The molecular basis of chloroquine block of the inward rectifier Kir2.1 channel

Abstract

Although chloroquine remains an important therapeutic agent for treatment of malaria in many parts of the world, its safety margin is very narrow. Chloroquine inhibits the cardiac inward rectifier K(+) current I(K1) and can induce lethal ventricular arrhythmias. In this study, we characterized the biophysical and molecular basis of chloroquine block of Kir2.1 channels that underlie cardiac I(K1). The voltage- and K(+)-dependence of chloroquine block implied that the binding site was located within the ion-conduction pathway. Site-directed mutagenesis revealed the location of the chloroquine-binding site within the cytoplasmic pore domain rather than within the transmembrane pore. Molecular modeling suggested that chloroquine blocks Kir2.1 channels by plugging the cytoplasmic conduction pathway, stabilized by negatively charged and aromatic amino acids within a central pocket. Unlike most ion-channel blockers, chloroquine does not bind within the transmembrane pore and thus can reach its binding site, even while polyamines remain deeper within the channel vestibule. These findings explain how a relatively low-affinity blocker like chloroquine can effectively block I(K1) even in the presence of high-affinity endogenous blockers. Moreover, our findings provide the structural framework for the design of safer, alternative compounds that are devoid of Kir2.1-blocking properties.

Fig. 1.

Chloroquine preferentially blocks outward current through Kir2.1 channels. (A) Effect of chloroquine on Kir2.1 channels expressed in HEK293 cells. Kir 2.1 current elicited by 4-sec pulses from a holding potential of −80 mV to test potentials from −140 to 0 mV, applied in 10-mV increments, in absence (control) and presence of chloroquine 3 and 30 μM. (B) Normalized current-voltage relationship for currents measured at the end of 4-sec pulses. (C) Currents elicited by action potential command signals as voltage protocol, before and after application of 1, 3, 10, and 30 μM chloroquine. (D) Concentration–response relationship for Kir2.1 current inhibition. Steady-state peak-current amplitudes (shown in C) for each concentration of chloroquine normalized to control. Mean values were plotted against chloroquine concentration and fitted with the Hill equation. Mean IC₅₀ was 8.7 ± 0.9 μM, and the Hill coefficient n_H = 1.0 (n = 5). CQ, chloroquine.

Fig. 2.

Unblock of chloroquine at voltages negative to E_K is voltage- and [K⁺]_o-dependent. (A) Normalized current transients elicited by voltage step from 0 to −140 mV in the presence of 10 μM chloroquine and 4, 20, and 75 mM extracellular K⁺. The curves superimposed on the current transients are monoexponential fits. The time constants of chloroquine unblock were 830 ± 103, 218 ± 15, and 14 ± 3 ms (mean ± SEM; n = 4–5) for 4, 20, and 75 mM extracellular K⁺, respectively. (B) Voltage-dependence of chloroquine unblock at various extracellular K⁺. I/O, inside-out patch.

Fig. 3.

Spermine does not protect Kir2.1 channels from chloroquine block. In excised, inside-out patches, the membrane was depolarized to +50 mV, followed by cytoplasmic surface application of spermine (10 μM), chloroquine (10 μM), and combination of spermine, followed by chloroquine. Membrane hyperpolarization to −80 mV elicited nearly instantaneous inward currents in the presence of spermine alone (rapid spermine unblock) but slowly increasing inward currents in the presence of chloroquine (slow chloroquine unblock) despite preapplication of spermine.

Fig. 4.

Ala-scanning mutagenesis of Kir2.1 transmembrane and cytoplasmic pore regions. (A) Ala-scanning mutagenesis of transmembrane pore (M2 segment) of Kir2.1 channel. The fraction of current inhibited by 30 μM chloroquine at +50 mV (excised inside-out patches) is plotted as a bar graph for WT and each of the Ala mutants. (B) Ala-scanning mutagenesis of the Kir2.1 cytoplasmic domain. Data generated as described in A. *, P < 0.0001, by using one-way ANOVA and Holm–Sidak multiple comparison procedure (SigmaStat v3.5; Systat). (C) Concentration–effect relationship for current inhibition of Kir2.1 cytoplasmic domain mutants by chloroquine. The IC₅₀ as determined by fits to a Hill equation was 1.1 ± 0.2 μM for Kir2.1 WT (n = 8), 2.8 ± 0.2 μM for D255A (n = 6), 15.1 ± 1.9 μM for F254A (n = 5), 100 ± 16 μM for E299A (n = 5), 307 ± 30 for D259A (n = 5), and 698 ± 49 for E224A (n = 6).

Fig. 5.

Docking of chloroquine within the Kir2.1 cytoplasmic domain. (A Left) Chemical structure of chloroquine. Quinoline ring and alkylamino nitrogen ionization sites are depicted by + sign. Atoms are colored as follows: nitrogen, blue; chlorine, red; carbon, green; hydrogen, gray. (Right) Transmembrane (KirBac1.1) and cytoplasmic domain (Kir2.1) highlighting positions in Kir2.1 critical for polyamine block; D172 (blue), E224 (red), E299 (yellow), and D259 (orange). (B) Enlarged view of dotted box from A illustrating a single Kir2.1 cytoplasmic domain subunit (25) and docking of chloroquine relative to positions E224, E299, and D259. The aromatic side chain of F254 is shown in ball and stick format, colored in orange. (C) Stereo view (“Top-down”) of the tetrameric Kir2.1 cytoplasmic domain showing chloroquine plugging the conduction pathway. F254 side chain depicted as in B.