Screening for the smooth muscle relaxants

本研究旨在探讨细胞骨架蛋白对嗜离子受体活性的影响。细胞松弛素和秋水仙碱具有破坏细胞骨架的作用，在细胞外作用时，它们会影响某些受体的活性，这一点多年来已经为人所知。因此，我们预计肌动蛋白和tubilin及其相关蛋白可能对电离性通道发挥调节作用。1996年，我们应用破坏肌动蛋白丝的细胞松弛素和稳定肌动蛋白丝的phalloidin，用膜片钳法记录了purinergic P_2x_2离子电流的变化。不幸的是，我们没有发现药剂的任何显著效果。1997年，我们将PC-12细胞进行RT-PCR，获得了编码p2x_2受体的cDNA。利用pET表达系统在大肠杆菌中表达该受体，研究了p2x_2与细胞骨架蛋白的相互作用。然而，转染该系统的大肠杆菌不产生P_2x_2。在1998年。我们利用背根神经节进行RT-PCR，获得辣椒素受体的cDNA编码，并将PCR产物插入pET表达系统。大肠杆菌转染系统后幸运地产生了辣椒素受体。我们用柱层析法纯化了大量的受体蛋白。通过与肌动蛋白丝一起沉淀，检测受体蛋白的肌动蛋白结合活性。本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.