European Institute for Molecular Imaging (EIMI), Roentgenstr 16, University of Muenster, 48149 Muenster, Germany; Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, University of Tübingen, Roentgenweg 13, 72076, Tuebingen, Germany.
University Hospital Muenster, Department of Neurology with Institute of Translational Neurology, Albert-Schweitzer-Campus 1, 48149 Muenster, Germany.
Biochem Pharmacol. 2024 Nov;229:116543. doi: 10.1016/j.bcp.2024.116543. Epub 2024 Sep 19.
The development of in vitro pharmacological assays relies on creating genetically modified cell lines that overexpress the target protein of interest. However, the choice of the host cell line can significantly impact the experimental outcomes. This study explores the functional characterization of P2X7 and P2X4 receptor modulators through cellular assays and advanced electrophysiological techniques. The influence of different host cell lines (HEK-293, HEK-293FT, and 1321N1) on the activity of reference agonists and antagonists targeting human and murine P2X4 and P2X7 receptors was systematically investigated, highlighting the significant impact of the host cell on experimental results. The 1321N1 cell line was identified as the preferred host cell line when investigating the human P2X4 receptor due to more consistent agonist activities, antagonist potencies, and a more stable assay signal window. Furthermore, a patch-clamp protocol that allows for the repetitive recording of ATP-mediated inward currents from isolated human CD4+ T-cells was established, revealing that both P2X7 and P2X4 receptors are crucial for immune cell regulation, positioning them as promising therapeutic targets for managing inflammatory disorders.
体外药理学测定的发展依赖于创建过表达目标蛋白的基因修饰细胞系。然而，宿主细胞系的选择会显著影响实验结果。本研究通过细胞测定和先进的电生理技术，探索了 P2X7 和 P2X4 受体调节剂的功能特征。系统研究了不同宿主细胞系（HEK-293、HEK-293FT 和 1321N1）对靶向人源和鼠源 P2X4 和 P2X7 受体的参考激动剂和拮抗剂活性的影响，突出了宿主细胞对实验结果的重大影响。在研究人源 P2X4 受体时，发现 1321N1 细胞系是首选宿主细胞系，因为其激动剂活性、拮抗剂效力更为一致，并且测定信号窗口更稳定。此外，还建立了一种能够从分离的人 CD4+T 细胞中重复记录 ATP 介导的内向电流的膜片钳方案，揭示了 P2X7 和 P2X4 受体在免疫细胞调节中都至关重要，将它们定位为管理炎症性疾病的有前途的治疗靶点。
