Expression and Characterization of alpha6beta2beta3 nicotinic receptors

α6β2β3 烟碱受体的表达和表征

• 批准号: 8697031
• 负责人: Brandon Jarrod Henderson
• 金额: $ 5.33万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-02 至 2015-03-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8697031
• 关键词: Acetylcholine; Affect; Affinity; Agonist; Biological Assay; Brain; Caliber; Cell Line; Cell membrane; Cells; Cessation of life; Characteristics; Charge; Chronic; Cigar; Cigarette; Detection; Development; Drosophila acetylcholine receptor alpha-subunit; Endoplasmic Reticulum; Esthesia; Evaluation; Exposure to; Fluorescence; Future; Gated Ion Channel; Goals; Health; Heart Diseases; In Vitro; Intervention; Ions; Kinetics; Knowledge; Ligands; Lung diseases; Measurement; Mediating; Mediator of activation protein; Medical; Membrane; Methods; Modeling; Mus; Mutation; Neurons; Nicotine; Nicotine Dependence; Nicotinic Receptors; Oral Tobacco; Parkinson Disease; Peripheral Nervous System; Permeability; Pharmaceutical Preparations; Photobleaching; Play; Point Mutation; Population; Preclinical Drug Evaluation; Process; Property; Pulmonary Emphysema; Regulation; Rewards; Role; Saline; Site; Synapses; System; Techniques; Therapeutic; Tobacco; United States; Up-Regulation; Vascular Diseases; Withholding Treatment; addiction; cost; desensitization; epibatidine; improved; in vivo; insight; motor control; mouse model; nigrostriatal dopaminergic pathway; novel; premature; public health relevance; receptor; receptor expression; response; single molecule; smoking cessation; stoichiometry

DESCRIPTION (provided by applicant): Alpha6* (alpha6-containing) nicotinic acetylcholine receptors (nAChRs) play an important role in nicotine addiction. Alpha6* nAChRs are found in just a few regions of the mammalian brain (the mesolimbic and nigrostriatal dopaminergic pathways) and mediate reward and motor control. Aim 1 develops and exploits a cell line expressing alpha6beta2beta3 nAChRs without the use of chimeric or concatameric subunits, which have both been required for the successful expression of this subtype in cell lines. Successful expression of alpha6beta2beta3 nAChRs will be aided by expressing two point mutations in the beta2 subunit, which have been found to enhance export from the endoplasmic reticulum (ER). In addition, a fluorescent marker for ER exit sites (Sec24D) will be used to identify cells with abundant ER exit sites which are a marker for high levels of alpha6beta2beta3 nAChR expression on the plasma membrane. Functional alpha6beta2beta3 will be characterized using electrophysiological techniques. A distinguishing factor that governs functional properties of nAChRs is the subunit stoichiometry of the assembled pentamers. Aim 2 therefore determines the subunit stoichiometry of alpha6beta2beta3 nAChRs using a novel technique involving zero-mode waveguides (ZMWs). ZMWs allow the single-molecule analysis of a single membrane receptor by seeding cells on nanofabricated apertures ~100 nm diameter. Combining fluorescent nAChR subunits and photobleaching techniques will allow the quantification of specific subunits in receptor assemblies. It is well known that some nAChRs (e.g., alpha4beta2) are upregulated when chronically stimulated by nicotine. This change in receptor number has been implicated as one of the mechanisms that is critical for nicotine addiction. To determine how alpha6* nAChRs may play a role in nicotine addiction, Aim 3 determines how these receptors are changed when exposed to nicotine by using mice that have fluorescent alpha6 nAChR subunits. The mice will be chronically exposed to nicotine and quantitative fluorescence will document the change in receptor number. The understanding of how nicotine, in comparison to other drugs (i.e., acetylcholine, epibatidine), modifies the functio and receptor levels of alpha6* nAChRs will provide novel insights regarding the mechanisms that mediate nicotine addiction. Characterization of alpha6beta2beta3 nAChRs, determination of alpha6beta2beta3 nAChR stoichiometry, and determination of upregulation of alpha6* nAChRs will advance the understanding of nicotine addiction and will further the development of novel smoking cessation therapeutics.

描述（由申请人提供）：Alpha6*（含α6）烟碱乙酰胆碱受体（nAChR）在尼古丁成瘾中发挥重要作用。 Alpha6* nAChR 仅存在于哺乳动物大脑的几个区域（中脑边缘和黑质纹状体多巴胺能通路），并介导奖赏和运动控制。目标 1 开发和利用表达 alpha6beta2beta3 nAChR 的细胞系，而不使用嵌合或多联体亚基，而这两种亚基都是在细胞系中成功表达该亚型所必需的。 α6β2β3 nAChR 的成功表达将通过在 beta2 亚基中表达两个点突变来帮助，这两个点突变已被发现可以增强内质网 (ER) 的输出。此外，ER 出口位点的荧光标记 (Sec24D) 将用于识别具有丰富 ER 出口位点的细胞，这些位点是质膜上高水平 alpha6beta2beta3 nAChR 表达的标记。将使用电生理学技术来表征功能性α6β2β3。控制 nAChR 功能特性的一个显着因素是组装的五聚体的亚基化学计量。因此，目标 2 使用涉及零模波导 (ZMW) 的新技术确定 alpha6beta2beta3 nAChR 的亚基化学计量。 ZMW 允许通过在直径约 100 nm 的纳米加工孔径上接种细胞来对单个膜受体进行单分子分析。结合荧光 nAChR 亚基和光漂白技术将允许对受体组件中的特定亚基进行定量。众所周知，一些 nAChR（例如 α4β2）在尼古丁的长期刺激下会上调。受体数量的这种变化被认为是尼古丁成瘾的关键机制之一。为了确定 alpha6* nAChR 如何在尼古丁成瘾中发挥作用，Aim 3 使用具有荧光 alpha6 nAChR 亚基的小鼠来确定这些受体在接触尼古丁时如何变化。小鼠将长期暴露于尼古丁，定量荧光将记录受体数量的变化。与其他药物（即乙酰胆碱、皮巴替丁）相比，了解尼古丁如何改变 α6* nAChR 的功能和受体水平将为介导尼古丁成瘾的机制提供新的见解。 α6β2β3 nAChR 的表征、α6β2β3 nAChR 化学计量的测定以及 α6* nAChR 上调的测定将增进对尼古丁成瘾的理解，并将进一步开发新型戒烟疗法。