Screening for the smooth muscle relaxants

本研究旨在研究细胞骨架蛋白对亲离子受体活性的影响。细胞松弛素和秋水仙素在细胞外应用时会影响某些受体的活性，而细胞松弛素和秋水仙素可以破坏细胞角质形成。因此，我们预测肌动蛋白和微管蛋白及其相关蛋白可能对电离通道起到调节作用。1996年，我们应用细胞松弛素(破坏肌动蛋白细丝)和鬼臼毒素(稳定肌动蛋白细丝)，并用膜片钳方法记录了嘌呤能P_2x_2离子电流的变化。遗憾的是，我们没有检测到这些药物的任何显著作用。1997年，我们通过RT-PCR方法获得了编码P_2X_2受体的cDNA。然后，我们尝试通过pET表达系统在E.Coli中表达该受体，以检测P_2X_2与细胞骨架蛋白的相互作用。但在1998年，用该系统转导的大肠杆菌不产生P_2X_2。通过对背根神经节进行RT-PCR，获得了编码辣椒素受体的cDNA，并将其插入到pET表达系统中。幸运的是，转化了该系统的大肠杆菌产生了辣椒素受体。我们通过柱层析对受体蛋白进行了大量的纯化。通过将其与肌动蛋白细丝一起作用，检测到该受体蛋白的肌动蛋白结合活性。这一初步实验将为研究细胞骨架蛋白对P_2X_2活性的影响提供线索。

This research aims to examine the effects of cytoskeletal proteins on the activity of ionotropic receptor. It has been known for many years that Cytochalasin and Colchicine, which destroy cytoskeltons, affected some receptor activities, when they were applied extracellularly. Therefore, we expected that actin and tubilin and their associated proteins may exert regulatory roles to the ionotropic channels.In 1996, we applied cytochalasin (which destroys actin filaments) and phalloidin (which stabilizes actin filaments) and recorded the changes in the ionic currents of purinergic P_2x_2 by the patch clamp method. Unfortunately we failed to detect any significant effects of the agents.In 1997, we obtained cDNA coding P_2x_2 receptor by subjecting PC-12 cells to RT-PCR method. We then tried to express the receptor in E.Coli by the pET expression system so that the interaction between P_2x_2 and cytoskeletal proteins was examined. However, E.Coli transfected with the system did not produce P_2x_2.In 1998. we obtained cDNA coding a capsicin receptor by subjecting dorsal root ganglion to RT-PCR, and inserted the PCR product to pET expression system. E.Coli transfected the system fortunately produced the capsicin receptor. We purified the receptor protein by the column chromatographies in a large amount. Actin-binding activity was detected for the receptor protein by precepitating it together with actin filaments. This preliminary experiment will provide a clue to examine the effect of cytoskeletal proteins on P_2x_2 activity.