Functional study of the effect of phosphatase inhibitors on KCNQ4 channels expressed in Xenopus oocytes.
OBJECTIVE
KCNQ4 channels play an important part in adjusting the function of cochlear outer hair cells. The aim of this study was to investigate the effects of ser/thr phosphatase inhibitors on human KCNQ4 channels expressed in Xenopuslaevis oocytes.


METHODS
Synthetic cRNA encoding human KCNQ4 channels was injected into Xenopus oocytes. We used a two-electrode voltage clamp to measure the ion currents in the oocytes.


RESULTS
Wild-type KCNQ4 expressed in Xenopus oocytes showed the typical properties of slow activation kinetics and low threshold activation. The outward K(+) current was almost completely blocked by a KCNQ4 blocker, linopirdine (0.25 mmol/L). BIMI (a PKC inhibitor) prevented the effects of PMA (a PKC activator) on the KCNQ4 current, indicating that PKC may be involved in the regulation of KCNQ4 expressed in the Xenopus oocyte system. Treatment with the ser/thr phosphatase inhibitors, cyclosporine (2 micromol/L), calyculin A (2 micromol/L) or okadaic acid (1 micromol/L), caused a significant positive shift in V(1/2) and a decrease in the conductance of KCNQ4 channels. The V(1/2) was shifted from -14.6+/-0.5 to -6.4+/-0.4 mV by cyclosporine, -18.8+/-0.5 to -9.2+/-0.4 mV by calyculin A, and -14.1+/-0.5 to -0.7+/-0.6 mV by okadaic acid. Moreover, the effects of these phosphatase inhibitors (okadaic acid or calyculin A) on the induction of a positive shift of V(1/2) were augmented by further addition of PMA.


CONCLUSIONS
These results indicate that ser/thr phosphatase inhibitors can induce a shift to more positive potentials of the activation curve of the KCNQ4 current. It is highly likely that the phosphatase functions to balance the phosphorylated state of substrate protein and thus has an important role in the regulation of human KCNQ4 channels expressed in Xenopus oocytes.
Su
Tzu-rong
TR
Antai Medical Care Cooperation Antai Tian-Sheng Memorial Hospital, Tong-Kang, Pintong, Taiwan, China.
Chen
Cay-huyen
CH
Huang
Shih-jen
SJ
Lee
Chun-yi
CY
Su
Mao-chang
MC
Chen
Gwan-hong
GH
Li
Shuan-yow
SY
Yang
Jiann-jou
JJ
Lin
Min-jon
MJ
D000200:Action Potentials / Q000187:drug effects; D000818:Animals; D016572:Cyclosporine / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D051656:KCNQ Potassium Channels / Q000187:drug effects*; D008301:Maleimides / Q000494:pharmacology; D008387:Marine Toxins; D019319:Okadaic Acid / Q000494:pharmacology; D009865:Oocytes / Q000378:metabolism; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors*; D026902:Potassium Channel Blockers / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011725:Pyridines / Q000494:pharmacology; D014982:Xenopus laevis
D007211:Indoles; D051656:KCNQ Potassium Channels; C117367:KCNQ4 protein, human; D008301:Maleimides; D008387:Marine Toxins; D010080:Oxazoles; D026902:Potassium Channel Blockers; D011725:Pyridines; D019319:Okadaic Acid; C059041:calyculin A; D016572:Cyclosporine; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; D010744:Phosphoric Monoester Hydrolases; C061017:linopirdine; C070515:bisindolylmaleimide I
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