Sabrina Brockmöller, Thomas Seeger, Franz Worek, Simone Rothmiller
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Therefore, Gibson cloning was used to generate transfer plasmids carrying the sequence of nAChR or chosen biological chaperones to support the nAChR folding in the cellular host. Viral transduction was used for stable integration of these transgenes in Chinese hamster ovary cells (CHO). Proteins were detected with Western blot, in-cell and on-cell Western, and the function of the receptor with voltage clamp analysis. We show that the internalization of nAChR into plasma membranes was sufficient for detection and function. Additional transgenic overexpression of biological chaperones did result in a reduced nAChR expression. Chemical chaperones, posttranslational modification supporting substances, and hypothermic conditions are well-suited supporting applications to increase the protein levels of different subunits. This study presents a stable and functional cell line that expresses human muscle-type nAChR and yields can be further increased using the chemical chaperone nicotine without affecting cell viability. The simplified access to this model system should enable numerous applications beyond drug development. 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引用次数: 0
摘要
人类肌肉型烟碱乙酰胆碱受体α12β1δε(nAChR)是一种复杂的跨膜受体,需要用于先天性肌无力综合征和多发性翼状胬肉综合征等疾病的药物筛选。直到今天,大多数模型仍在使用来自加利福尼亚鱼雷的 nAChR。表达功能性人类肌肉型 nAChR 的简单可重现细胞系统仍然缺失。本研究解决了这一问题,并进一步检验了不同的生物和化学伴侣、翻译后修饰支持物质以及低温培养能够提高 nAChR 产量的假设。因此,利用吉布森克隆技术生成携带 nAChR 序列或所选生物伴侣的转运质粒,以支持 nAChR 在细胞宿主中折叠。病毒转导用于在中国仓鼠卵巢细胞(CHO)中稳定整合这些转基因。通过 Western 印迹、细胞内和细胞外 Western 检测蛋白质,并通过电压钳分析检测受体的功能。我们的研究表明,nAChR 内化到质膜中足以进行检测和发挥功能。生物伴侣素的额外转基因过表达确实导致了 nAChR 表达的减少。化学伴侣、翻译后修饰辅助物质和低温条件是提高不同亚基蛋白水平的理想辅助应用。本研究提出了一种表达人类肌肉型 nAChR 的稳定、功能性细胞系,使用化学伴侣物尼古丁可进一步提高产量,而不影响细胞活力。通过简化这一模型系统的获取途径,可在药物开发之外实现多种应用。图解摘要。
Recombinant cellular model system for human muscle-type nicotinic acetylcholine receptor α12β1δε.
The human muscle-type nicotinic acetylcholine receptor α12β1δε (nAChR) is a complex transmembrane receptor needed for drug screening for disorders like congenital myasthenic syndromes and multiple pterygium syndrome. Until today, most models are still using the nAChR from Torpedo californica electric ray. A simple reproducible cellular system expressing functional human muscle-type nAChR is still missing. This study addressed this issue and further tested the hypothesis that different chaperones, both biological and chemical, and posttranslational modification supporting substances as well as hypothermic incubation are able to increase the nAChR yield. Therefore, Gibson cloning was used to generate transfer plasmids carrying the sequence of nAChR or chosen biological chaperones to support the nAChR folding in the cellular host. Viral transduction was used for stable integration of these transgenes in Chinese hamster ovary cells (CHO). Proteins were detected with Western blot, in-cell and on-cell Western, and the function of the receptor with voltage clamp analysis. We show that the internalization of nAChR into plasma membranes was sufficient for detection and function. Additional transgenic overexpression of biological chaperones did result in a reduced nAChR expression. Chemical chaperones, posttranslational modification supporting substances, and hypothermic conditions are well-suited supporting applications to increase the protein levels of different subunits. This study presents a stable and functional cell line that expresses human muscle-type nAChR and yields can be further increased using the chemical chaperone nicotine without affecting cell viability. The simplified access to this model system should enable numerous applications beyond drug development. GRAPHICAL ABSTRACT.
期刊介绍:
Cell Stress and Chaperones is an integrative journal that bridges the gap between laboratory model systems and natural populations. The journal captures the eclectic spirit of the cellular stress response field in a single, concentrated source of current information. Major emphasis is placed on the effects of climate change on individual species in the natural environment and their capacity to adapt. This emphasis expands our focus on stress biology and medicine by linking climate change effects to research on cellular stress responses of animals, micro-organisms and plants.