State-dependent blockade of human ether-a-go-go-related gene (hERG) K(+) channels by changrolin in stably transfected HEK293 cells

Abstract

Aim: To study the effect of changrolin on the K(+) channels encoded by the human ether-a-go-go-related gene (hERG).

Methods: hERG channels were heterologously stably expressed in human embryonic kidney 293 cells, and the hERG K(+) currents were recorded using a standard whole-cell patch-clamp technique.

Results: Changrolin inhibited hERG channels in a concentration-dependent and reversible manner (IC(50)=18.23 mumol/L, 95% CI: 9.27-35.9 mumol/L; Hill coefficient=-0.9446). In addition, changrolin shifted the activation curve of hERG channels by 14.3+/-1.5 mV to more negative potentials (P<0.01, n=9) but did not significantly affect the steady-state inactivation of hERG (n=5, P>0.05). The relative block of hERG channels by changrolin was close to zero at the time point of channel opening by the depolarizing voltage step and quickly increased afterwards. The maximal block was achieved in the inactivated state, with no further development of the open channel block. In the "envelope of tails" experiments, the time constants of activation were found to be 287.8+/-46.2 ms and 174.2+/-18.4 ms, respectively, for the absence and presence of 30 mumol/L changrolin (P<0.05, n=7). The onset of inactivation was accelerated significantly by changrolin between -40 mV and +60 mV (P<0.05, n=7).

Conclusion: The results demonstrate that changrolin is a potent hERG blocker that preferentially binds to hERG channels in the open and inactivated states.

Figure 1

Chemical structure of changrolin [2,6-bis(pyrrolidin-1-ylmethyl)-4-(quinazolin-4-ylamino) phenol].

Figure 2

Inhibition of hERG channels by changrolin. (A) Representative current traces recorded from the same cell under control conditions and after superfusion with changrolin (10 μmol/L, 30 μmol/L and 100 μmol/L). (B) Concentration-response relationship of the effects of changrolin on hERG peak tail currents (n=7). The IC₅₀ was 18.23 μmol/L (95% CI: 9.27–35.9 μmol/L) with a Hill coefficient of −0.9446. (C) Time course of hERG tail current inhibition by 30 μmol/L changrolin (n=6).

Figure 3

Effects of changrolin on the voltage dependence of hERG channel activation. Control measurement (A) and the inhibitory effect of 30 μmol/L changrolin (B) are shown in the same cell. (C) The corresponding activating current amplitude at the end of the first test pulse is shown as a function of the test pulse potentials. Following the application of 30 μmol/L changrolin, the peak current amplitude was reduced by 42.1%±7.7% (n=6). (D) The tail current amplitude as a function of the preceding test pulse potentials. The application of 30 μmol/L changrolin reduced the peak tail current amplitude by 59.9%±3.1% (n=6). (E and F) Normalized activating and tail currents in the absence and presence of 30 μmol/L changrolin. Changrolin shifted the peak activating currents by 10 mV toward more negative potentials. The activation curves of hERG tail currents were also shifted by 13.3±2.2 mV toward more negative potentials (P<0.01).

Figure 4

Changrolin shifted the hERG current activation curves to more negative potentials. The activation of the hERG current curves was studied in detail using a two-step protocol (lower panel) that gave rise to large inward tail currents. (A and B) A representative experiment in the absence and presence of 30 μmol/L changrolin. (C) Peak inward tail currents were normalized to the maximum currents, plotted as a function of the preceding test pulse potential and fitted to a Boltzmann equation to obtain activation curves. Changrolin caused a significant shift in the mean half-maximal activation voltage by 14.3±1.5 mV toward more negative potentials (P<0.01, n=9).

Figure 5

Changrolin blocked hERG channels that were in the open and inactivated states. To determine if hERG channels are blocked by changrolin in the open or the closed states, the voltage was stepped from −80 mV to 0 mV for 7.5 s to induce a large activation current. Shown are overlay experiments (A) for control and after incubation with 100 μmol/L changrolin (for 5 min, without intermittent test pulses). (B) The normalized relative block was plotted versus time after a voltage step to 0 mV, indicating a rapid open channel block without a major blockade of closed channels. Similar results were obtained in five experiments. (C) Inhibition of inactivated channels by 30 μmol/L changrolin. To investigate whether hERG channels were blocked by changrolin in the inactivated state, a holding potential of –80 mV kept hERG channels in their closed state while a 4000-ms test pulse to +80 mV led to channel inactivation, which was followed by channel opening at 0 mV. The corresponding normalized relative block during the test pulse to 0 mV is shown in (D). Maximum inhibition was achieved at the beginning of the second pulse, and no further time-dependent blockade occurred upon channel opening during the second voltage step. Similar results were obtained in six experiments.

Figure 6

Development of hERG channel blockade during depolarization was assessed using the “envelope of tails” protocol (lower panel). (A and B) Representative original current traces of the hERG channel, elicited by the “envelope of tails” protocol in the absence and presence of 30 μmol/L changrolin. (C) The peak tail current elicited by a repolarizing step to −50 mV was plotted as a function of the test pulse duration. Solid lines were obtained by fitting with a single exponential function. (D) The pooled time constants of hERG current activation in the absence and presence of 30 μmol/L changrolin were 287.8±46.2 ms and 174.2±18.4 ms, respectively (differences were considered significant, with ^bP<0.05, n=7).

Figure 7

Effects of changrolin on hERG current inactivation. (A) Time constants of inactivation were investigated using a three-stage voltage protocol (inset). The original traces in the absence and presence of 30 μmol/L changrolin are shown. (B) Voltage dependence of the time constants for the onset of inactivation in the absence and presence of changrolin. The onset of inactivation was accelerated significantly at potentials between −40 mV and +60 mV (differences were considered significant with ^bP<0.05, n=7). (C) Representative current traces of steady-state inactivation were elicited by the protocol (inset), and the region of interest is magnified for clarity. (D) The inactivating outward current amplitude measured at +60 mV was normalized and fitted to a Boltzmann function to obtain steady-state inactivation curves. Only a small shift was observed in the steady-state inactivation curves without significance (n=5, P>0.05).