Visinin-like protein-1 modulation of nicotinic receptors

摘要。据世界卫生组织称，烟草使用是世界上可预防死亡的主要原因。 Organization.在美国，吸烟导致的死亡人数约占死亡人数的五分之一， 每年有48万人死亡。努力戒烟的烟草使用者通常会复发。2018年，大约55%的成年人 在美国，吸烟者在过去一年内尝试戒烟;只有大约8%成功戒烟 6-12个月。因此，有必要采用新的方法来提高尼古丁戒烟的成功率。 尼古丁是一种与尼古丁使用障碍（NUD）高度相关的化合物，它与神经元烟碱乙酰胆碱结合 受体（nAChRs）激活大脑奖励和正强化回路。此外，尼古丁的使用导致 胆碱能张力失衡部分由nAChR α4β2亚型失调引起。神经元α4β2 亚型表达对烟碱激动剂高、低敏感性的两种亚型（HS（α4β2）2β2和LS （α4β2）2α4）。尼古丁上调功能性α4β2 nAChRs并富集质膜 HS（α4β2）2β2亚型的表达。这些变化会导致大脑的过度敏感 （中皮质边缘）电路，同时提高多巴胺水平，加强奖励行为，有助于 当然。恢复胆碱能张力，使其敏感性降低，并与尼古丁前水平相当，可能是 在开发治疗NUD的有效方法中至关重要。关于伴侣知之甚少 α4β2 nAChR亚型的蛋白调节，一个关键的建议使命，和伴侣蛋白的能力， 抵消尼古丁驱动的α4β2亚型失衡。为了开始回答这些问题，我们建议 定义视见素样蛋白-1（VILIP-1）对受体生理学功能作用的基础，相互作用位点， 和VILIP-1驱动的α4β2 nAChR质膜表达的变化。我们的初步数据 显示VILIP-1减弱α4β2 nAChR功能并增强质膜表达。该等相关 研究结果使我们假设，通过一个小的 分子可能是治疗NUD的一种新方法。将VILIP-1/α4β2 nAChR相互作用位点定义为 这里提出的，将消除阻碍科学和临床NUD工作的关键障碍，除了其他 受α4β2 nAChR失调影响的病症。我们将通过以下方式实现本提案的目标： 用活细胞共聚焦显微镜进行受体功能的电生理学研究。本申请的资助将 为PI Weltzin的进展提供基础支持，成为一个独立的，建立 研究人员通过展示项目的可资助性和可行性，分析进展，初步数据生成， 和资深作者出版物。她的导师团队包括Miwa博士和Whiteaker博士， 专家此外，该提案将为代表性不足的学生提供实践研究支持， 生物医学科学这项建议所支持的活动将维持和丰富研究环境 在阿拉斯加大学费尔班克斯。

ABSTRACT. Tobacco use is the world’s leading cause of preventable death, according to the World Health Organization. Within the US, cigarette smoking cases approximately one in five deaths, accounting for more than 480K deaths each year. Tobacco users who strive to quit usually relapse. In 2018, approximately 55% of adults in the US who smoked tobacco had made a quit attempt within the past year; only roughly 8% successfully quit for 6-12 months. Because of this, novel approaches are necessary to improve nicotine cessation success rates. Nicotine, a compound highly involved in nicotine use disorder (NUD), binds to neuronal nicotinic acetylcholine receptors (nAChRs) to activate brain reward and positive reinforcement circuits. Further, nicotine use results in cholinergic tone imbalance caused, in part, by dysregulation of the nAChR α4β2 subtype. The neuronal α4β2 subtype expresses in two isoforms with high and low sensitivity to nicotinic agonist (HS (α4β2)2β2 and LS (α4β2)2α4, respectively). Nicotine upregulates functional α4β2 nAChRs and enriches plasma membrane expression of the HS (α4β2)2β2 isoform. These changes result in hypersensitive brain reward (mesocorticolimbic) circuits while enhancing dopamine levels that reinforce rewarding behavior, contributing to NUD. Restoring the cholinergic tone to be less sensitive and comparable to pre-nicotine levels, is likely to be critical in the development of an efficacious approach to treating NUD. Little is known regarding chaperone protein regulation of α4β2 nAChR isoforms, a key proposal mission, and the ability of chaperone proteins to counteract nicotine-driven α4β2 isoform imbalance. To begin to answer some of these questions, we propose to define the basis of visinin-like protein-1 (VILIP-1) functional effects on receptor physiology, the interaction site, and VILIP-1-driven changes in α4β2 nAChR plasma membrane expression. Our preliminary data consistently show VILIP-1 attenuates α4β2 nAChR function and enhances plasma membrane expression. These underlying findings led us to our hypothesis that leveraging the interactions between VILIP-1 and α4β2 nAChRs by a small molecule may be a novel approach to treating NUD. Defining the VILIP-1/α4β2 nAChR interaction site, as proposed here, will remove critical barriers that stymie scientific and clinical NUD work, in addition to other conditions affected by α4β2 nAChR dysregulation. We will pursue the aims of this proposal by combining electrophysiology studies of receptor function with live cell confocal microscopy. Funding of this application will provide foundational support for PI Weltzin’s progress towards becoming an independent and established researcher by demonstrating project fundability and feasibility, assay advancement, preliminary data generation, and senior-author publications. Her mentorship team includes Drs. Miwa and Whiteaker, established nAChR experts. In addition, the proposal will provide hands-on research support for underrepresented students in biomedical science. Activities supported by this proposal will sustenance and enrich the research environment at the University of Alaska Fairbanks.