Namovic Marian T, Jarvis Michael F, Donnelly-Roberts Diana
Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, Illinois, USA.
Curr Protoc Pharmacol. 2012 Jun;Chapter 9:Unit 9.15.. doi: 10.1002/0471141755.ph0915s57.
Adenosine triphosphate (ATP) activates two receptor superfamilies, metabotropic P2Y and ionotropic P2X receptors. The P2X receptors are nonselective cation channels that are widely expressed on excitable cells including neurons, glia, and smooth muscle cells. The protocols in this unit are useful for evaluating ligands that interact with P2X receptors on native cells or that are cloned and expressed in recombinant heterologous cell systems. Calcium imaging methods are described for the pharmacological characterization of fast or slowly desensitizing P2X receptors. While these methods are readily applicable to a wide variety of ligand-gated ion channels, the protocols provided herein detail how they can be used to measure activation of homomeric P2X3 (fast desensitizing) and heteromeric P2X2/3 (slowly desensitizing) receptors. Appropriate agonists and/or calcium dyes can be substituted to assess activity at other P2X receptor subtypes. An additional protocol is provided for measuring P2X7 receptor-mediated pore formation in THP-1, a native human acute monocytic leukemia cell line that can be used to study homomeric P2X7 (non-desensitizing) receptors that are expressed on macrophages and microglial cells.
三磷酸腺苷（ATP）激活两个受体超家族，即代谢型P2Y受体和离子型P2X受体。P2X受体是一种非选择性阳离子通道，广泛表达于包括神经元、神经胶质细胞和平滑肌细胞在内的可兴奋细胞上。本单元中的实验方案可用于评估与天然细胞上的P2X受体相互作用的配体，或在重组异源细胞系统中克隆和表达的配体。文中描述了用于快速或缓慢脱敏的P2X受体药理学特征分析的钙成像方法。虽然这些方法很容易应用于多种配体门控离子通道，但本文提供的实验方案详细说明了如何用它们来测量同源性P2X3（快速脱敏）和异源性P2X2/3（缓慢脱敏）受体的激活情况。可以替换合适的激动剂和/或钙染料来评估其他P2X受体亚型的活性。还提供了另一个实验方案，用于测量THP-1细胞中P2X7受体介导的孔形成，THP-1是一种天然的人类急性单核细胞白血病细胞系，可用于研究巨噬细胞和小胶质细胞上表达的同源性P2X7（非脱敏）受体。
