， 保守）与非激动剂结合nAChR α（-）亚基界面的相互作用，以及不同的结果 产生于每个原毒素/nAChR界面处的相互作用的细节。新的初步数据还表明， 在体细胞尼古丁戒断中具有明确作用的嘴侧-IPN（IPR）GABA神经元共表达α3β4*- 和α4β2*-nAChR，以及高水平的α5亚基和lynx 1 mRNA。因此，它们代表了 一个优秀的，依赖性相关的，天然的系统，用于研究nAChR群体α5亚基 关联，以及lynx 1如何调节IPN中这些相同的nAChR群体。因此，我们理想地 用于比较相同定义的nAChR人群中lynx 1调节的功能结局， 天然神经元和体外模型，增强了对这些系统中发现的验证和解释。 我们将通过结合来自多学科实验的精确实验数据来追求这个机会。 先进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 首次建立了一个通用的框架，以了解原毒素调节剂如何产生 多种nAChR亚型和亚型的功能结果，在天然神经元和体外 表达系统。它还将确保保持科学的严谨性，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nAChR功能的调节限于特定的脑区域或细胞类型。原毒素/nAChR 因此，相互作用可能代表有希望的新药物靶点。通过探索自然和地点， 原毒素/nAChR相互作用，这项研究有望消除科学和 与尼古丁成瘾相关的临床工作，以及受尼古丁受体功能影响的其他疾病。

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.