Acta Pharmacologica Sinica (2009) 30: 1220–1226; doi: 10.1038/aps.2009.117

Acta Pharmacologica Sinica (2009) 30: 1220–1226; doi: 10.1038/aps.2009.117; published online 24 August 2009

Original Article

Functional study of the effect of phosphatase inhibitors on KCNQ4 channels expressed in Xenopus oocytes

Tzu-rong SU¹, Cay-huyen CHEN², Shih-jen HUANG², Chun-yi LEE², Mao-chang SU³, Gwan-hong CHEN⁴, Shuan-yow LI⁴, Jiann-jou YANG⁴, Min-jon LIN^4,^*

¹Antai Medical Care Cooperation Antai Tian-Sheng Memorial Hospital, Tong-Kang, Pintong, Taiwan, China; ²Department of Anesthesiology, Buddhist Tzu Chi General Hospital, Taipei branch, Taiwan, China; ³Department of Otolaryngology, Chung Shan Medical University Hospital, Taichung, Taiwan, China; ⁴Department of Biomedical Sciences, Chung Shan Medical University, Taichung, Taiwan, China

Aim: 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 Xenopus laevis 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 µmol/L), calyculin A (2 µmol/L) or okadaic acid (1 µmol/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.

Conclusion: 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.

Keywords: KCNQ4 channel; phosphatase inhibitor; calyculin A; okadaic acid; protein kinase C; phosphorylation; electrophysiology; Xenopus oocyte

Prof Thomas J JENTSCH is acknowledged for kindly supplying human KCNQ4 cDNA. These studies were supported by grants from the National Science Council, Taiwan (NSC 96-2320-B-040-013), CSMU-TSMH-097-007, and TCRD-TPE-95-39.

* To whom correspondence should be addressed.
Emal mjl@csmu.edu.tw
Received 2009- 02-25 Accepted 2009-07-02

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