Study of the mechanism of different regulation of Kir current in two expressions systems by PKC / 中国药理学通报

ABSTRACT

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 PIP2 hydrolysis.Following the same line,the inability of PKC inhibition of Kir 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 PLC?1PH domain known to be able to bind PIP2 specifically. There was no significant change of PIP2 level on COS-7 cell membrane after longtime treatment of PMA,whereas again,the activation of M1 receptor by ACh induced a significant change in the PIP2 level.These results were in perfect agreement with the electrophysiological results.Conclusions PMA,through activation of PKC,inhibited Kir 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.