Garcia-Guzman M, Soto F, Gomez-Hernandez J M, Lund P E, Stühmer W
Department of Molecular Biology of Neuronal Signals, Max-Planck Institute for Experimental Medicine, Göttingen, Germany.
Mol Pharmacol. 1997 Jan;51(1):109-18. doi: 10.1124/mol.51.1.109.
We isolated a cDNA from human brain encoding a purinergic receptor that shows a high degree of homology to the rat P2X4 receptor (87% identity). By fluorescence in situ hybridization, the human P2X4 gene has been mapped to region q24.32 of chromosome 12. Tissue distribution analysis of human P2X4 transcripts demonstrates a broad expression pattern in that the mRNA was detected not only in brain but also in all tissues tested. Heterologous expression of the human P2X4 receptor in Xenopus laevis oocytes and human embryonic kidney 293 cells evoked an ATP-activated channel. Simultaneous whole-cell current and Fura-2 fluorescence measurements in human embronic kidney 293 cells transfected with human P2X4 cDNA allowed us to determine the fraction of the current carried by Ca2: this was approximately 8%, demonstrating a high Ca2+ permeability. Low extracellular Zn2+ concentrations (5-10 microM) increase the apparent gating efficiency of human P2X4 by ATP without affecting the maximal response. However, raising the concentration of the divalent cation (> 100 microM) inhibits the ATP-evoked current in a non-voltage-dependent manner. The human P2X4 receptor displays a very similar agonist potency profile to that of rat P2X4 (ATP > > 2-methylthio-ATP > or = CTP > alpha, beta-methylene-ATP > dATP) but has a notably higher sensitivity for the antagonists suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid, and bromphenol blue. Chimeric constructs between human and rat isoforms as well as single-point mutations were engineered to map the regions responsible for the different sensitivity to suramin and pyridoxal-phosphate-6-azophenyl-2'4'-disulfonic acid.
我们从人脑中分离出一个编码嘌呤能受体的cDNA，该受体与大鼠P2X4受体具有高度同源性（同一性为87%）。通过荧光原位杂交，人P2X4基因已被定位到12号染色体的q24.32区域。人P2X4转录本的组织分布分析表明其表达模式广泛，因为不仅在脑中检测到了mRNA，在所有测试组织中也都检测到了。人P2X4受体在非洲爪蟾卵母细胞和人胚肾293细胞中的异源表达引发了ATP激活的通道。在用人类P2X4 cDNA转染的人胚肾293细胞中同时进行全细胞电流和Fura-2荧光测量，使我们能够确定由Ca2+携带的电流比例：约为8%，表明其Ca2+通透性较高。低细胞外Zn2+浓度（5 - 10 microM）可提高ATP对人P2X4的表观门控效率，而不影响最大反应。然而，提高二价阳离子浓度（> 100 microM）会以非电压依赖性方式抑制ATP诱发的电流。人P2X4受体与大鼠P2X4受体的激动剂效力谱非常相似（ATP >> 2 - 甲硫基 - ATP ≥ CTP > α,β - 亚甲基 - ATP > dATP），但对拮抗剂苏拉明、磷酸吡哆醛 - 6 - 偶氮苯基 - 2',4' - 二磺酸和溴酚蓝的敏感性明显更高。构建了人和大鼠异构体之间的嵌合构建体以及单点突变体，以确定对苏拉明和磷酸吡哆醛 - 6 - 偶氮苯基 - 2',4' - 二磺酸不同敏感性的区域。
