Cryo-EM Structure of the Open Human Ether-à-go-go-Related K+ Channel hERG

Abstract

The human ether-à-go-go-related potassium channel (hERG, Kv11.1) is a voltage-dependent channel known for its role in repolarizing the cardiac action potential. hERG alteration by mutation or pharmacological inhibition produces Long QT syndrome and the lethal cardiac arrhythmia torsade de pointes. We have determined the molecular structure of hERG to 3.8 Å using cryo-electron microscopy. In this structure, the voltage sensors adopt a depolarized conformation, and the pore is open. The central cavity has an atypically small central volume surrounded by four deep hydrophobic pockets, which may explain hERG's unusual sensitivity to many drugs. A subtle structural feature of the hERG selectivity filter might correlate with its fast inactivation rate, which is key to hERG's role in cardiac action potential repolarization.

Keywords: K(+) channel; cryo-EM; drug-induced Long QT; hERG; hERG block; inactivation; structure; voltage-dependent gating.

Figure 1. A hERG construct for cryo-EM structure determination

(A) The primary structure of hERG with domains indicated in different colors. Amino acid residues between 140–351 and 870–1006 were removed in the hERG_T construct. (B) and (C) Representative whole-cell voltage family recordings in transfected HEK-293T cells of (B) hERG_WT and (C) the hERG_T construct. Bath solution contained 20 mM K⁺ and pipette solution contained 120 mM K⁺. Voltage protocol is illustrated at the bottom of (C). Cell membrane was held at −80 mV, depolarized to −80 mV to 60 mV for 2 s in 10 mV steps, and then repolarized back to −80 mV. The open arrows denote the steady state currents during depolarization and the closed arrows denote the tail currents upon repolarization. Dashed lines indicate the 0 current level. (D) Normalized tail currents (mean ± SEM) of hERG_WT (blue triangles, n = 6) and hERG_T (black circles, n = 11) are plotted as a function of the depolarization step voltage and fitted with Boltzmann equation (solid lines. blue, hERG_WT; black, hERG_T. See STAR Methods). Fitted half activation voltage V_1/2 and apparent valence z are shown beside the plots. (E) Dose response characteristics of hERG_T to astemizole (black circles, n=3, mean ± SEM) and dofetilide (blue triangles, n=3, mean ± SEM). IC₅₀ values from rectangular hyperbolar fit (solid lines. blue, dofetilide; black, astemizole, see STAR Methods) are shown in the figure.

Figure 2. Overall architecture of the hERG channel

(A) Local resolution of reconstructed density map as estimated using software “blocres” (Heymann and Belnap, 2007) with 20 voxel box size and 0.5 Fourier Shell Correlation (FSC) cutoff. Density map is colored according to the local resolution using UCSF Chimera (Pettersen et al., 2004). (B) hERG overall architecture viewed down the four-fold axis from the extracellular side. hERG channel is represented as semi-transparent molecular surface with ribbon representation for one subunit. Domains are colored as in figure 1A. (C) Stereo view of the hERG channel from the side. See also figures S1, S2 and S5.

Figure 3. hERG channel is in the open conformation

(A) The central pore generated with program “hollow” (Ho and Gruswitz, 2008) is shown as yellow surface. Only two of the opposing hERG subunits are shown as blue ribbons for clarity. (B) The radius of the transmembrane pore (calculated with “HOLE” software (Smart et al., 1993)) plotted against the displacement from the top of the selectivity filter (blue: hERG, cyan: KvChim (PDB ID 2R9R), gray: EAG1 (PDB ID 5K7L)). Positions of constriction site residues Q476 in EAG1 and its counterpart Q664 in hERG are indicated with arrows. (C) Stereo view of two opposing subunits of hERG (blue) and EAG1 (gray) overlaid. hERG and EAG1 tetramers were aligned using the selectivity filter and the pore helix. Secondary structure elements are labeled in bold. Constriction site residues (hERG Q664 and EAG1 Q476) are shown as sticks. “Gating hinge” glycines (hERG G648 and EAG1 G460) are shown as spheres.

Figure 4. Conformation of the hERG voltage sensor

Stereo view of the hERG voltage sensor. Gray lines indicate the membrane-solution interfaces. Basic residues (labeled in blue) at positions K1 to R5 and the gating charge transfer center residues (red labels for acidic residues and green for the aromatic) are shown as sticks. Secondary structure elements are labeled in bold.

Figure 5. Local chemistry of a putative drug-binding site

(A) Sequence alignment of selected K⁺ channels near the putative drug-binding site. Conserved residues are shown in bold. Residues related to drug binding are colored in yellow with arrows indicating the amino acid residue numbers in hERG. (B) EM density map of the putative drug-binding site is shown as blue mesh and molecular model shown as sticks. Residues related to drug binding are highlighted in yellow. Only one subunit is shown for clarity. (C) Overlay of hERG (blue) and KvChim (cyan) near the central cavity with drug binding related residues shown as sticks. (D) Internal molecular surface around the central cavity of hERG is represented as translucent surface colored by eletrostatic potential according to the scale shown. Residues related to drug binding are shown as sticks on the otherwise ribbon representation of the channel. (E) Internal molecular surface around the central cavity of KvChim represented similarly as in (D). See also figure S3.

Figure 6. Selectivity filter rearrangements accompanying hERG rapid inactivation

Side view of the cryo-EM density maps (translucent surfaces) of the selectivity filters of (A) hERG_T (blue), (B) EAG1 (gray) and (C) a non-inactivating mutant hERG_Ts S631A (green). “*” indicate the positions of the selectivity filter aromatic residues (F627 in hERG, F439 in EAG1). Only two opposing subunits are shown for clarity. (D) Overlay of the equivalent aromatic residues in the selectivity filter shown as sticks, hERG_T F627 (blue), hERG_Ts S631A F627 (green), EAG1 F439 (grey), KcsA Y78 (yellow) and KvChim Y373 (cyan). (E) Density maps of F627 in hERG_T (blue mesh) and F439 in EAG1 (grey mesh) viewed from the extracellular side along the four-fold axis. Stick models are shown for one subunit of each channel. Arrows highlight the counter-clockwise rotation of this residue. (F) Overlay of the density maps and model of residue F627 in hERG_T (blue) and hERG_Ts S631A (green) in a similar way as in (E). (G) and (H) Representative whole-cell voltage family recordings in HEK-293T cells of (G) hERG_T and (H) a non-inactivating mutant hERG_T S631A. See also figures S1, S4–S6.