The Natural Flavone Acacetin Blocks Kv4.3 Current by Interacting With P-Loop Filter Helix of the Channel

Abstract

Background and objective: We have recently demonstrated that the natural flavone acacetin is an atrial-selective compound that inhibits ultra-rapid delayed rectifier potassium current (IKur) and transient outward potassium current (Ito) in human atrial myocytes, and also acetylcholine-activated potassium current (IK.ACh). It increased atrial effective refractory period and effectively prevented atrial fibrillation (AF) in anesthetized dogs without prolonging QT interval of ECG. The present study was designed to investigate the potential molecular determinants of hK4.3 channels that encode human cardiac Ito. Methods: Cell culture, mutagenesis and whole-cell patch voltage-clamp techniques were used in the present study. Results: It was found acacetin inhibited hKv4.3 current in HEK 293 cells stably expressing Kv4.3 gene (KCND3) in a concentration-dependent manner. The current inhibition with an increase of time-to-peak and inactivation time constant of the current, suggesting an open channel blockade. However, the stimulation pause during drug administration revealed a strong tonic blocking property. This effect induced a use- or frequency-dependent inhibition at lower concentrations (1 and 3 M), but not at high concentrations. The IC50 of acacetin for inhibiting hKv4.3 was reduced from 6.09 M at 0.2 Hz to 5.80, 4.55, 3.96, and 3.65 M respectively at 1, 2, 3, and 4 Hz. The mutagenesis study showed that the channel blockade by acacetin was dramatically reduced in hKv4.3 mutant T366A and T367A (IC50, 197.8 M for T366A and 166.1 M for T367A) of the P-loop helix, and IC50 was also reduced in V392A, I395A, and V399A (IC50: 25.9 M, 24.1 M, and 9.5 M) of the S6 domain. Conclusion: These results demonstrate the novel information that acacetin is a tonic and open channel blocker of hKv4.3 by binding to T365 and T366 of the P-loop helix, and also interacts with V392, I395, and V399 of the S6 domain of hKv4.3 channels. The use- and rate-dependent blocking property of hKv4.3 by acacetin indicates that this natural compound could exert a strong suppressive effect in the treatment of tachycardiac arrhythmia diseases.