Chloroquine blocks a mutant Kir2.1 channel responsible for short QT syndrome and normalizes repolarization properties in silico

Abstract

Short QT Syndrome (SQTS) is a novel clinical entity characterized by markedly rapid cardiac repolarization and lethal arrhythmias. A mutation in the Kir2.1 inward rectifier K+ channel (D172N) causes one form of SQTS (SQT3). Pharmacologic block of Kir2.1 channels may hold promise as potential therapy for SQT3. We recently reported that the anti-malarial drug chloroquine blocks Kir2.1 channels by plugging the cytoplasmic pore domain. In this study, we tested whether chloroquine blocks D172N Kir2.1 channels in a heterologous expression system and if chloroquine normalizes repolarization properties using a mathematical model of a human ventricular myocyte. Chloroquine caused a dose- and voltage-dependent reduction in wild-type (WT), D172N and WT-D172N heteromeric Kir2.1 current. The potency and kinetics of chloroquine block of D172N and WT-D172N Kir2.1 current were similar to WT. In silico modeling of the heterozygous WT-D172N Kir2.1 condition predicted that 3 microM chloroquine normalized inward rectifier K+ current magnitude, action potential duration and effective refractory period. Our results suggest that therapeutic concentrations of chloroquine might lengthen cardiac repolarization in SQT3.

Fig. 1

Concentration-response relationships for chlorquine inhibition of WT and mutant Kir2.1 current. A, Representative WT and mutant Kir2.1 currents elicited by action potential command signals as voltage protocol, before (black traces) and after application of 0.3 (red traces) and 3 (blue traces) µM chloroquine. B, Concentration-response relationship for WT and mutant Kir2.1 current inhibition. Steady-state peak-current amplitudes (shown in A) for each concentration of chloroquine normalized to control. Mean values were plotted against chloroquine concentration and fitted with the Hill equation (IC₅₀ was 1.4 ± 0.1 µM with a Hill coefficient of 1.3 for WT, 1.2 ± 0.1 µM with a Hill coefficient of 1.1 for D172N and 1.5 ± 0.2 µM with a Hill coefficient of 1.1 for WT-D172N Kir2.1 current (n = 5 cells).

Fig. 2

Chloroquine preferentially blocks outward current through WT and mutant Kir2.1 channels. Top panel, WT and mutant Kir 2.1 current elicited by 4 s pulses from a holding potential of −80 mV to test potentials from −120 to −20 mV, applied in 10 mV increments, in absence and presence of chloroquine 10 µM. B, Normalized current-voltage relationship for currents measured at the end of 4 s pulses for control and indicated concentrations of chloroquine.

Fig. 3

Block of D172N Kir2.1 current by intracellular chloroquine is voltage-dependent. A, Effect of intracellular application of chloroquine on D172N Kir2.1 current recorded in excised inside-out patches. Currents from a single cell elicited by 1 s step depolarization between +40 and −80 mV in control and after application of 1 and 10 µM chloroquine. Dashed lines define zero current levels. B, Averaged steady-state I-V curves in the absence and presence of chloroquine at the indicated concentrations recorded in excised inside-out patches. C, Relative current-voltage relationships obtained for block of D172N Kir2.1 currents by chloroquine at the indicated concentrations. The continuous lines show fits with Woodhull equation, yielding an apparent Kd (at 0 mV) of 34 ± 11 µM with an apparent valence (z) of 1.6 ± 0.3 (n = 5 cells).

Fig. 4

Onset of and recovery from chloroquine block of D172N Kir2.1 current is voltage-dependent. A, Representative current traces recorded in inside-out patches elicited by voltage steps to +80, +70 and +60 mV in presence of 10 µM chloroquine. Lines representing monoexponential fits to data are superimposed over raw current traces. B, The time constants of chloroquine block (τ) were derived by fitting current traces (as in A) to a monoexponential function. The rate constant of block (1/τ) is plotted vs. chloroquine concentration for various voltages. C. D172N current traces recorded in whole-cell configuration (4 mM external K⁺) elicited by hyper-polarizing voltage steps between −120 and −100 mV in the presence of 10 µM chloroquine. D. The time constants of chloroquine unblock (τ unblock) were determined by monoexponential fits to D172N Kir2.1 current elicited by hyperpolarizing voltage steps to membrane potentials negative to the reversal potential for potassium in 4 mM external K⁺ (whole-cell configuration) and 150 mM symmetrical K⁺ (excised inside-out patches). Data represent the mean ± SEM of 4 cells.

Fig. 5

Left panel, simulated transmembrane voltages of an epicardial myocyte for (A) WT, (B) D172N and (C) WT-D172N simulations at stimulation frequencies of 1 Hz under control conditions and the indicated chloroquine concentrations. The stimulus was applied at t = 50ms. Right panels, simulated I_K1 elicited during action potential for WT, D172N and WT-D172N simulations at indicated chloroquine concentrations.

Fig. 6

Transmembrane voltages and I_K1 for WT and heterozygous (WT-D172N) conditions. The effect of 2 and 3 µM chloroquine on WT-D172N is shown.