Comparative studies on the moleular architecture of insitol-trisphosphate receptor and ryanodine receptor and their intracellular distribution

肌醇-1,4,5-三磷酸受体（IP_3R）和红嘌呤受体（RyaR）均可形成配体门控的Ca^<2+> -通道，并在细胞内信号转导中发挥重要作用。虽然已经确定了这两种受体的总氨基酸序列，但对这些受体的组织学研究仍然不足，对它们在细胞或组织中的三维分布也没有实质性的研究。在结构方面，通过负染色或低温电镜对四聚体Rya-R粒子的分子结构进行了广泛的研究，从而重建了其三维结构。然而，尽管对IP_3-R进行了许多类似的研究，但到目前为止，还没有人成功地获得该受体分子结构的具体信息。作者一直将其专业的速冻深蚀刻复制电镜技术应用于各种蛋白质结构的研究。利用这种技术，人们可以获得溶液或细胞/组织中大分子的对比度非常高的图像，具有非常高的时间和空间分辨率，并能很好地保存三维结构。因此，我们成功地获得了IP_3R在小脑浦肯野细胞树突中的原位图像。我们将其与RyaR进行了比较，惊奇地发现IP_3R的物理尺寸远远小于两种受体的分子量之比与RyaR的实际尺寸计算的值。我们还发现，在一定的缺氧条件下，四聚体IP_3R在细胞中原位形成非常规则的二维分子阵列。要了解这种阵列的生理作用，还需要进一步的研究。确定受体分子的高分辨率结构以确定与各种配体相互作用的位点也非常重要。

Both inositol-1,4,5-trisphosphate receptor (IP_3R) and ryanodine-receptor (RyaR) form ligand-gated Ca^<2+> -channel and play a crucial role in intracellular signal transduction. Though the total amino-acid sequence was already determined for both, histological studies on these receptors still remain insufficient and nothing substantial has been done on their three-dimensional distribution in cells or tissues. On the structure side, the molecular architecture of tetrameric Rya-R particle has been extensively studied either by negative staining or cryo-electron microscopy, leading to the reconstruction of its three-dimensional structure. Despite many efforts similarly done for IP_3-R,however, nobody has successfully obtained, so far, the concrete information on the molecular architecture of that receptor. The author has been applying his expertised technique of quick-freeze deep-etch replica electron microscopy to the studies of various kinds of protein architecture. With this technique, one obtain very conrasty images of macromolecules in solution or in cells/tissues with very high time-and spacial-resolution, with nicely preserved three-dimensional organization. Thus, we could succesfully obtain the images of IP_3R in situ in the dendrites of cerebellar Purkinje cells. We compared them with those of RyaR and were surprized to find that the physical size of IP_3R is by far smaller than the value calculated from the ratio of molecular weights of two receptors and the actual size of RyaR.We also found that tetrameric IP_3R forms a very regular two-dimensional molecular array in situ in the cell, under certain anoxemic conditions. Further studies must be done to understand the physiological role of such array. It is also of great importance to determine the higher-resolution structure of the receptor molecules to define the sites of interacton with variuos ligands.