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

Abstract

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 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.

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.

Figure 1

The expressed currents of KCNQ4 were blocked by the addition of linopirdine on Xenopus oocytes. (A) Typical current traces were recorded from an oocyte cell expressing KCNQ4 channels with a voltage step protocol as follows: the oocyte was clamped at −60 mV for 3 s, and the channel was activated by 2-s command steps from −100 mV to +60 mV in 10-mV increments, followed by a 1-s step to −30 mV. The calibration scale of all current traces is in the upper right corner of (A): 2 s and 1 μA. The calibration scale of the voltage step protocol is shown between (A) and (B): 2 s and 50 mV. (B) The expressed KCNQ4 current was almost completely blocked by the application of linopirdine (0.25 mmol/L) after 5 min. (C) This inhibitory effect of linopirdine on the KCNQ4 current was reversible by washout. (D) The summarized curves of tail current-voltage relationships are shown in the absence (•) and presence (□) of linopirdine. The control curves, with oocytes of RNase-free water injection (○) and without injection (△) are also shown for comparison. (E) Time course effect of linopirdine on KCNQ4 tail-current (•). Treatment with linopirdine and washout are indicated with arrows. The control curve (without chemical treatment; ○) showed little run-down during the 50-min recording. The time course of the tail-current amplitude was measured at a tail potential of −30 mV (from a step command of 30 mV).

Figure 2

BIMI, a PKC inhibitor, can antagonize the effect of PMA on KCNQ4 channels. (A) The amplitudes of control KCNQ4 currents (upper traces in A) were inhibited by the application of PMA (2 μmol/L) (bottom traces in A). (B) The inhibitory effect of PMA on the KCNQ4 current was attenuated by pretreatment with BIMI (2 μmol/L) (upper traces: BIMI alone; bottom traces: BIMI+PMA). The voltage step protocol of (A) and (B) is as indicated in the middle of (A). Calibration scale of all current traces: 2 s and 1 μA. Calibration scale of the voltage step protocol: 2 s and 50 mV. (C) The midpoint potential of the conductance-voltage curve (V_1/2) was shifted significantly to a more positive value after treatment with PMA (before PMA: • after PMA: ○). (D) Representative time courses of KCNQ4 tail-current during application of 2 μmol/L PMA (•) or vehicle (○). The effect of PMA was reversible by washout, as indicated by arrows. (E) The shift effect of V_1/2 produced by PMA was attenuated by pretreatment with BIMI (BIMI alone: ▴ BIMI+PMA: △). V_1/2 was obtained from the conductance-voltage curves, which were fitted using a two-state Boltzmann equation as described in the Materials and Methods.

Figure 3

Effect of phosphatase inhibitor cyclosporine (2 μmol/L) on the KCNQ4 current expressed in Xenopus oocytes. (A) The control KCNQ4 currents (upper current traces) were significantly inhibited by the addition of cyclosporine (2 μmol/L; lower current traces). Calibration scale of current traces: 2 s and 1 μA (upper right corner). Calibration scale of voltage step protocol: 2 s and 50 mV (between two current traces). (B) The V_1/2 was shifted significantly to a more positive value after the treatment with cyclosporine (control: • cyclosporine treatment: ○).

Figure 4

Effects of phosphatase inhibitors (calyculin A and okadaic acid) and the combination of phosphatase inhibitor with PMA on the KCNQ4 currents. Typical recording traces of KCNQ4 are shown in figures (A)(F). The traces in Figures (A)–(C) and Figures (D)–(F) are recorded from the same oocyte, respectively. Tail current amplitudes of control KCNQ4 (A) were significantly inhibited by the addition of 2 μmol/L calyculin A (B) and, subsequently, addition of PMA (2 mol/L) caused a further decrease in the current amplitudes (C). The effect of okadaic acid (1 μmol/L) on the KCNQ4 current amplitudes was similar to that of calyculin A (D: before the treatment with okadaic acid; E: after the treatment with okadaic acid; F: okadaic acid plus 2 mol/L PMA). All static values of current amplitudes are shown in the results section. Calibration scales of all current traces are shown in the upper right corners of (A) and (D): 2 s and 2 μA. The voltage-step protocol is shown below (A). Figures (G) and (H) show conductance-voltage (G–V) curves of KCNQ4. Both calyculin A and okadaic acid can produce a positive shift of the half voltage of the GV curve (•: before the treatment in Figs G and H; △: after treatment with calyculin A in Figure G; ○: after treatment with okadaic acid in Figure H). Subsequently, the further addition of PMA after the phosphatase inhibitor can produce a more positive shift of V1/2 (□: calyculin A plus PMA and okadaic acid plus PMA in Figures G and H, respectively).