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Article in Chinese | WPRIM | ID: wpr-564731


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.

Article in Chinese | WPRIM | ID: wpr-555995


Aim To visualize the dynamics of PtdIns(4,5)P 2 hyd ro lysis and resynthesis, and modulate it by pharmacological agents wortmannin, LiC l, U73122 and neomycin. Methods We used a fusion construct of g reen fluorescent protein(GFP) with the PH domain of phospholipase C ?1(PL C ?1PH)(PLC ?1PH-GFP) known to bind PtdIns(4,5)P 2 specifically , and laser-scanning confocal microscopy to trace PtdIns(4,5)P 2 translocatio n. Results Stimulation of endogenous P 2Y receptors by ATP in CHO cells induced a reversible PLC ?1PH-GFP translocation, indicating Pt dIns(4,5)P 2 hydrolysis through the receptor-mediated phospholipase C (PLC) ac tivation. Wortmannin and LiCl did not affect the translocation of PLC ?1PH -GFP from plasma membrane to cytosol but blocked the recovery after the translo cation. The transient translocation from plasma membrane was blocked by the PLC inhibitor U73122 but was not affected by another PLC inhibitor neomycin. However , in the absence of PLC ?1PH-GFP expression, neomycin inlibited the recep tor-induced PLC hydrolysis of PtdIns(4,5)P 2.Conclusion PLC ?1PH-GFP can be used as a valuable fluorescence probe to visualize the dyn amic change of PtdIns(4,5)P 2 in living cells. Wortmannin, LiCl, U73122 and neo mycin have distinct modulation effects on PtdIns(4,5)P 2 metabolism. PLC ?1 PH,when bound to PtdIns(4,5)P 2,prevents neomycin from inhibiting PLC hydro lyzing PtdIns(4,5)P 2.

Article in Chinese | WPRIM | ID: wpr-555992


Aim To study the modulation of KCNQ2/3 potassium cha nn els by extracellular pH.Methods In vitro transcription was used to synthesize cRNA of KCNQ2/3 potassium channels.The cRNA was injected into Xenopus oocytes to express the KCNQ2/3 channel.The modulation of KCNQ2/3 potass ium channels by extracellular pH was studied by two electrodes voltage clamp tec hniques.Results KCNQ2/3 currents were inhibited and current-vo ltage relationship of activation were shifted to the right with decreased extrac ellular pH. pH modulation of KCNQ2/3 currents was voltage dependent,with a more pronounced effect at more negative potentials above the activation threshold (-60 mV). Extracelluar pH also decreased activation and deactivation kinetics of KCNQ2/3 currents.Conclusion KCNQ2/3 channels, known to contr ibute to neuronal excitability, were modulated by extracelluar pH. The profound effects of the extracelluar pH exerted on KCNQ2/3 channel may play an important role during physiology neuronal activity and pathological events such a s epileptic seizures, cerebral ischemia and shock etc.

Article in Chinese | WPRIM | ID: wpr-678719


Kir2 1. Moreover, drop of pHi reduced the M 1 induced inhibition of Kir3 1/3 4 currents, and enhanced the desensitization of M 2 induced Kir3 1/3 4 activation. CONCLUSION The basal currents and M receptor induced currents of Kir3 1/3 4 can be regulated by intracellular pH. These changes may play some important roles in pathophysiological conditions like cardiac ischemia.