Inhibition of the hERG Potassium Channel by a Methanesulphonate-Free E-4031 Analogue

Figures

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  Figure 1. Structures of E-4031 and E-4031-17. Upper panel shows E-4031, asterisk highlights the methanesulphonate group. Lower panel shows E-4031-17.

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  Figure 2. Concentration and time-dependence of IhERG inhibition by E-4031 and E-4031-17. (Ai) Representative traces of WT IhERG using the voltage protocol illustrated (lower panel) in Control (black) and after 10 min of superfusion with 30 nM E-4031 (grey). Protocol was applied with a start-to-start interval of 12 s. Concentration-response relationships for fractional inhibition of WT IhERG tails at −40 mV by E-4031. (n ≥ 5 for each point) to give IC50 and nH values reported in the Results section (Aii). (B) Representative traces of WT IhERG in Control (black) and after 10 min of superfusion with 30 nM E-4031-17 (grey). Protocol shown in lower panel (Bi). Concentration-response relationships for fractional inhibition of WT IhERG tails at −40 mV by E-4031-17 (n = 5–8 for each point), to give IC50 and nH values reported in the Results section (Bii). (C) Representative IhERG traces elicited by a sustained depolarisation from −80 to 0 mV for 10 s are shown in (Ci) in Control and immediately following equilibration in 300 nM E-4031. Four more runs of the protocol were applied in the presence of the drug; only sweeps 1 and 5 are shown for clarity. (Cii) Mean (±S.E.M.) data from experiment…

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  Figure 3. Effects of E-4031 and E-4031-17 at different test voltages (A) Representative WT IhERG traces elicited by the I–V protocol illustrated (lower panel) in Control (Ai) and in the presence of 30 nM E-4031 (Aii). The membrane potential was depolarised from a holding potential of −80 mV to a range of different test potentials (−40 mV to +60 mV) in 10 mV increments. This was followed by repolarisation to −40 mV, which elicited resurgent IhERG tails used to assess current-voltage (I-V) relationships (start-to-start interval = 12 s). (B) Normalised I–V relationships for hERG Itails in Control (black) and in the presence of 30 nM E-4031 (grey). Peak tail currents before and after application of 30 nM E-4031 were normalised to the peak tail current amplitude in Control (n = 5, ‘&’ signifies p < 0.05; 2 Way ANOVA with Bonferroni post hoc test). Experimental points were fitted with a Boltzmann function (Equation (3), Methods). Control V0.5 = −25.0 ± 1.6 mV, k = 4.3 ± 0.8, and 30 nM E-4031 V0.5 = −27.8 ± 2.0 mV, k = 4.6 ± 2.1. (n = 5). (C) Voltage-dependence of IhERG block by E-4031 (black dotted line), with superimposed voltage-dependent activation relationships for IhERG in Control (black…

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  Figure 4. IhERG inactivation and effects of E-4031 and E-4031-17 (A) Voltage protocol used to evaluate WT IhERG inactivation is shown in (Ai). Membrane potential was depolarised from a holding potential of −80 mV to +40 mV for 500 ms to activate and inactivate IhERG. This was followed by a very brief (2 ms) repolarising pulse to a range of potentials (−140 to +50 mV in 10 mV increments for each pulse). This phase enabled IhERG rapidly to recover from inactivation; the extent of recovery was dependent on the repolarising test potential. The membrane potential was then stepped to +40 mV for 100 ms to assess IhERG availability. (Aii) shows plots describing voltage-dependence of IhERG inactivation in Control (black circles, black dashed line) and in the presence of 30 nM E-4031 (grey circles, grey dashed line). Data were corrected for deactivation, then fitted with Equation (4) Methods. V0.5 = −52.4 ± 1.1 mV, k = 20.7 ± 0.9 in Control and V0.5 = −73.7 ± 0.9 mV, k = 20.9 ± 0.8 in the presence of 30 nM E-4031 (p < 0.05, paired t-test, n = 6). (Aiii) Voltage-dependence of IhERG inactivation in Control (black circles, black dashed line) and in the presence of 100 nM E-4031-17 (grey circles, grey…

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  Figure 5. Effects of mutations at the base of the pore helix on actions of E-4031 and E-4031-17. (A) Representative WT (Ai) and T623A (Aii) hERG inward Itails elicited at −120 mV in 94 mM [K+]e in Control and in the presence of 300 nM E-4031 (Aii). The traces and voltage protocol (lower panels) are expanded at the end of the depolarising step to +20 mV and the beginning of the hyperpolarising step to −120 mV for clarity. (B) Representative WT (Bi) and T623A (Bii) IhERG traces similar to (A) but with 300 nM E-4031-17. (C) Bar charts showing means ±S.E.M. normalised inward Itails at −120 mV after 8 min in Control (black) or in the presence of 300 nM E-4031 (Ci) or 300 nM E-4031-17 (Cii). Values are expressed as IE-4031/IControl and IE-4031-17/IControl, and represent the relative tail current remaining after 8 min of superfusion with drug and after rundown correction. ‘**’ and ‘****’ represent p ˂ 0.005 and p ˂ 0.0001, respectively; Unpaired t-test, n ≥ 5. (D) Representative WT (Di) and S624A (Dii) hERG Itails elicited at −40 mV using the ‘standard protocol’ in Control (Di) and in the presence of 300 nM E-4031 (Dii). (E) Representative WT (Ei) and S624A (Eii) IhERG traces similar to (D) but…

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  Figure 6. Effects of mutations of S6 aromatic residues on actions of E-4031 and E-4031-17. (A) Representative WT (Ai) and Y652A (Aii) hERG Itails elicited at −40 mV using the ‘standard protocol’ in Control and in the presence of 300 nM E-4031. (B) Representative WT (Bi) and Y652A (Bii) IhERG traces similar to (A) but with 300 nM E-4031-17. (C) Bar charts showing means ±S.E.M. normalised Itails at −40 mV after 8 min in Control (black) or in the presence of 300 nM E-4031 (Ci) or 300 nM E-4031-17 (Cii). Values are expressed as IE-4031/IControl or IE-4031-17/IControl, and represent the relative tail current remaining after 8 min of superfusion with drug and after rundown correction. ‘***’ and ‘****’ represent p ˂ 0.0005 and p ˂ 0.0001, respectively; Unpaired t-test, n ≥ 5. (D) Representative WT (Di) and F656V (Dii) hERG Itails elicited at −40 mV using the ‘standard protocol’ in Control and in the presence of 300 nM E-4031. (E) Representative WT (Ei) and F656V (Eii) IhERG traces similar to (D) but with 300 nM E-4031-17. (F) Bar charts showing means ±S.E.M. normalised Itails at −40 mV after 8 min in Control (black) or in the presence of 300 nM E-4031 (Fi) or 300 nM E-4031-17 (Fii). Values are…

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  Figure 7. Effect of the S5 F557L mutation on actions of E-4031 and E-4031-17 (A) Representative WT (Ai) and F557L (Aii) hERG Itails elicited at −40 mV using the ‘standard protocol’ in Control and in the presence of 300 nM E-4031. (B) Representative WT (Bi) and F557L (Bii) IhERG traces similar to (A) but with 300 nM E-4031-17. (C) Bar charts showing means ±S.E.M. normalised Itails at −40 mV after 8 min in Control (black) or in the presence of 300 nM E-4031 (Ci) or 300 nM E-4031-17 (Cii). Values are expressed as IE-4031/IControl or IE-4031-17/IControl, and represent the relative tail current remaining after 8 min of superfusion with drug and after rundown correction. ‘‘****’ represents p ˂ 0.0001; Unpaired t-test, n ≥ 5. (D) Normalised current (IE-4031/IControl) measured after steady state block by 300 nM E-4031 (n ≥ 5; error bars represent S.E.M). (E) Normalised current (IE-4031-17/IControl) measured after steady state block by 300 nM E-4031-17 (n ≥ 5; error bars represent S.E.M). Bars in (D,E) shaded grey indicates currents that were recorded in High [K+]e (as shown in Figure 5).