Rudolf Boehm Institute of Pharmacology and Toxicology, University of Leipzig, Leipzig, Germany.
J Biol Chem. 2011 Apr 1;286(13):11067-81. doi: 10.1074/jbc.M110.198879. Epub 2011 Jan 24.
P2X7 receptors have emerged as potential drug targets for the treatment of medical conditions such as e.g. rheumatoid arthritis and neuropathic pain. To assess the impact of pharmaceuticals on P2X7, we screened a compound library comprising approved or clinically tested drugs and identified several compounds that augment the ATP-triggered P2X7 activity in a stably transfected HEK293 cell line. Of these, clemastine markedly sensitized Ca(2+) entry through P2X7 to lower ATP concentrations. Extracellularly but not intracellularly applied clemastine rapidly and reversibly augmented P2X7-mediated whole-cell currents evoked by non-saturating ATP concentrations. Clemastine also accelerated the ATP-induced pore formation and Yo-Pro-1 uptake, increased the fractional NMDG(+) permeability, and stabilized the open channel conformation of P2X7. Thus, clemastine is an extracellularly binding allosteric modulator of P2X7 that sensitizes P2X7 to lower ATP concentrations and facilitates its pore dilation. The activity of clemastine on native P2X7 receptors, Ca(2+) entry, and whole-cell currents was confirmed in human monocyte-derived macrophages. Similar effects were observed in murine bone marrow-derived macrophages. Consistent with the data on recombinant P2X7, clemastine augmented the ATP-induced cation entry and Yo-Pro-1 uptake. In accordance with the observation that P2X7 controls the cytokine release from LPS-primed macrophages, we found that clemastine augmented the IL-1β release from LPS-primed human macrophages. Collectively, these data point to a sensitization of the recombinantly or natively expressed human P2X7 receptor toward its physiological activator, ATP, possibly leading to a modulation of macrophage-dependent immune responses.
P2X7 受体已成为治疗某些疾病（例如类风湿性关节炎和神经病理性疼痛）的潜在药物靶点。为了评估药物对 P2X7 的影响，我们筛选了一个包含已批准或临床测试药物的化合物库，并确定了几种可增强稳定转染的 HEK293 细胞系中 ATP 触发的 P2X7 活性的化合物。其中，氯马斯汀显著增强了 P2X7 对较低 ATP 浓度的钙内流。细胞外而非细胞内给予氯马斯汀可快速且可逆地增强非饱和 ATP 浓度下 P2X7 介导的全细胞电流。氯马斯汀还加速了 ATP 诱导的孔形成和 Yo-Pro-1 摄取，增加了 NMDG（+）通透性分数，并稳定了 P2X7 的开放通道构象。因此，氯马斯汀是一种细胞外结合的 P2X7 别构调节剂，可使 P2X7 对较低的 ATP 浓度敏感，并促进其孔扩张。在人单核细胞衍生的巨噬细胞中证实了氯马斯汀对天然 P2X7 受体、钙内流和全细胞电流的作用。在鼠骨髓来源的巨噬细胞中也观察到了类似的效果。与重组 P2X7 上的研究数据一致，氯马斯汀增强了 ATP 诱导的阳离子内流和 Yo-Pro-1 摄取。与 P2X7 控制 LPS 预刺激的巨噬细胞细胞因子释放的观察结果一致，我们发现氯马斯汀增强了 LPS 预刺激的人巨噬细胞中 IL-1β 的释放。总的来说，这些数据表明，氯马斯汀使重组或天然表达的人 P2X7 受体对其生理激活剂 ATP 的敏感性增加，可能导致对巨噬细胞依赖性免疫反应的调节。
