Molecular Pharmacology of ATP Receptor using Atomic Force Microscopy

用原子力显微镜观察ATP受体/通道(P2X受体)的结构。从昆虫来源的Sf9细胞表达系统制备的受体蛋白在空气中观察到高度约4 nm的扁平岛状结构。为了在水中观察，获得了单个受体蛋白的图像，它们的高度约为4 nm。当在水中加入ATP时，受体蛋白形成聚集体的比例增加，这表明在ATP的存在下，蛋白质的结构发生了变化。当在离子强度较高的水中进行观察时，可以获得在ATP存在下蛋白质密集堆积的二维晶状图像。这种紧密堆积的蛋白质可以提供更高的分辨率，因为探针沿z轴的移动减少了。实际上，受体蛋白的表面可以以纳米级的分辨率进行分析。分析提供了以下信息：1)蛋白质由直径为10 nm的单位结构组成，2)单位结构中间有孔结构，3)每个单位由几个(或三个)亚单位组成。在没有ATP的情况下，不能获得如此密集的图像，这表明当受体蛋白没有被ATP占据时，它们的结构并不统一。当ATP受体拮抗剂苏拉明与三磷酸腺苷共存时，可获得密集排列的图像，但表面的单元结构不明显。这一结果表明：1)苏拉明并不是简单地被ATP取代，2)苏拉明结合受体蛋白的结构与ATP结合受体蛋白的结构不同。综上所述，这些发现表明，原子力显微镜是一种强大的工具，可以用来分析受体蛋白和配体之间的相互作用。

The structure of ATP receptor/channel (P2X receptor) was observed using atomic force microscopy. When receptor proteins prepared from insect-derived Sf9 cell expression system were observed in air, flat island-like structures of about 4 nm in height were seen. For observation in water, images of individual receptor proteins were obtained, and their height was found to be about 4 nm. When ATP was added to the water, the ratio of receptor proteins forming aggregations was increased, suggesting that the structure of the proteins are changed in the presence of ATP. When observation was made in the water with a higher ionic strength, two-dimensional crystal-like images where the proteins were densely packed in the presence of ATP were obtained. Such densely packed proteins can provide higher resolution because of reduced movement of probes along the z-axis. Actually, the surface of the receptor proteins could be analyzed with nanometer resolution. The analysis provided the following information : 1) the proteins were composed of a unit structure with 10 nm in diameter, 2) a pore structure is found in the middle of the unit structure, and 3) each unit consists of several (maybe three) subunits. Such densely packed images were not obtained in the absence of ATP, suggesting that the structure of the receptor proteins is not uniform when they are not occupied by ATP. When suramin, an antagonistat ATPreceptors, was applied in the presence of ATP, densely packed images were obtained, but the unit structure in the surface was not obvious. This result suggests that : 1) suramin does not simply replace with ATP, and 2) the structure of suramin-bound receptor proteins is different from that of ATP-bound ones. In summary, these findings indicate that atomic force microscopy is a powerful tool for "nano" level analysis of interactions between receptor proteins and ligands.