Identification and Characterization of a Selective Allosteric Antagonist of Human P2X4 Receptor Channels
Identification and Characterization of a Selective Allosteric Antagonist of Human P2X4 Receptor Channels
复制标题
DOI：
10.1124/mol.114.096222
复制
发表时间：
2015-04-01
影响因子：
3.6
通讯作者：
Seguela, Philippe
中科院分区：
文献类型：
--
作者：
Ase, Ariel R.;Honson, Nicolette S.;Seguela, Philippe
P2X4是一个atp门控的非选择性阳离子通道，对钙具有高度渗透性。越来越多的证据表明，这种同源嘌呤受体在几种神经元和免疫细胞类型中表达，与慢性疼痛和炎症有关。目前缺乏明确的药理学工具来询问或调节P2X4功能，这促使我们继续寻找选择性拮抗剂。在化合物文库的高通量筛选中，我们鉴定出苯脲BX430(1-（2,6-二溴-4-异丙基-苯基）-3-（3-吡啶基）尿素，分子量= 413)，对p2x4介导的人钙摄取具有拮抗剂特性。膜片钳电生理证实细胞外BX430具有亚微摩尔效价（IC50 = 0.54 mu M）直接抑制P2X4电流。BX430具有高度选择性，对所有其他P2X亚型（即P2X1-P2X3、P2X5和P2X7）几乎没有功能影响，IC50是其10-100倍。由于BX430是斑马鱼P2X4的有效拮抗剂，但对大鼠和小鼠P2X4同源物没有影响，因此发现了意想不到的物种差异。BX430存在时ATP对人P2X4的浓度-响应曲线显示出不可逾越的阻断，表明其作用机制是非竞争性的变构机制。通过荧光染料摄取实验，我们观察到BX430也有效抑制atp诱导的和伊维菌素增强的P2X4孔扩张诱导的膜通透性。最后，在单细胞钙成像中，我们验证了其对分化为巨噬细胞的人THP-1细胞表面天然P2X4通道的选择性抑制作用。总之，这种配体提供了一种新的分子探针来评估P2X4在炎症和神经性疾病中的特定作用，在这些疾病中，ATP信号已被证明是功能失调的。
P2X4 is an ATP-gated nonselective cation channel highly permeable to calcium. There is increasing evidence that this homomeric purinoceptor, which is expressed in several neuronal and immune cell types, is involved in chronic pain and inflammation. The current paucity of unambiguous pharmacological tools available to interrogate or modulate P2X4 function led us to pursue the search for selective antagonists. In the high-throughput screen of a compound library, we identified the phenylurea BX430 (1-(2,6-dibromo-4-isopropyl-phenyl)-3-(3-pyridyl) urea, molecular weight = 413), with antagonist properties on human P2X4-mediated calcium uptake. Patch-clamp electrophysiology confirmed direct inhibition of P2X4 currents by extracellular BX430, with submicromolar potency (IC50 = 0.54 mu M). BX430 is highly selective, having virtually no functional impact on all other P2X subtypes, namely, P2X1-P2X3, P2X5, and P2X7, at 10-100 times its IC50. Unexpected species differences were noticed, as BX430 is a potent antagonist of zebrafish P2X4 but has no effect on rat and mouse P2X4 orthologs. The concentration-response curve for ATP on human P2X4 in the presence of BX430 shows an insurmountable blockade, indicating a noncompetitive allosteric mechanism of action. Using a fluorescent dye uptake assay, we observed that BX430 also effectively suppresses ATP-evoked and ivermectin-potentiated membrane permeabilization induced by P2X4 pore dilation. Finally, in single-cell calcium imaging, we validated its selective inhibitory effects on native P2X4 channels at the surface of human THP-1 cells that were differentiated into macrophages. In summary, this ligand provides a novel molecular probe to assess the specific role of P2X4 in inflammatory and neuropathic conditions, where ATP signaling has been shown to be dysfunctional.