Role of nAChR stoichiometry in nicotine-induced upregulation

nAChR 化学计量在尼古丁诱导的上调中的作用

• 批准号: 8214490
• 负责人: Christopher I Richards
• 金额: $ 2.27万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8214490
• 关键词: Acetylcholine; Affect; Appearance; Behavior; Binding; Cell Culture Techniques; Cell membrane; Cells; Cessation of life; Characteristics; Chronic; Coupled; Development; Docking; Drug Exposure; Endocytosis; Endoplasmic Reticulum; Equilibrium; Event; Exhibits; Exocytosis; Exposure to; Fluorescence; Fluorescence Resonance Energy Transfer; Health; Heart Diseases; Image; Individual; Lead; Ligands; Lung diseases; Malignant Neoplasms; Measurement; Measures; Membrane; Membrane Protein Traffic; Methods; Microscopy; Midbrain structure; Monitor; Mus; Neurons; Nicotine; Nicotine Dependence; Nicotinic Receptors; Pattern; Pharmaceutical Preparations; Phase; Photobleaching; Play; Population; Population Distributions; Process; Proteins; Pulmonary Emphysema; Resolution; Retrieval; Role; Route; Smoking; Spatial Distribution; Testing; Time; Tobacco use; Up-Regulation; Variant; Vascular Diseases; Vesicle; Withholding Treatment; addiction; base; behavior change; cytisine; erythroidine; improved; insight; neuroblastoma cell; public health relevance; receptor; receptor sensitivity; research study; residence; response; reticulum cell; single molecule; single-molecule FRET; smoking cessation; stoichiometry; time use; trafficking

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.

描述（由申请人提供）：烟碱乙酰胆碱受体（nAChR）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 超过其他亚型，更具体地说是高敏感性 (HS) 化学计量 (2:3) 而非低敏感性 (LS) 化学计量 (3:2)，上调导致膜上 HS 受体数量增加。该提案研究了 1422 受体的膜传递和回收机制，以确定运输和周转在尼古丁诱导的上调中所起的作用。此外，将通过单一受体分辨率研究尼古丁诱导的这些受体化学计量的变化，以直接可视化它们在上调中的作用。我们将通过观察 1422 的胞吐作用和内吞作用活性的变化，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可能来自两种不同的活动：尼古丁与膜受体的结合直接影响在膜上的停留时间或 1422 的两种化学计量具有不同的停留时间，尼古丁优先上调化学计量，具有更长的寿命，从而在膜上呈现出增加的停留时间。将使用全内反射显微镜（TIRFM）和荧光共振能量转移（FRET）在单囊泡和单受体水平上研究尼古丁对此行为的影响以及它如何诱导差异运输到小鼠神经母细胞瘤细胞和培养神经元的亚细胞区域。尼古丁诱导的受体比率重新分布（HS 与 LS）显然在上调中发挥着重要作用。第1422章 被人欺负了这些研究将是第一个在单分子水平上直接测量和可视化这两种化学计量的研究。尼古丁诱导的 nAChR 运输和膜停留时间变化的表征与不同亚细胞区域的化学计量识别特别相关，将提高我们对成瘾的理解，促进新的、更有效的戒烟治疗的开发。 公共健康相关性：烟草消费每年导致数以百万计的癌症死亡，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，开发适度吸烟的方法是全球健康的当务之急，而了解导致尼古丁成瘾的过程对于戒烟疗法的开发至关重要。这里的研究将集中于尼古丁如何影响内质网和细胞膜的受体活性，特别是因为它与受体化学计量和亚细胞定位有关。