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

Abstract

npg Introduction KCNQ4, a novel potassium channel, plays a part in regulating the membrane potential and function of various cell types in the body [1] . The KCNQ4 current is a low-threshold, slow activating and noninactivating current that is expressed in the outer hair cells of the cochlea, brain, and heart. Mutations in KCNQ4 give rise to an inherited syndrome of deafness [2] . Therefore, the regulatory pathways of KCNQ4 channels play an important role in adjusting the function of outer hair cells. The KCNQ gene subfamily is composed of five K + channels, KCNQ1 to KCNQ5. The KCNQ4 channel was included in the Kv nomenclature as Kv 7.4 (voltage-gated potassium channel subunit Kv7.4) [3] . The heteromers of KCNQ2/KCNQ3 underlie the neuronal M-current, which modulates neuronal excitability. Many intracellular messengers, eg, PIP2, IP3, diacylglycerol (DAG), calmodulin, calcineurin, activators/inhibitors of PKC, tyrosine kinases, and myosin light chain kinase, have been reported to modulate M currents [4][5][6] . Moreover, the A-kinase-anchoring protein AKAP150, which binds PKC, facilitates the inhibition of KCNQ2 current [7] . Analysis of recombinant KCNQ2 channels suggests that targeting of PKC through association with AKAP150 is important for inhibition. However, the effect of PKC activation on KCNQ channels is still controversial [8] . The inhibition of the metabolism of DAG by DAG kinase blockers cannot mimic the effect of muscarinic modulation by muscarinic agonist, and it has been suggested 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 ...

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