Abstract
The human ether-a-go-go-related gene (hERG) potassium channel conducts rapid delayed rectifier potassium currents (IKr) and contributes to phase III cardiac action potential repolarization. Drugs inhibit hERG channels by binding to aromatic residues in hERG helixes. Berberine (BBR) has multiple actions, and its hydrogenated derivative dihydroberberine (DHB) is a potential candidate for developing new drugs. Previous studies have demonstrated that BBR blocks hERG channels and prolongs action potential duration (APD). Our present study aimed to investigate the effects and mechanism of DHB on hERG channels. Protein expression and the hERG current were analyzed using western blotting and patch-clamp, respectively. DHB inhibited the hERG current concentration-dependently after instantaneous perfusion, accelerated channel inactivation by directly binding tyrosine (Tyr652) and phenylalanine (Phe656), and decreased mature (155-kDa) and simultaneously increased immature (135-kDa) hERG expression, respectively. This suggests disruption of forward trafficking of hERG channels. Besides, DHB remarkably reduced heat shock protein 90 (Hsp90) expression and its interaction with hERG, indicating that DHB disrupted hERG trafficking by impairing channel folding. Meanwhie, DHB enhanced the expression of cleaved activating transcription factor-6 (ATF-6), a biomarker of unfolded protein response (UPR). Expression of calnexin and calreticulin, chaperones activated by ATF-6 to facilitate channel folding, were also increased, which indicating UPR activation. Additionally, the degradation rate of mature 155-kDa hERG increased following DHB exposure. In conclusion, we demonstrated that DHB acutely blocked hERG channels by binding the aromatic Tyr652 and Phe656. DHB may decrease hERG plasma membrane expression through two pathways involving disruption of forward trafficking of immature hERG channels and enhanced degradation of mature hERG channels. Furthermore, forward trafficking was disrupted by impaired channel folding associated with altered interactions between hERG proteins and chaperones. Finally, trafficking inhibition activated UPR, and mature hERG channel degradation was increased by DHB.
Highlights
The human ether-a-go-go-related gene potassium channel conducts the rapid delayed rectifier potassium current (IKr), thereby contributing to phase III repolarization of the cardiac action potential [1]
The maximum % of Dimethyl sulfoxide (DMSO) in final experiment test solutions was 1%. human ether-a-go-go-related gene (hERG) currents were evoked through a 4-s repolarizing step to -50 mV, following a 2-s depolarization step with a 10 mV stepwise increase from -60 to 40 mV, which was initiated after a holding potential of -80 mV. hERG activation curves were fitted by Boltzmann sigmoidal after standardization of tail currents
Drug-induced cardiotoxic events are significantly involved in the QT prolongation caused by hERG channel deficiency [19]
Summary
The human ether-a-go-go-related gene (hERG) potassium channel conducts the rapid delayed rectifier potassium current (IKr), thereby contributing to phase III repolarization of the cardiac action potential [1]. Functional impairment of the hERG channel can result in inherited and acquired long QT syndrome type 2 (LQT2), caused by mutations of the hERG or off-target effects of diverse therapeutic agents, respectively [2]. Increasing attention has been focused on the cardiac safety evaluation of new drugs [4]. Current paradigms of cardiac safety assessment consider hERG channel inhibition a potential risk concern, and new drug approval departments regularly require developers to provide data on the effects of drugs on hERG channel before they are marketed [6]
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