Stereoselective block of the hERG potassium channel by the Class Ia antiarrhythmic drug disopyramide

Figures

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  Fig. 1. Effects of S(+) and R(−) Disopyramide on WT IhERG. Representative IhERG traces before (control) and after achieving steady-state block by S(+) disopyramide (A) and R(−) disopyramide (B) with the voltage protocol shown underneath. C shows concentration-response relation for S(+) and R(−) disopyramide (n = 5–7 for S(+) and n = 4–6 for R(−) disopyramide). D, E show rightwards shift of the concentration–response relation for S(+) disopyramide (D) and R(−) disopyramide (E) in 94 mM[K+]e Tyrode’s solution(n = 5–7 for both S(+) and R(−) disopyramide) compared with 4 mM [K+]e Tyrode’s

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  Fig. 2. Effects of mutant N588K and N588E on IhERG inhibition by S(+) and R(−) Disopyramide. Representative traces of N588K IhERG in the absence and presence of S(+) disopyramide (Ai) and R(−) disopyramide (Bi), elicited by the voltage protocol shown in the lower panel. Aii, Bii. Concentration–response relations for N588K IhERG block compared with that for WT hERG for S(+) disopyramide (Aii, n = 4–7 for N588K) and R(−) disopyramide (Bii, n = 4–8 for N588K) or compared with each other (C). Di and Dii concentration–response relations for N588E IhERG block compared with WT for S(+) disopyramide (Di, n = 5–8 for N588E) and R(−) disopyramide (Dii, n = 4–8 for N588E)

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  Fig. 3. Effects of S6 aromatic mutants Y652A and F656A on S(+) and R(−) disopyramide block of IhERG. Representative traces of Y652A IhERG in the absence and presence of S(+) disopyramide (Ai) and R(−) disopyramide (Bi), elicited by the voltage protocol shown in the lower panel. Aii, Bii. Concentration–response relations for Y652A IhERG block compared with WT for S(+) disopyramide (Aii, n = 5–11 for Y652A) and R(−) disopyramide (Bii, n = 5–7 for Y652A). Representative traces of F656A IhERG in the absence and presence of S(+) disopyramide (Ci) and R-disopyramide (Cii), elicited by the whole voltage protocol (left) or on expanded timescale (right), shown in the lower panels. Di, Dii. Concentration–response relations for F656A IhERG block compared with WT for S(+) disopyramide (Di, n = 5–9 for F656A) and R(−) disopyramide (Dii, n = 5–8 for F656A).

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  Fig. 4. Effects of S6 tandem dimer D-Y652A (td [wt: Y652A]) on S(+) and R(−) disopyramide block of IhERG. (Ai) Tandem dimers D-Y652A (td[wt:Y652A]) are shown as a concatenated molecule of the monomer WT(N-terminal) and monomer Y652A (C-terminal) with a short linker. (Aii) When functional channels incorporating dimers assemble, mutation sites should be located on subunits in the diagonal position of the tetrameric channel, WT subunits are indicated by ovals and the mutated subunits by rectangles. (Bi) Upper left inset panel shows the assembly of D-Y652A, oval represents WT monomer with residue Y in position 652, and rectangle represents Y652A mutant monomer with residue A in position 652. Lower left panel represents the possible contribution of residues to the binding on the subunits. Filled oval represent enantiomers interacting with residues on the subunits (solid lines indicate positive interactions, and dashed lines indicate possible interactions). Representative traces of tandem dimer Y652A IhERG in the absence and presence of S(+) disopyramide (Bi) and R(−) disopyramide (Ci), elicited by the voltage protocol shown in the lower panel. (Bii, Cii). Concentration–response relations for…

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  Fig. 5. Repositioning of aromatic residues on an N588K background. (A) Schematic showing sequences of S5-pore and S6 domain regions to show location of N588K and repositioning N588K-up or down mutants. (B-D) effects of repositioning S6 aromatic residue Y652 and F656 in attenuated inactivation N588K channels on IhERG blocking potency of R(−) disopyramide. Representative traces of N588K-Y-up IhERG in the absence and presence of R(-) disopyramide (Bi, n = 5–8 for N588K-Y-up), elicited by the voltage protocol shown in the lower panel and its concentration-response relation (Bii), Representative traces of N588K-Y-down IhERG in the absence and presence of R(−) disopyramide (Ci, n = 5–6 for N588K-Y-down), elicited by the voltage protocol shown in the lower panel and its concentration-response relation (Cii). Representative traces of N588K-F-up IhERG in the absence and presence of R(−) disopyramide (Di, n = 5–8 for N588K-F-up), elicited by the voltage protocol shown in the lower panel and its concentration–response plot (Dii)

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  Fig. 6. Stereoselective interactions of disopyramide enantiomers within a hERG pore homology model. Each panel is a snapshot of the interaction of a disopyramide enantiomer obtained during 200 ns MD runs within a fully hydrated, membrane-incorporated MthK-based hERG pore homology model (see Supplementary Figs. 6 and 7 for the structure of the membrane system and starting structures of each disopyramide enantiomer within the pore cavity). Disopyramide enantiomers are represented as yellow sticks and hERG pore amino acid side chains are Y652 (pink), F656 (blue) and S624 (green). Discrete drug-pore interactions as defined in [44] are shown as black (aromatic stacking), blue (cation-pi) and green (hydrogen bond) dotted lines. The location of the tertiary aliphatic amino group in or near the binding site for a hydrated K+ ion below the selectivity filter is indicated with a blue star. The purple sphere is a K+ ion in the 4-position of the selectivity filter. The Y652 side chains that interact with disopyramide lie on adjacent pore subunits in both S(+) and (R−) bound structures. Stereo views of these binding modes are shown in Supplementary Figs. 8 and 9