Role of nAChR stoichiometry in nicotine-induced upregulation

描述（由申请人提供）：尼古丁乙酰胆碱受体（nAChRs）的上调在尼古丁成瘾中起着不可或缺的作用。选择性地促进1422而不是其他亚型，更具体地说，高敏感性（HS）化学计量（2:3）高于低敏感性（LS）化学计量（3:2），上调导致膜上HS受体的数量增加。本研究旨在研究1422受体的膜传递和膜回收机制，以确定其转运和转换在尼古丁诱导的上调中所起的作用。此外，尼古丁诱导的这些受体化学计量学的变化将通过单受体分辨率进行研究，以直接观察它们在上调中的作用。我们将通过观察1422的胞吐和内吞活性的变化，重点关注内质网（ER）的运输如何随特定受体亚型和化学计量学的变化而变化。观察到尼古丁存在时膜停留时间的增加主要是由于两种不同的活动：尼古丁与膜受体的结合直接影响膜上的停留时间，或者1422的两种化学计量量具有不同的停留时间，尼古丁优先上调化学计量量，其寿命更长，使膜上的停留时间增加。尼古丁对这种行为的影响，以及它如何诱导小鼠神经母细胞瘤细胞和培养神经元的亚细胞区域的差异运输，将在单囊泡和单受体水平上使用全内反射显微镜（TIRFM）和荧光共振能量转移（FRET）进行研究。尼古丁诱导的受体比例再分配（HS vs. LS）显然在上调中起重要作用。与乙酰胆碱、胱氨酸和DH2E相比，了解尼古丁如何改变1422在细胞膜上的转运和插入不同亚细胞区域后的停留时间，将有助于对驱动上调的机制有新的认识。这些研究将是第一次在单分子水平上直接测量和可视化这两种化学计量。表征尼古丁诱导的nAChR运输和在膜上停留时间的变化与不同亚细胞区域的化学计量学鉴定特异性相关，将提高我们对成瘾的理解，促进开发新的更有效的戒烟治疗方法。

DESCRIPTION (provided by applicant): Upregulation of nicotinic acetylcholine receptors (nAChRs) plays an integral role in nicotine addiction. Selectively promoting 1422 over other subtypes and more specifically the high sensitivity (HS) stoichiometry (2:3) over the low sensitivity (LS) stoichiometry (3:2), upregulation leads to an increased population of HS receptors at the membrane. This proposal investigates the mechanisms of membrane delivery and retrieval of 1422 receptors to determine the role trafficking and turnover play in nicotine induced upregulation. Additionally, nicotine-induced changes in the stoichiometry of these receptors will be investigated with single receptor resolution to directly visualize their role in upregulation. We will focus on how trafficking out of the endoplasmic reticulum (ER) varies with specific receptor subtypes and stoichiometries by observing variations in exocytosis and endocytosis activity of 1422. Observations of increased membrane dwell time in the presence of nicotine could primarily result from two distinct activities: binding of nicotine to the membrane receptor directly influences dwell time at the membrane or the two stoichiometries of 1422 have different dwell times and nicotine preferentially upregulates the stoichiometry with a longer lifetime giving the appearance of an increasing residence time at the membrane. The effects of nicotine on this behavior and how it induces differential trafficking to subcellular regions of mouse neuroblastoma cells and cultured neurons will be investigated using total internal reflection microscopy (TIRFM) and fluorescence resonance energy transfer (FRET) at both the single vesicle and single receptor level. Nicotine- induced redistribution of receptor ratios (HS vs. LS) clearly plays an important part in upregulation. Understanding how nicotine, in comparison to acetylcholine, cytisine, and DH2E, modifies 1422 trafficking to the membrane and residence time after insertion in different subcellular region will yield new insight into the mechanisms that drive upregulation. These studies will be the first to directly measure and visualize the two stoichiometries at the single molecule level. Characterization of nicotine-induced changes in nAChR trafficking and residence time at the membrane specifically correlated to stoichiometric identification in different subcellular regions will improve our understanding of addiction, facilitating the development of new and more effective smoking cessation treatments. PUBLIC HEALTH RELEVANCE: Tobacco consumption contributes to millions of cancer deaths every year and is also a leading factor in many other heart and lung diseases such as emphysema and vascular disease. Clearly developing methods to moderate smoking is a worldwide health imperative, and understanding the processes that lead to nicotine addiction will be crucial in the development of smoking cessation therapies. The studies here will focus on how nicotine affects receptor activity at the endoplasmic reticulum and the cell membrane particularly as it relates to receptor stoichiometries and subcellular localization.