Expression and Characterization of alpha6beta2beta3 nicotinic receptors

描述(申请人提供)：α6*(含α6)烟碱型乙酰胆碱受体(NAChRs)在尼古丁成瘾中起重要作用。α6*nAChRs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的多巴胺能通路)，并参与奖赏和运动控制。目的1开发和利用一种表达α6beta2beta3nAChRs的细胞系，而不使用嵌合或串联亚基，这两种亚基都是在细胞系中成功表达该亚型所必需的。α6β2β3 nAChRs的成功表达将通过表达Beta2亚单位中的两个点突变来帮助，这两个点突变已被发现可以促进内质网(ER)的输出。此外，ER出口部位的荧光标记(Sec24D)将用于识别具有丰富的ER出口部位的细胞，这是质膜上高水平表达α6β2β3 nAChR的标志。将使用电生理技术对功能α6β2β3进行表征。决定nAChRs功能性质的一个显著因素是组装的五聚体的亚单位化学计量比。因此，目的2使用一种涉及零模波导(ZMW)的新技术来确定α6β2β3 nAChRs的亚基化学计量比。ZMW通过在直径约100 nm的纳米孔径上播种细胞，可以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，可以对受体组件中的特定亚基进行定量。众所周知，一些nAChRs(例如，α4beta2)在尼古丁的长期刺激下上调。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*nAChRs如何在尼古丁成瘾中发挥作用，Aim 3通过使用带有荧光alpha6 nAChR亚单位的小鼠，确定了这些受体在暴露于尼古丁时的变化。小鼠将长期暴露在尼古丁中，定量荧光将记录受体数量的变化。与其他药物(如乙酰胆碱、表巴替丁)相比，理解尼古丁如何改变α6*nAChRs的功能和受体水平，将为研究尼古丁成瘾的调节机制提供新的见解。α6β2β3 nAChRs的表征、α6β2β3 nAChR化学计量学的测定和α6*nAChRs上调的测定将促进人们对尼古丁成瘾的认识，并将进一步推动新型戒烟疗法的发展。

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.