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Functional characterization of naturally expressed G-protein-coupled receptors in mammalian cells using the automated high-throughput pharmacological system HT-PS 100

机译:使用自动化高通量药理系统HT-PS 100在哺乳动物细胞中天然表达的G蛋白偶联受体的功能表征

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Abstract: In studying the molecular mechanics of stimulation of a receptor and mechanisms of the receptor's interaction with ligands, the widely sued approach is to characterize dose- dependent functional responses of stimulation or inhibition of the receptor. Many GPCRs respond to the stimulation by transient changes in cytoplasmic calcium. A time trace of the ligand-evoked 'calcium signal' visualizes the sequence of signaling events taking place after the initial receptor stimulation. It is more important to know how those events depend on the ligand concentration. This type of data provides ligand affinity profiles together with information about mechanisms of the receptor/ligand interaction - competitive, non-competitive antagonism, full or partial stimulation. We have developed an automated system, HT-PS 100, for registering continuous concentration-dependent functional responses in real time at the rate of 2 min per dose-response curve. The flow-through fluidics prepares a concentration gradient of the compound and sequentially mixes it with another reagent, agonist or antagonist, and finally with cells. The resulting 'real time' concentration dependent signal is registered with a fluorescence detector. By monitoring calcium mobilization with Fura-2, we have functionally and mechanistically characterized a variety of G protein-coupled receptors, cholinergic, histaminergic, purinergic, endothelin, and bradykinin, endogenously expressed in different cell lines, SK-N-MC, TE671 and DDT$- 1$/MF-2. !12
机译:摘要:在研究刺激受体的分子力学以及受体与配体相互作用的机理时,广泛使用的方法是表征刺激或抑制受体的剂量依赖性功能反应。许多GPCR通过细胞质钙的瞬时变化来响应刺激。配体诱发的“钙信号”的时间轨迹显示了在初始受体刺激后发生的信号事件的序列。更重要的是要知道这些事件如何取决于配体浓度。此类数据提供配体亲和力概况以及有关受体/配体相互作用机理的信息-竞争性,非竞争性拮抗作用,完全或部分刺激。我们开发了一个自动化系统HT-PS 100,用于实时记录连续的浓度依赖性功能响应,每条剂量反应曲线的速率为2分钟。流通式流体技术可制备化合物的浓度梯度,然后将其与另一种试剂,激动剂或拮抗剂混合,最后与细胞混合。产生的“实时”浓度相关信号被荧光检测器记录。通过监测Fura-2的钙动员作用,我们在功能上和机理上表征了多种G蛋白偶联受体,胆碱能,组胺能,嘌呤能,内皮素和缓激肽,它们在不同的细胞系,SK-N-MC,TE671和DDT $ -1 $ / MF-2。 !12

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