Screening for the smooth muscle relaxants

本研究旨在探讨细胞骨架蛋白对离子型受体活性的影响。细胞松弛素和秋水仙碱是一种破坏细胞膜的物质，当它们被细胞外应用时，会影响某些受体的活性。1996年，我们应用细胞松弛素（cytochalasin，破坏肌动蛋白丝）和鬼笔环肽（phalloidin，稳定肌动蛋白丝），用膜片钳方法记录了嘌呤能P_2x_2离子电流的变化。1997年，我们用RT-PCR方法从PC-12细胞中获得了编码P_2x_2受体的cDNA。然后，我们尝试用pET表达系统在大肠杆菌中表达P_2x_2受体，以检测P_2x_2与细胞骨架蛋白的相互作用。1998年，用该系统转染大肠杆菌，但不产生P_2x_2。我们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PCR产物插入pET表达系统。幸运的是，大肠杆菌转染的系统产生了辣椒素受体。我们通过柱层析大量纯化了受体蛋白。肌动蛋白结合活性检测的受体蛋白preepitating它与肌动蛋白丝。本实验为进一步研究细胞骨架蛋白对P_2x_2活性的影响提供了线索。

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.