Structural basis for the antiarrhythmic blockade of a potassium channel with a small molecule

Abstract

The acetylcholine-activated inward rectifier potassium current ( I_KACh) is constitutively active in persistent atrial fibrillation (AF). We tested the hypothesis that the blocking of I_KACh with the small molecule chloroquine terminates persistent AF. We used a sheep model of tachypacing-induced, persistent AF, molecular modeling, electrophysiology, and structural biology approaches. The 50% inhibition/inhibitory concentration of I_KACh block with chloroquine, measured by patch clamp, was 1 μM. In optical mapping of sheep hearts with persistent AF, 1 μM chloroquine restored sinus rhythm. Molecular modeling suggested that chloroquine blocked the passage of a hydrated potassium ion through the intracellular domain of Kir3.1 (a molecular correlate of I_KACh) by interacting with residues D260 and F255, in proximity to I228, Q227, and L299. ¹H ¹⁵N heteronuclear single-quantum correlation of purified Kir3.1 intracellular domain confirmed the modeling results. F255, I228, Q227, and L299 underwent significant chemical-shift perturbations upon drug binding. We then crystallized and solved a 2.5 Å X-ray structure of Kir3.1 with F255A mutation. Modeling of chloroquine binding to the mutant channel suggested that the drug's binding to the pore becomes off centered, reducing its ability to block a hydrated potassium ion. Patch clamp validated the structural and modeling data, where the F255A and D260A mutations significantly reduced I_KACh block by chloroquine. With the use of numerical and structural biology approaches, we elucidated the details of how a small molecule could block an ion channel and exert antiarrhythmic effects. Chloroquine binds the I_KACh channel at a site formed by specific amino acids in the ion-permeation pathway, leading to decreased I_KACh and the subsequent termination of AF.-Takemoto, Y., Slough, D. P., Meinke, G., Katnik, C., Graziano, Z. A., Chidipi, B., Reiser, M., Alhadidy, M. M., Ramirez, R., Salvador-Montañés, O., Ennis, S., Guerrero-Serna, G., Haburcak, M., Diehl, C., Cuevas, J., Jalife, J., Bohm, A., Lin,Y.-S., Noujaim, S. F. Structural basis for the antiarrhythmic blockade of a potassium channel with a small molecule.

Keywords: IKACh; atrial fibrillation; potassium inward rectifier.

Figure 2.

Effect of chloroquine on persistent AF in isolated Langendorff-perfused hearts. A) Ex vivo epicardial and endocardial mapping setup includes 3 synchronized CCD cameras: cameras 1 and 2 for the epicardial imaging of the LA and RA and camera 3 coupled to an endoscope advanced through the left ventricle into the LA for endocardial PLA imaging. B) APD₇₅ and APD₅₀ at constant 2.5 Hz pacing in PLA, LA, and RA, before and after the application of 1 µM chloroquine (n = 5). C) Representative DF maps of the PLA, LA, and RA in baseline AF and 2 and 4 min after chloroquine 1 µM perfusion. After 4 min, the heart spontaneously reverted to a 1.2 Hz SR. D) Time course of the average DF in the 5 hearts, 30 min before and after chloroquine perfusion. The black × marks indicate the time of spontaneous AF cardioversion in each of the hearts. E) Average maximum DF right before chloroquine application at baseline and immediately before AF termination. *P < 0.05, **P < 0.01.

Figure 1.

Determination of the IC₅₀ for chloroquine and tertiapinQ block of I_KACh in HEK293 cells stably transfected with Kir3.1 and Kir3.4. A) Confocal microscopy. Fluorescence staining of I_KACh proteins. Upper: Live HEK293 cells transfected with mCherry. TertiapinQ (TPQ) ATTO-488 does not show staining. Lower: HEK293 cells, transfected with mCherry and stably expressing Kir3.1/Kir3.4, show robust staining of the cell membranes by tertiapinQ ATTO-488. Laser and detector settings were identical in both cases. B) I_KACh recorded in HEK293 cells expressing Kir3.1/3.4 in response to a ramp in the absence and the presence of 1 μM chloroquine (CQ) or 0.1 μM tertiapinQ. C) Dose-response curve of I_KACh block by chloroquine, IC₅₀ = 1.02 μM, R² = 0.96; TertiapinQ, IC₅₀ = 0.07 μM, R² = 0.94.

Figure 3.

Effect of tertiapinQ on persistent AF in isolated Langendorff-perfused hearts. A) APD at APD₇₅ and APD₅₀ at constant 2.5 Hz pacing in PLA, LA, and RA, before and after the application of 0.2 µM tertiapinQ (n = 4). B) Representative DF maps of the PLA, LA, and RA in baseline AF and 2 and 4 min after 0.1 µM tertiapinQ perfusion. After 4 min, the heart spontaneously reverted to a 0.8 Hz SR. C) Time course of the average DF in 4 hearts, 30 min before and after TertiapinQ perfusion. The black × marks indicate the time of spontaneous AF cardioversion in each of the hearts. D) Average maximum DF right before TertiapinQ application at baseline and immediately before AF termination. *P < 0.05, **P < 0.01.

Figure 4.

Docking of chloroquine in the ion-permeation pathway of the Kir3.1 channel. A, B) Two lowest energy poses. Top: magnified view of the binding poses of chloroquine (cyan sticks) in Kir3.1. The D260 and F255 residues from each of the 4 Kir3.1 subunits are shown in green and orange sticks, respectively. A) The amine nitrogen of chloroquine forms a hydrogen bond (red line) with the side chain of D260 in 1 subunit, whereas the aminoquinoline ring of chloroquine is involved in an aromatic–aromatic interaction with the phenylalanine ring of F255 in the adjacent subunit. B) The amine nitrogen of chloroquine hydrogen bonds (red line) the carbonyl oxygen of F255 in 1 subunit, whereas the aminoquinoline ring of chloroquine is involved in an aromatic–aromatic interaction with the phenylalanine ring of F255 in the opposing subunit. Middle, bottom: van der Waals representations of the channel bound to chloroquine (cyan), viewed from the intracellular and extracellular sides, respectively.

Figure 5.

Chloroquine binding to the isolated and purified Kir3.1 intracellular domain studied with ¹H ¹⁵N HSQC NMR. A) Examples of resonance peaks of residues W323, K49, I228, and L299 obtained by a ¹H ¹⁵N NMR TROSY-HSQC sequence. The black contours are of the Kir3.1 protein alone, and the red contours are after addition of chloroquine. The resonance peaks of W323 and K49 did not undergo significant shifts, suggesting that these residues are not involved in the binding of chloroquine to the channel. The peaks of I228 and L229 underwent significant shifts, indicating that the chemical environment of these residues have changed upon chloroquine binding to the channel. B) An intracellular bird’s-eye view of the tetrameric Kir3.1 is shown in gray ribbons, whereas the residues that underwent significant CSPs in the NMR experiments are shown as red sticks. C) A longitudinal representation of the tetrametric channel, with the front 2 subunits removed, exposing the amino acids (Q227, I228, F255, and L299) that form a binding pocket for chloroquine (white circle) in the aqueous vestibule. D) Placement of docked chloroquine in the binding pocket suggested by NMR. Longitudinal view of chloroquine (cyan sticks) docked into the Kir3.1 channel from Fig. 4 with the front subunits removed, and the residues that underwent significant shifts in the NMR experiment are shown in red. E) Magnified view of the boxed area in D showing agreement between the molecular docking of chloroquine and the binding pocket suggested by NMR, with chloroquine sitting in the NMR-suggested binding site. F) Chloroquine in cyan sticks is shown with the amino acids that underwent CSPs in NMR, depicted in red sticks.

Figure 6.

X-Ray crystallography structure of Kir3.1 with F255A mutation at 2.5 Å resolution and docking of chloroquine in the mutant channel structure. A) Left: electron density map for Kir3.1 F255A tetrameric channel after molecular replacement. The difference (F_o-F_c) electron density map, around the area of residue 255, is contoured in red at −3 σ. Right: magnified view of the residue 255 region from 1 subunit highlighting the red negative-density blob, which indicates the absence of residue F255. B) Docking of chloroquine into the ion-permeation pathway of the F255A mutant Kir3.1 channel. Left: magnified view of the binding pose of chloroquine (cyan sticks) in Kir3.1. Chloroquine binding is off centered, with the amine nitrogen of chloroquine forming a hydrogen bond with residue D260. The F255 residues from each of the 4 Kir3.1 subunits are shown in red sticks. Right) van der Waals representation of the F255A mutant channel bound to chloroquine (cyan), viewed from the intracellular side.

Figure 7.

Estimation of chloroquine’s ability to block the WT and F255A mutant Kir3.1 channel using voxelation. A) Intracellular, magnified view of the WT (left) and F255A mutant (middle) channel’s aqueous pore with docked chloroquine in cyan from Figs. 4 and 6, respectively. Right: A longitudinal view of the channel’s ribbon structure, with the orange box indicating the area of the channel represented by the voxelation experiments of B and C. B) Voxelated WT channel’s ion-permeation pathway in gray, with the front subunit removed for clarity. Chloroquine is in cyan. The different snapshots show a spherical voxelated probe colored in purple, with a radius = 2.5 Å traveling through the channel, starting toward the extracellular portion and getting blocked by chloroquine. C) The voxelated F255A mutant ion-permeation pathway is in gray, with the front subunit removed for clarity. Chloroquine is in cyan. The different snapshots show a spherical, voxelated probe colored in purple, with a radius = 2.5 Å traveling down the channel, starting toward the extracellular portion, without being affected by chloroquine.

Figure 8.

F255A and D260A mutations reduce the ability of chloroquine to block I_KACh. A) Confocal microscopy. Fluorescence staining of I_KACh proteins. Top) Live HEK293 cells transfected with mCherry, WT Kir3.1, and Kir3.4. Middle (bottom): cells transfected with mCherry, Kir3.1 F255A or D260A, and Kir3.4, where tertiapinQ ATTO-488 showed robust staining of the cell membranes. B) BaCl₂-sensitive I_KACh currents elicited in WT Kir.31/Kir3.4-, F255A Kir3.1/Kir3.4-, or D260A Kir3.1/Kir3.4-transfected cells, in response ramps from −140 to +30 mV, before and after addition of 1 μM CQ. C, left: Dose-response curves for the effect of chloroquine on the BaCl₂-sensitive inward current measured at −120 mV. IC₅₀ for WT: 0.8 μM, R² = 0.81, n = 11; F255A: 3.2 μM, R² = 0.75, n = 10; and D260: 2.8 μM, R² = 0.9, n = 10. P < 0.01 for WT vs. F255A and D260. Right: dose-response curves for outward current measured at +20 mV. IC₅₀ for WT: 1.1 μM, R² = 0.83, n = 7; F255A: 4.2 μM, R² = 0.65, n = 7; and D260: 3.8 μM, R² = 0.89, n = 7. P < 0.01 for WT vs. F255A and D260. D) TertiapinQ (70 nM) block of WT (46.2 ± 5.1%, n = 5) and F255A (52.3 ± 11.3%, n = 5; P = 0.6) Kir3.1/ Kir3.4 currents at −120 mV.

Figure 9.

Modeling the binding of chloroquine to the Kir3.1 transmembrane domain. A) Chloroquine docking in the aqueous region of the transmembrane domain in the homology model for Kir3.1 using the PDB of Kir3.2 (PDB ID: 3SYO). Chloroquine (in cyan) binds the transmembrane domain of the channel at residue D173. Right: the lowest binding-affinity pose viewed from the intracellular side. Chloroquine’s amine group hydrogen bonds (red line) the negative side chain of D173. B) Voxelated version of the channel (in red) overlaying the Kir3.1 ribbon structure (gray). C) Voxelated channel (red) with the corresponding z-axis scale along the length of the channel axis (starting at 0 Å at the intracellular entrance of the intracellular vestibule). D) Aqueous vestibule’s cross-sectional area calculated as a function of the channel axis. The narrowest region is at 37.25 Å, with a cross-sectional area of 8.25 Ų.

Figure 10.

A) Slice of the voxelated channel at 37.25 Š(red beads) with the surrounding corresponding residues T306, G307, and M308 as sticks (left) and as van der Waals radii (right). B) Voxelated chloroquine (cross-sectional area 46.6 Ų) in blue, on top of the G-loop.