PKC对两种表达系统中内向整流性钾通道调节的不同及机制研究

Study of the mechanism of different regulation of Kir current in two expressions systems by PKC

目的分别在蛙卵细胞和COS-7细胞中表达Kir2.3通道,观察PKC的激动剂PMA对两种细胞中表达的Kir2.3通道电流调节的不同,并通过共聚焦显微镜扫描方法研究PIP2水解在PKC调节这两种细胞中Kir通道的作用。方法用显微注射方法和磷酸钙介导的质粒DNA细胞转染法分别将Kir2.3通道表达于蛙卵细胞和培养的COS-7细胞中,用双电极电压钳方法和全细胞电流膜片钳记录法检测PMA对两种细胞中表达的Kir2.3通道电流的作用。用共聚焦显微镜扫描法检测细胞膜PIP2水解。结果在蛙卵细胞的双电极电压钳实验中,PMA能够明显抑制蛙卵细胞中表达的Kir2.3通道的电流,而PMA不能抑制COS-7细胞中表达的Kir2.3通道的电流。激光共聚焦扫描的方法结果发现PMA不能使COS-7细胞膜上的PIP2水解,这一结果同细胞电生理的结果相一致。提示了PIP2的水解在PKC对Kir通道的调节过程中起重要的作用。结论PMA通过激活PKC抑制表达于蛙卵细胞中的Kir2.3电流而对表达于COS-7细胞中的Kir2.3电无作用;PMA可促进蛙卵细胞中的PIP2水解,而对COS-7细胞中的PIP2无影响;因此,PIP2的水解在PKC对Kir通道的调节中可能起着重要的作用。

Aim To study the regulatory effects of PMA, a PKC activator, on Kir 2.3 channel function expressed in Xenopus oocytes and COS-7 cells, and PIP2 involvement in these regulations. Methods Kir 2.3 channel was expressed in Xenopus oocytes and COS-7 cell by RNA microinjection and DNA transfection using calcium phosphate precipitate, respectively. Two-electrode-voltage-clamp and whole-cell patch clamp were used to record the Kir 2. 3 current in Xenopus oocytes and COS-7 cell. The PIP2 hydrolysis was detected by confocal microscopy. Results PMA significantly inhibited Kir 2. 3 current in Xenopus oocytes. But PMA had no effect on the Kir 2.3 current expressed in COS- 7 cell,in which activation of M1 receptor, however, induced a significant inhibition of Kir 2. 3 current. It was reported recently that PMA could trigger the PIP2 hydrolysis in membrane of oocytes. Thus PKC inhibition of Kir 2. 3 current seen in oocytes could be the result of PIP： hydrolysis. Following the same line, the inability of PKC inhibition of Kit 2. 3 current seen in COS-7 cells would suggest PKC could not induce PIP2 hydrolysis in these cells. This hypothesis was tested by monitoring the PIP2 level in COS-7 cell membrane by confocal microscopy. Dynamic changes in membrane PIP2 level were imaged using GFP fluorescence signal that had been tagged to the PLCδ1PH domain known to be able to bind PIP2 specifically. There was no significant change of PIP2 level on COS - 7 cell membrane after long time treatment of PMA, whereas again, the activation of M1 receptor by ACh induced a significant change in the PIPE level. These results were in perfect agreement with the eleetrophysiologieal results. Conclusions PMA, through activation of PKC, inhibited Kit 2.3 current expressed in Xenopus oocytes but not in COS-7 cells. Similarly PMA induced significant reduction in membrane PIP2 level in Xenopus oocytes but not in COS-7 cells. PIP2 hydrolysis plays an important role in PKC- induced inhibition of the Kir channel currents.