Functional consequences of methionine oxidation of hERG potassium channels

Abstract

Reactive species oxidatively modify numerous proteins including ion channels. Oxidative sensitivity of ion channels is often conferred by amino acids containing sulfur atoms, such as cysteine and methionine. Functional consequences of oxidative modification of methionine in human ether à go-go related gene 1 (hERG1), which encodes cardiac I(Kr) channels, are unknown. Here we used chloramine-T (ChT), which preferentially oxidizes methionine, to examine the functional consequences of methionine oxidation of hERG channels stably expressed in a human embryonic kidney cell line (HEK 293) and native hERG channels in a human neuroblastoma cell line (SH-SY5Y). ChT (300 microM) significantly decreased whole-cell hERG current in both HEK 293 and SH-SY5Y cells. In HEK 293 cells, the effects of ChT on hERG current were time- and concentration-dependent, and were markedly attenuated in the presence of enzyme methionine sulfoxide reductase A that specifically repairs oxidized methionine. After treatment with ChT, the channel deactivation upon repolarization to -60 or -100 mV was significantly accelerated. The effect of ChT on channel activation kinetics was voltage-dependent; activation slowed during depolarization to +30 mV but accelerated during depolarization to 0 or -10mV. In contrast, the reversal potential, inactivation kinetics, and voltage-dependence of steady-state inactivation remained unaltered. Our results demonstrate that the redox status of methionine is an important modulator of hERG channel.

Fig. 1

Time- and concentration-dependent effect of ChT on hERG current in HEK 293 cells. A, hERG activating and outward tail currents obtained in the absence and presence of ChT (300 µM). The hERG current was activated by applying a 3-s depolarization step to 0 mV from a holding potential of −80 mV and the outward tail current was recorded during a 4-s repolarization pulse at −50 mV (see inset in A). Pulses were applied once every 15 s during superfusion with ChT. B, pooled data (n = 6–7) showing the time- and concentration- dependent inhibition of hERG tail current. The tail currents were normalized to the respective initial value at time t = 0 s. Straight lines connect the data points.

Fig. 2

Effect of ChT on native hERG current in SH-SY5Y neuroblastoma cells. A, An example of superimposed current traces from a SH-SY5Y neuroblastoma cell before (black) and 300 sec after (grey) application of 300 µM ChT. hERG currents were elicited by 1-sec depolarization to +50 mV from a holding potential of −60 mV. The characteristic inward current was measured during the repolarization phase at −120 mV. B, Pooled data (n = 5) showing the time-dependent inhibition of hERG tail current. The tail current was normalized to the respective initial control value.

Fig. 3

Effect of MSRA on ChT-mediated inhibition of hERG current in HEK 293 cells. In panels A, B, and C, hERG activating and tail currents were recorded in the absence and presence of ChT using the same voltage protocol as shown in Fig. 1A. Current recording was started at least 3 min after obtaining access to whole-cell configuration. ChT (300 µM) was perfused for 1 min and then washed out for 3 to 4 min. A, normal internal solution was used. B, DTT (4 mM) was present in the internal solution. C, bMSRA (15 µg/ml) and DTT (4 mM) were included in the internal solution. D, pooled data showing the time-dependent change of tail currents under the conditions shown in A-C. Tail currents were normalized to the respective initial current size at time t = 0 s. n = 5–10. * P < 0.05 vs (ChT + DTT).

Fig. 4

Effect of ChT on hERG activation kinetics in HEK 293 cells. A, Activation time course of hERG current (at +30 mV) assessed by an envelope of tails test (see inset for clamp protocol) before (left) and after exposure to ChT for 1 min (300 µM) (right). B, pooled data (n = 7) for the activation time courses, which were plots of the tail currents recorded at −100 mV against the activation pulse (+30 mV) duration. C, Activation time course of hERG current (at 0 mV) assessed by an envelope of tails test (see inset for clamp protocol) before (left) and after exposure to ChT for 1 min (300 µM) (right). D, Pooled data (n = 6) for the activation time courses, which were plots of the tail currents recorded at −100 mV against the activation pulse (0 mV) duration. The time course was fit with a single exponential function.

Fig. 5

Current-voltage relationships of hERG K⁺ current in HEK 293 cells before and after ChT application. hERG current was activated by applying voltage pulses for 3 s from −60 to +40 mV (10-mV increments). Outward tail current was recorded upon repolarization to −60 mV for 4 sec. Holding potential was −80 mV. Representative current traces before (A) and after application of 300 µM ChT for 1 min (B). C, pooled current-voltage plot for activating current measured at the end of the depolarizing voltage steps (n = 9). Straight lines connect the data points. D, Pooled current-voltage plot for peak outward tail current (activation curve). The activation curve was fit with a Boltzmann function. The slope factor was increased by ChT (5.5 ± 0.8 vs 6.2 ± 0.3 mV, n = 9, P = 0.005) but the V_1/2 (−28.7 ± 1.0 vs −29.8 ± 0.8 mV, n = 9) was not altered by ChT (P > 0.05).

Fig. 6

Effect of ChT on hERG channel inactivation in HEK 293 cells. A, Representative traces showing the time course of hERG channel inactivation before (left) and after 1-min application of ChT (300 µM) (right). A three-pulse protocol was used to assess inactivation kinetics (see inset). B, Inactivation time constant plotted as a function of test voltage. The time constants were determined by fitting a single exponential function to the inactivating current (in A). At all voltages, the time constants were not significantly influenced by ChT (n = 7). Straight lines connect the data points. C, Representative data showing steady-state inactivation of hERG channels before (left) and after 1-min application of ChT (300 µM) (right). Currents were elicited by the protocol shown in inset in which inactivation was allowed to relax to steady-state during 20-ms test pulses to potentials ranging from −120 to +10 mV. D, Steady-state inactivation curves. Peak outward currents elicited by the second step to +20 mV were corrected for channel closing and plotted as a function of the preceding test pulse potential. Inactivation curve was fit with a Boltzmann function. The estimated peak amplitudes were 15.0 ± 2.5 and 7.4 ± 1.4 nA (n = 6).

Fig. 7

ChT alters hERG tail current kinetics in HEK 293 cells. A, tail currents recorded at −60 mV following a depolarizing step to 0 mV before (control) and after exposure to ChT (300 µM for 1 min). B, Comparison of scaled representative tail currents recorded at −60 mV before and after treatment with ChT. The black lines are double exponential fit of the scaled tail currents plotted in grey. C and D, Pooled data (n = 7) showing changes in the double exponential parameters caused by ChT. Fractional amplitude for the fast or slow component was calculated respectively using the formula: A1/(A1 +A2) or A2/(A1 +A2), where A1 is the amplitude of the fast component of the tail and A2 the amplitude of the slow component.

Fig. 8

ChT does not alter the reversal potential of hERG current in HEK 293 cells. Cells were depolarized from a holding potential of −80 mV to +20 mV for 2 s followed by repolarizing in 5-mV increments to different voltages, which bracketed the reversal potential (see inset in B). A, Representative data obtained from a cell before treatment with ChT. B, Data from the same cell after treatment with ChT. Only part of the activating and tail currents is shown in both panels. The reversal potential was measured as the zero intercept of the linear fit to the instantaneous tail currents at voltages bracketing the reversal potential.