Rosiglitazone inhibits Kv4.3 potassium channels by open-channel block and acceleration of closed-state inactivation

Abstract

Background and purpose: Rosiglitazone is a widely used oral hypoglycaemic agent, which improves insulin resistance in type 2 diabetes. Chronic rosiglitazone treatment is associated with a number of adverse cardiac events. The present study was designed to characterize the effects of rosiglitazone on cloned K(v)4.3 potassium channels.

Experimental approach: The interaction of rosiglitazone with cloned K(v)4.3 channels stably expressed in Chinese hamster ovary cells was investigated using whole-cell patch-clamp techniques.

Key results: Rosiglitazone decreased the currents carried by K(v)4.3 channels and accelerated the current inactivation, concentration-dependently, with an IC(50) of 24.5 µM. The association and dissociation rate constants for rosiglitazone were 1.22 µM(-1)·s(-1) and 31.30 s(-1) respectively. Block by rosiglitazone was voltage-dependent, increasing in the voltage range for channel activation; however, no voltage dependence was found in the voltage range required for full activation. Rosiglitazone had no effect on either the deactivation kinetics or the steady-state activation of K(v)4.3 channels. Rosiglitazone shifted the steady-state inactivation curves in the hyperpolarizing direction, concentration-dependently. The K(i) for the interaction between rosiglitazone and the inactivated state of K(v)4.3 channels was 1.49 µM, from the concentration-dependent shift in the steady-state inactivation curves. Rosiglitazone also accelerated the kinetics of the closed-state inactivation of K(v)4.3 channels. Rosiglitazone did not affect either use dependence or recovery from inactivation of K(v)4.3 currents.

Conclusion and implications: Our results indicate that rosiglitazone potently inhibits currents carried by K(v)4.3 channels by interacting with these channels in the open state and by accelerating the closed-state inactivation of K(v)4.3 channels.

Figure 1

(A) Concentration-dependent effect of rosiglitazone on K_v4.3 channel currents following a 500 ms depolarizing pulse to +40 mV from a holding potential of −80 mV at 10 s intervals. The inset shows the first 50 ms of the current recordings on an expanded time scale. (B) Concentration–response curve for rosiglitazone was fitted to the Hill equation to obtain the IC₅₀ value. Data are expressed as means ± SE.

Figure 2

Time-dependent block of K_v4.3 channels by rosiglitazone. (A) The fractional block was calculated by subtracting the ratio of the current before and after addition of the drug from unity. This procedure was carried out at four different drug concentrations. The time courses of inhibition were fit to a biexponential function that yielded the concentration-dependent time constants. (B) The inverse of the fast components of the time constants were plotted against the rosiglitazone concentrations. The solid line represents the least-squares fit of the data to the following equation: 1/τ_Block=k₊₁[D]+k₋₁. Data are expressed as means ± SE.

Figure 3

Effect of rosiglitazone on current–voltage relationships. The representative whole-cell K_v4.3 channel current traces under control conditions (A) and in the presence of rosiglitazone (B). The inset shows the first 50 ms of the current recordings on an expanded time scale. (C) Current–voltage curves for rosiglitazone-induced K_v4.3 currents. The data were taken from the peak amplitude of K_v4.3 currents under control conditions and in the presence of rosiglitazone. (D) The integral of the current in the presence of rosiglitazone was normalized to that of the control at each voltage. In the voltage range for channel activation between −20 and +20 mV, the block of K_v4.3 channels increased and was significantly different from inhibition at −20 mV (n = 6, *P < 0.05). The dotted line represents the activation curve of K_v4.3 channels under control conditions. Data are expressed as means ± SE.

Figure 4

Effect of rosiglitazone on the deactivation kinetics of K_v4.3 channels. Tail currents were obtained at −60 mV after 8 ms depolarizing pulses to +40 mV at 10 s intervals in the absence and presence of rosiglitazone. The solid lines represent the single exponential fit and the dotted line represents zero current.

Figure 5

Effect of rosiglitazone on the steady-state activation of K_v4.3 channels. (A) The representative tail current traces of the K_v4.3 channels in the absence and presence of rosiglitazone. The inset shows the tail currents on an expanded time scale. (B) The normalized currents were plotted as a function of the test potentials, and the resulting curves were well fit to the Boltzmann equation. Data are expressed as means ± SE.

Figure 6

Effect of rosiglitazone on the steady-state inactivation of K_v4.3 channels. (A) Representative K_v4.3current traces in the absence and presence of rosiglitazone were recorded using a two-pulse protocol. Normalized currents of the K_v4.3 channels, which were the currents after the second pulse relative to the current recorded after the first pulse, are shown as a function of the holding potentials. (B) The plot of exp (ΔV/k) against rosiglitazone concentrations. The V_1/2 and k values were obtained from the steady-state inactivation curves. The concentration-dependent shift in the midpoint (ΔV) was determined as the difference between V_1/2 values in control conditions and at 30 and 100 µM rosiglitazone (n = 6). The affinity of rosiglitazone for the inactivated state of Kv4.3 can be calculated on the basis of this shift: −ΔV/k = ln[(1 + D/K_i)/(1 + D/K_R)] where D is the drug concentration, and K_i and K_R are the apparent dissociation constants for the inactivated and resting states respectively (Bean et al., 1983). If one assumes that K_R is very large, the following equation can be used to calculate K_i: exp (−ΔV/k) = D/K_i+ 1. The solid line represents the linear fit of the data: exp (ΔV/k) = 0.67 [rosiglitazone]− 3.09, where [rosiglitazone] represents the rosiglitazone concentration. K_i, the reciprocal of the slope, was calculated from this fit. Data are expressed as means ± SE.

Figure 7

Effects of rosiglitazone on the kinetics of K_v4.3 channels during closed-state inactivation. (A) K_v4.3 currents were recorded at +40 mV using a double-pulse protocol. The control pulse was applied from a membrane potential of −100 mV. (B) The current amplitudes evoked by the second pulse, relative to the amplitude resulting from the initial control pulse, were plotted against the duration of the conditioning pulse. The data were well fit to a single exponential function (n = 9). Data are expressed as means ± SE.

Figure 8

Effect of rosiglitazone on use-dependent inhibition of K_v4.3 channels. (A) K_v4.3 current traces obtained from depolarizing pulses at 1 or 2 Hz in the absence and presence of rosiglitazone. (B) Plot of normalized currents as a function of the number of pulses. The peak amplitudes of the current after each pulse were normalized to the peak amplitude of the current obtained after the first pulse. Data are expressed as means ± SE.

Figure 9

Effects of rosiglitazone on recovery from inactivation of K_v4.3 channels. (A) A double-pulse protocol was used to characterize the recovery of K_v4.3 channels from inactivation in the absence and presence of rosiglitazone. (B) The solid lines represent the single exponential fit of the peak amplitude of the K_v4.3 currents as a function of the interpulse interval. Data are expressed as means ± SE.