Modulating the voltage sensor of a cardiac potassium channel shows antiarrhythmic effects

Abstract

Cardiac arrhythmias are the most common cause of sudden cardiac death worldwide. Lengthening the ventricular action potential duration (APD), either congenitally or via pathologic or pharmacologic means, predisposes to a life-threatening ventricular arrhythmia, Torsade de Pointes. I_Ks (KCNQ1+KCNE1), a slowly activating K⁺ current, plays a role in action potential repolarization. In this study, we screened a chemical library in silico by docking compounds to the voltage-sensing domain (VSD) of the I_Ks channel. Here, we show that C28 specifically shifted I_Ks VSD activation in ventricle to more negative voltages and reversed the drug-induced lengthening of APD. At the same dosage, C28 did not cause significant changes of the normal APD in either ventricle or atrium. This study provides evidence in support of a computational prediction of I_Ks VSD activation as a potential therapeutic approach for all forms of APD prolongation. This outcome could expand the therapeutic efficacy of a myriad of currently approved drugs that may trigger arrhythmias.

Keywords: C28; IKs; KCNQ1; antiarrhythmia; voltage sensor domain.

Fig. 1.

Effects of C28 on KCNQ1 opening. (A) Cryo-EM structure of KCNQ1 (Protein Database Bank [PDB] entry: 6uzz) docking with C28 (cyan, yellow, and red sticks). G219C (green) was covalently labeled with Alexa488 in VCF experiments. S1 to S6: transmembrane helices. The VSD (S1 to S4) and the PD (S5 to S6) are from neighboring subunits. (B) C28 molecule. (C) Currents of KCNQ1 (black) and with C28 (10 µM, red) at various test voltages (see D). The potentials before and after test pulses were −80 and −40 mV, respectively. (D) Steady-state current-voltage relations with current amplitudes at the end of test pulses shown in C. P > 0.05 between control and C28 at all voltages, unpaired Student’s t test. (E) G–V relations. Solid lines are fits to the Boltzmann relation with V_1/2 and slope factor (millivolts) for control: −41.4 ± 1.4 and 9.3 ± 1.3 and for 10 µM C28: −87.7 ± 1.4 and 13.3 ± 1.4. (F) The change of V_1/2 of G–V relations depends on C28 concentration, with EC₅₀ of 7.6 µM. (G) Reversal potential measurements. KCNQ1 channels were activated at 40 mV and then currents were tested at −100 to 20 mV. Holding potential: −80 mV. Control: black, with C28 (10 µM): red. (H) Peak tail currents in relation to the test voltages. The currents reversed at potentials (millivolts): −58.2 ± 0.8 for control (black) and −57.1± 1.1 for 10 µM C28 (red). All data in this figure and subsequent figures are mean ± SEM, n = 3 to 15 unless otherwise specified.

Fig. 2.

Effects of C28 on I_Ks opening. (A) I_Ks (black) and with C28 (10 µM, red) at various voltages (see B). The potentials before and after test pulses were −80 and −40 mV, respectively. (B) Steady-state current-voltage relations. P < 0.05 between control and C28 at voltages −50 to +20 mV, unpaired Student’s t test. (C) G–V relations. Solid lines are fits to the Boltzmann relation with V_1/2 and slope factor (millivolts): 26.4 ± 0.6 and 13.1 ± 0.5 for control, and −10.4 ± 1.3 and 17.3 ± 1.3 for 10 µM C28. (D) G–V shifts in response to C28. EC₅₀ is 5.9 µM. (E) I_Ks deactivation. The channels were activated at 60 mV and then tested at −100 to 40 mV. Control: black, with C28 (10 µM): red. (F) Voltage dependence of activation (Act) and deactivation (Dea) time constants of I_Ks in control and 10 µM C28. The time constants were obtained by exponential fitting to current traces. Solid lines are exponential fits to the data. P < 0.05 between control and C28 at all voltages, unpaired Student’s t test.

Fig. 3.

C28 enhances KCNQ1 VSD activation. (A) Currents of psWT KCNQ1 (black) and with C28 (1.5 µM, red) at various test voltages (see B). The potentials before and after test pulses were −80 and −40 mV, respectively. (B) G–V relations. Solid lines are fits to the Boltzmann relation with V_1/2 and slope factor (millivolts): −50.4 ± 0.9 and 13.9 ± 0.7 for psWT, and −59.7 ± 1.1 and 15.2 ± 1.4 for C28. n = 7. (C) Fluorescence change of psWT in response to voltage pulses to 40 mV (−80 mV before and −40 mV after the test pulse, pulse interval: 40 s) altered direction upon C28 (1.5 µM) application. (D) Fluorescence changes of psWT KCNQ1 (black) and with C28 (1.5 µM, red) in response to the voltage protocols from the same oocyte in A. (E) Normalized fluorescence–voltage relation. The curves are fits with double Boltzmann functions (F1 and F2) with the V_1/2 and the slope factor. V_1/2 (millivolts): F1 −51.9 ± 1.1 and F2 49.1 ± 2.0 for psWT; F1 −60.6 ± 1.4 and F2 39.1 ± 3.3 for C28. Slope factor (millivolts): F1 12.2 ± 0.5 and F2 36.0 ± 0.8 for psWT, and F1 21.5 ±1.4 and F2 26.5 ±3.1 for C28. n = 5. The statistical significance of differences in V_1/2 between C28 and control were tested using unpaired Student’s t test; P < 0.001 for G–V (B), and P < 0.005 and P < 0.05 for F1 and F2, respectively (E).

Fig. 4.

Interactions of C28 with the VSD of KCNQ1. (A and B) C28 alters KTQ channel activation. KTQ is a fusion protein between the two-pore–domain channel TASK3 and KCNQ1 VSD (Inset). (A) Currents were elicited by voltage pulses from −100 to 80 mV with 20 mV increments from a holding potential of −80 mV. (B) G–V and Boltzmann fits (solid lines) with V_1/2 and slope factor (millivolts): 53.1 ± 2.5 and 18.4 ± 2.5 for control; 14.9± 8.2 and 32.8 ± 8.0 for C28. (C and D) C28 has no effect on KTV channel activation. KTV is a fusion protein between TASK3 and Kv1.2 VSD (Inset). In C, currents were elicited by voltage pulses from −100 to 40 mV with 20 mV increments. In D, G–V and Boltzmann fits (solid lines) with V_1/2 and slope factor (millivolts) for control (21.7 ± 4.0 and 18.5 ± 4.2) and for C28 (19.2± 3.8 and 22.5 ± 4.0). (E) Residues interacting with C28 based on molecular docking to the cryo-EM structure of human KCNQ1 (PDB entry: 6uzz). The S3 to S4 loop was missing in the cryo-EM structure, and we built it using MODELER (55). (F) G–V shift of mutant I_Ks in response to C28. Each data point was averaged from recordings in three to seven oocytes. The curve for WT is the same as in Fig. 2D. (G) Maximal G–V shift and EC₅₀ for each mutation and WT.

Fig. 5.

Effects of C28 on KCNQ1 channels in the AO and IO states. (A) Current traces of KCNQ1 with mutations E160R-R237E, which arrested the VSD in the activated state and stabilized the channel at the AO state (36). Currents were recorded without (black) and with (red) 10 µM C28 at various voltages (see B). The voltage before and after the test pulses were −80 and −40 mV, respectively. (B) Steady-state current-voltage relations of KCNQ1 E160R-R237E mutant. (C) Current traces of KCNQ1 with mutations F167R-Q234E-D202N, which arrested the VSD at the intermediate state, thus the channels were stabilized at the IO state (10). Currents were recorded without (black) and with (red) 10 µM C28 at various voltages (see D). The voltage before and after the test pulses were −80 and −40 mV, respectively. (D) Current-voltage relations of KCNQ1 F167R-Q234E-D202N mutant. P < 0.05 in B but P > 0.05 in D between control and C28 at all voltages, using unpaired Student’s t test.

Fig. 6.

C28 enhances I_Ks and stabilizes APs in GP ventricular and atrial myocytes. (A) I_Ks currents, measured as the Chromanol 293B sensitive currents (SI Appendix, Fig. S8 A and B), of control (black) and with C28 (100 nM, red) at various voltages (see B) from a ventricular myocyte. The voltage before and after the test pulses were −40 and −20 mV, respectively. (B) I_Ks G–V relations in various C28 concentrations. The lines are fits to Boltzmann equation. (C) V_1/2 of G–V relations versus C28 concentration. (D) C28 (100 nM) on APs in control and in the presence of PI-103 (1 µM) and Moxifloxacin (Moxi, 100 µM). (E) Effects of C28 on APD. *P < 0.05, n = 5 to 44. (F) APs of a GP atrial myocyte recorded in control, C28 (100 nM and 10 µM), and after washout (Wash). The stimulus was 180 pA in amplitude and 10 ms in duration at 1 Hz frequency. (G) Effects of C28 on atrial APD. *P < 0.05 compared to control, n = 9 to 12.