Molecular Pharmacology of ATP Receptor using Atomic Force Microscopy

原子力显微镜观察ATP受体/通道（P2X受体）的结构。在空气中观察由昆虫源性Sf9细胞表达系统制备的受体蛋白时，可观察到高约4 nm的扁平岛状结构。在水中观察，获得了单个受体蛋白的图像，发现其高度约为4 nm。当ATP加入水中时，受体蛋白形成聚集的比例增加，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，可以获得蛋白质在ATP存在下密集堆积的二维晶体状图像。由于减少了探针沿z轴的移动，这种密集排列的蛋白质可以提供更高的分辨率。实际上，受体蛋白的表面可以用纳米分辨率进行分析。分析结果表明：1)蛋白质由直径为10 nm的单元结构组成，2)在单元结构的中间发现一个孔结构，3)每个单元由几个（可能是三个）亚基组成。在没有ATP的情况下无法获得如此密集的图像，这表明当受体蛋白不被ATP占据时，它们的结构是不均匀的。当ATP拮抗剂苏拉明（suramin）在ATP存在的情况下应用时，可以获得密集的图像，但表面的单位结构不明显。这表明：1)苏拉明不是简单地用ATP代替，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。总之，这些发现表明原子力显微镜是一种强大的“纳米”级分析受体蛋白和配体之间相互作用的工具。

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.