Interactions between amiodarone and the hERG potassium channel pore determined with mutagenesis and in silico docking

Abstract

The antiarrhythmic drug amiodarone delays cardiac repolarisation through inhibition of hERG-encoded potassium channels responsible for the rapid delayed rectifier potassium current (IKr). This study aimed to elucidate molecular determinants of amiodarone binding to the hERG channel. Whole-cell patch-clamp recordings were made at 37°C of ionic current (IhERG) carried by wild-type (WT) or mutant hERG channels expressed in HEK293 cells. Alanine mutagenesis and ligand docking were used to investigate the roles of pore cavity amino-acid residues in amiodarone binding. Amiodarone inhibited WT outward IhERG tails with a half-maximal inhibitory concentration (IC50) of ∼45nM, whilst inward IhERG tails in a high K(+) external solution ([K(+)]e) of 94mM were blocked with an IC50 of 117.8nM. Amiodarone's inhibitory action was contingent upon channel gating. Alanine-mutagenesis identified multiple residues directly or indirectly involved in amiodarone binding. The IC50 for the S6 aromatic Y652A mutation was increased to ∼20-fold that of WT IhERG, similar to the pore helical mutant S624A (∼22-fold WT control). The IC50 for F656A mutant IhERG was ∼17-fold its corresponding WT control. Computational docking using a MthK-based hERG model differentiated residues likely to interact directly with drug and those whose Ala mutation may affect drug block allosterically. The requirements for amiodarone block of aromatic residues F656 and Y652 within the hERG pore cavity are smaller than for other high affinity IhERG inhibitors, with relative importance to amiodarone binding of the residues investigated being S624A∼Y652A>F656A>V659A>G648A>T623A.

Keywords: Amiodarone; Amiodarone hydrochloride (PubChem CID: 441325); Antiarrhythmic; I(Kr); Long QT; QT interval; hERG.

Graphical abstract

Fig. 1

Effect of amiodarone on WT I_hERG. (A, B) Representative current traces show outward (A) or inward WT I_hERG tail (B) in control (normal 4 mM [K⁺]_e Tyrode’s) solution and after 10 min application of 100 nM amiodarone (AMIOD), the current was evoked by the protocol shown in the lower panel and is shown on an expanded time-scale (denoted by the boxed area) in (B). Tail currents recorded at −40 mV or −120 mV were used to assess amiodarone inhibition. (C) Concentration response curves for outward and inward WT I_hERG tail inhibition by amiodarone in normal 4 mM [K⁺]_e and 94 mM [K⁺]_e Tyrode’s (high [K⁺]_e)_. Data were fitted with a Hill-equation (n ⩾ 5 cells per data-point). For IC₅₀ and h values refer to Section 3, also see . (D) Representative current traces in control (normal 4 mM [K⁺]_e Tyrode’s) solution and in 100nM amiodarone, overlying the applied AP voltage command.

Fig. 2

The time-dependence of inhibition of I_hERG by amiodarone. (A) Is a schematic representation of paired pulse voltage protocol used to elicit currents shown in (B) (Bi) shows representative current traces elicited during and following both 5 ms and 500 ms (n = 7) or (Bii) both 10 ms and 500 ms (n = 6, right) steps to +40 mV, in the absence and presence of 600 nM amiodarone (applied for 3 min in the absence of pulsing). (C) The bar chart displays the mean fractional block of I_hERG tails following the different duration steps to +40 mV. ^**p < 0.001 compared to 500 ms step, one way ANOVA followed by Bonferroni’s post test (5 ms, n = 7; 10 ms, n = 6; 500 ms, n = 13).

Fig. 3

Alanine-scanning mutagenesis of hERG to define binding sites for amiodarone. (A) Sequence alignment for hERG and the MthK channel, highlighting the pore helix and S6 transmembrane domains. The residues of hERG analysed in this study by Ala-scanning mutagenesis are underlined. Bottom pair highlight amino acid identities (-), strong similarities (:). Amino acids in red text have side chains facing the pore cavity of the MthK structure. Note that the last four residues of S6 in the MthK structure italicised (INRE) are not seen in the crystal structure and aren’t included in the hERG model. (B) Example traces showing I_hERG inhibition of WT or mutants in transient transfected HEK293 cells, I_hERG was recorded before (control) and after achieving steady-state block of current with 600 nM amiodarone. Voltage protocols are shown in each lower panel. (C) Normalised current (I_AMIOD/I_control) measured after steady-state block by 600nM amiodarone (n = 5–6 for each point; error bars, ±SEM). A value of 1 indicates no current inhibition by amiodarone (^**p < 0.001 compared to its WT, one way ANOVA followed by Bonferroni’s post test).

Fig. 4

Effect of S6 mutations on amiodarone inhibition of I_hERG. Representative traces from Y652A (A), F656A (B), G648A (C) and V659A (D) before (Control) and after achieving steady-state block by amiodarone, with the voltage protocol underneath. Lower panel shows concentration–response relation for the mutant (black) and its corresponding WT control (grey), yielding the IC₅₀ and h values in section 3. (For all, n ⩾ 5 cells per data-point). Note that for some data-points in (A), (B), (C) the SEM values are small and obscured by the symbols.

Fig. 5

Effect of pore helix mutations on amiodarone inhibition of I_hERG. Representative traces from T623A (A) and S624A (B) before (Control) and after achieving steady-state block by amiodarone, with the voltage protocol underneath. Lower panel shows concentration–response relation for the mutant (black) and its corresponding WT control (grey), yielding the IC₅₀ and h values in Section 3. (For all, n ⩾ 5 cells per data-point.) Note that for some data-points the SEM values are small and obscured by the symbols.

Fig. 6

Representative low energy score docking output for amiodarone in the MthK-based hERG pore homology model. (A) Amiodarone is shown in relation to the amino acid residues described in the text: blue: F656, pink: Y652; green: T623, S624, V625. These residues are also annotated in (B) which highlights the set of interactions between amiodarone and specific amino acid side chains including two pi-stacking interactions between F656 and amiodarone aromatic rings, and two cation–pi interactions and one hydrogen bond involving the protonated amino group and Y656 side chains. The location of the aliphatic amino group near the internal binding site for a K⁺ ion is indicated by the blue star. Stabilisation of the protonated amino group in this location may be enhanced by the hydroxyl side chain groups of S624. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Comparison of experimental and computational analysis of amiodarone block. (A) Low energy score structure of amiodarone (wheat coloured space filling representation) docked into the MthK-based hERG model extended to include the S5 helix. One subunit of the model is rendered as a Connelly surface coloured as a heat map according to amino acids whose mutation to Ala attenuates drug binding as defined in panel (B). K⁺ ions in the S1 and S3 positions of the selectivity filter are pink spheres. (B) One subunit of the hERG model extracted from panel (A) and coloured to define residues whose Ala mutation attenuates amiodarone block by: 17–22-fold (deep red); approx. 10-fold (pale red); 5–7-fold (mauve); the latter group comprised T623 and G648 (Table 2), however G648 lies behind Y652 and is hidden in this view. (C) The same subunit coloured according to residues that make interactions (as defined in [32]) with amiodarone in low energy docked states. Annotations in panel C define residues that make direct interactions with drug in docking and whose mutation to Ala attenuates drug block (except for A653 which was not mutated experimentally). Annotations in panel (B) define residues whose Ala mutation attenuates amiodarone block but which do not make direct interaction with drug in low energy score docked states. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)