Role of nAChR stoichiometry in nicotine-induced upregulation

描述(由申请人提供)：烟碱型乙酰胆碱受体(NAChRs)的上调在尼古丁成瘾中起着不可或缺的作用。相对于其他亚型，更具体地说，高敏感性(HS)化学计量比(2：3)高于低敏感性(LS)化学计量比(3：2)，上调1422导致膜上HS受体数量增加。本研究旨在探讨1422受体的膜转运和回收机制，以确定转运和周转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比的变化将以单一受体分辨率进行研究，以直接直观地显示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，重点研究内质网(ER)的运输如何随着特定的受体亚型和化学计量比的变化而变化。在尼古丁存在下观察到的膜停留时间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的两个化学计量比具有不同的停留时间，尼古丁优先上调化学计量比，寿命更长，表现为在膜上的停留时间增加。我们将利用全内反射显微镜(TIRFM)和荧光共振能量转移(FRET)在单个囊泡和单个受体水平上研究尼古丁对这一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向亚细胞区域的差异转运。尼古丁诱导的受体比例重分布(HS与LS)显然在上调中起着重要作用。理解尼古丁与乙酰胆碱、胱氨酸和DH2 E相比如何改变1422的膜转运和插入不同亚细胞区域后的停留时间，将为推动上调的机制提供新的见解。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个化学计量比。表征尼古丁诱导的nAChR转运和膜停留时间的变化与不同亚细胞区域的化学计量识别相关，将提高我们对成瘾的理解，促进新的、更有效的戒烟治疗的发展。 公共卫生相关性：烟草消费每年导致数百万癌症死亡，也是许多其他心肺疾病，如肺气肿和血管疾病的主要因素。显然，开发控制吸烟的方法是全球健康的当务之急，了解导致尼古丁成瘾的过程将对开发戒烟疗法至关重要。这里的研究将集中在尼古丁如何影响内质网和细胞膜上的受体活性，特别是当它与受体化学计量学和亚细胞定位有关时。

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.