Mutant MiRP1 subunits modulate HERG K+ channel gating: a mechanism for pro-arrhythmia in long QT syndrome type 6

Abstract

Mutations in KCNE2, which encodes the minK-related protein 1 (MiRP1), are associated with an increased risk of arrhythmias; however, the underlying mechanisms are unknown. MiRP1 is thought to associate with many K+ channel alpha-subunits, including HERG K+ channels, which have a major role in suppressing arrhythmias initiated by premature beats. In this study we have investigated in Chinese hamster ovary (CHO) cells at 37 degrees C the effects of co-expressing HERG K+ channels with either wild-type (WT) MiRP1 or one of three mutant MiRP1 subunits, T8A, Q9E and M54T. The most significant effects of MiRP1 subunits on HERG channels were a more negative steady-state activation for HERG + T8A MiRP1 and a more positive steady-state activation for HERG + M54T MiRP1 compared to either HERG + WT MiRP1 or HERG alone. All three mutants caused a significant slowing of deactivation at depolarised potentials. T8A MiRP1 also caused an acceleration of inactivation and recovery from inactivation compared to HERG + WT MiRP1. During ventricular action potential clamp experiments there was a significant decrease in current in the early phases of the action potential for HERG + WT MiRP1 channels compared to HERG alone. This effect was not as prominent for the mutant MiRP1 subunits. During premature action potential clamp protocols, the T8A and Q9E mutants, but not the M54T mutant, resulted in significantly larger current spikes during closely coupled premature beats, compared to HERG + WT MiRP1. At longer coupling intervals, all three mutants resulted in larger current spikes than HERG alone or HERG + WT MiRP1 channels. It is therefore possible that augmentation of HERG currents in the early diastolic period may be pro-arrhythmic.

Figure 3. Effect of T8A MiRP1 subunits on inactivation of HERG

A, inactivation at +20 mV for HERG + WT MiRP1 (thin trace) and HERG + T8A MiRP1 (thick trace). B, recovery from inactivation at -60 mV for HERG + WT MiRP1 (thin trace) and HERG + T8A MiRP1 (thick trace). C, summary of voltage dependence of rates of inactivation for HERG + WT (□) compared to HERG + MiRP1 T8A (▪).

Figure 1. Effect of mutant MiRP1 subunits on steady-state activation of HERG

Steady-state activation (A) and steady-state inactivation (B) curves for HERG alone (▪) and HERG co-expressed with WT (□), T8A (•), Q9E (○) or M54T (▴) MiRP1 subunits. The protocols used to record currents for activation and inactivation are illustrated in the insets (see text for further details). C, summary of the V_1/2 of activation (▪) and inactivation (□) for HERG alone and HERG expressed with WT or mutant MiRP1 subunits. Data are means ± S.E.M. (n = 4-7).

Figure 2. Effect of M54T MiRP1 subunits on deactivation of HERG

A, deactivation at -60 and -120 mV for HERG + WT MiRP1 (thin trace) and HERG + M54T MiRP1 (thick trace). B, voltage dependence of the rate constants of deactivation (plotted on a semi-logarithmic scale) for HERG + WT and HERG + M54T MiRP1.

Figure 4. Effect of mutant MiRP1 subunits on HERG currents recorded during ventricular AP waveforms

Typical current trace recorded during a ventricular AP waveform for HERG alone (thin trace) compared to: HERG + WT MiRP1 (A), HERG + T8A MiRP1 (B), HERG + Q9E MiRP1 (C), and HERG + M54T MiRP1 (D). All currents were normalised relative to the maximum current recorded during the AP waveform. Dotted lines indicate zero current line.

Figure 5. Effects of mutant MiRP1 subunits on HERG currents recorded during premature ventricular AP waveforms

A, typical examples of currents recorded from HERG alone, HERG + WT MiRP1 and HERG + T8A MiRP1, during premature ventricular AP clamp protocols where the premature stimulus was introduced at the point of APD₉₀ - 20 ms, APD₉₀, APD₉₀ + 20 ms, APD₉₀ + 50 ms and APD₉₀ + 100 ms (voltage waveform shown above). All current traces have been normalised to the maximum current during the premature depolarisation. B, the magnitude of the transient outward current recorded during premature ventricular AP clamp protocols plotted versus the coupling interval for the premature beat for HERG alone (▪, n = 4), and HERG co-expressed with WT (□, n = 3), T8A (•, n = 3), Q9E (○, n = 3) or M54T (▴, n = 3) MiRP1 subunits. Current magnitude has been normalised to the maximum transient outward current response. The coupling interval was defined relative to APD₉₀ of the ventricular AP waveform, which in these experiments was 300 ms. For purposes of clarity, error bars are not shown.

Figure 6. Summary of effects of mutant MiRP1 subunits on HERG currents recorded during premature ventricular AP waveforms

A, the magnitude of the peak outward current spike recorded during premature ventricular AP clamp protocols (I_prem) normalised to the peak current recorded during the preceding AP waveform (I_AP). Numbers above the bars indicate (1) significantly different from HERG + WT MiRP1 (ANOVA, P < 0.05) and (2) significantly different from HERG alone (ANOVA, P < 0.05). B and C, the normalised values of the peak current (I_prem/I_AP) recorded during premature ventricular AP clamp protocols plotted versus the coupling interval for the premature beat: HERG co-expressed with WT (□, n = 3), T8A (•, n = 3) or Q9E (○, n = 3)MiRP1 subunits (B); and HERG co-expressed with WT (□, n = 3), HERG alone (▪, n = 4) or M54T (▴, n = 3) MiRP1 subunits (C). The coupling interval was defined relative to APD₉₀ of the ventricular AP waveform, which in these experiments was 300 ms. For purposes of clarity, error bars are not shown.