Inhibitory effects and mechanism of dihydroberberine on hERG channels expressed in HEK293 cells

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.

Fig 1. Inhibitive effect of immediate perfusion of DHB on hERG currents.

(A) Examples of current traces in control and DHB-treated groups were recorded using illustrated voltage clamp protocol. (B) Normalized I–V relationships for tail current with and without various concentrations of DHB (1, 10 and 100 μM). DHB significantly decreased hERG current. *P < 0.05, **P < 0.01 vs. control, n = 10. (C) Half-maximal inhibitory concentration (IC50) of DHB against hERG current (n = 10).

Fig 2. Immediate effects of DHB on hERG channel kinetics.

(A) Voltage-dependent activation curves for control and 10 μM DHB-treated group, curves were best fit to a Boltzmann function. (B) Representative current tracing of steady-state inactivation (left and right, for control and 10 μM DHB-treated groups, respectively) using a two-pulse protocol. (C) Normalized steady-state inactivation curves of the control and following exposure to 10 μM DHB. (D) Voltage clamp protocol and representative current tracing for onset of hERG channel inactivation. (E) Voltage clamp protocol and representative current tracing for the recovery from inactivation. (F) Time constant for onset of inactivation and recovery from inactivation before and after exposure to 10 μM DHB. Smooth curves were best fitted of the data to a Boltzmann function. *P < 0.05 vs. control, n = 10.

Fig 3. Effects of DHB on the expressions of the hERG protein and transcription facor Sp1 after 24 h incubation.

(A) Downregulation and upregulation of 155-kDa and 135-kDa hERG expression, respectively after treatment with different concentrations of DHB (1 μM and 10 μM) for 24 h. *P < 0.05 and ## P< 0.01 vs. control, n = 8. (B) Western blot results of Sp1 expression after 24 h incubation with DHB. The Sp1 expression was unchanged, n = 6.

Fig 4. DHB inhibits hERG current but does not affect the hERG channel kinetics after 24 h incubation.

(A) Examples of hERG current traces under control and DHB-treated conditions were recorded using the voltage protocol shown in Fig 1A. (B) Voltage-dependent activation curves for the control and DHB-treated groups. (C) Effects of DHB on inactivation curve after 24 h treatment. (D) Effects of DHB on time constant for onset of inactivation and the recovery from inactivation after 24 h treatment. Traces in B-D were recorded using protocols shown in Fig 2. n = 10.

Fig 5. Phe656 and Tyr652 binding mediate acute inhibition of hERG channel.

(A-C) Examples of hERG current traces and I–V curves of WT-hERG and mutant hERG current of control and acutely treated DHB groups. (D) Statistical graph of currents. WT-hERG but not Y652A-hERG or F656V-hERG current decreased. *P < 0.05 vs. control, n = 8.

Fig 6. Reduction in hERG expression after 10 μM DHB incubation was mediated by Tyr652 binding.

(A) Western blot results and (B) Statistical analysis of WT-hERG and mutant hERG protein of control and DHB-treated groups. Mature WT-hERG and F656V-hERG but not Y652A-hERG decreased after DHB treatment, *P < 0.05, ##P < 0.01 vs. control, n = 6. (C) Examples of hERG current traces and I–V curves of WT-hERG and mutant hERG current of control and incubation treated DHB groups. (D) Statistical graph of currents. WT-hERG and F656V-hERG but not Y652A-hERG current decreased. **P < 0.01, #P < 0.05 vs. control, n = 6.

Fig 7. DHB disrupts interactions between hERG and chaperones.

(A) Western blot analysis of Hsp70 and Hsp90 after 24 h incubation with 1 μM and 10 μM DHB. Hsp90 expression was concentration-dependently reduced while Hsp70 expression did not change. *P < 0.05 vs. control, n = 4. (B) Immunoprecipitation results show increased and decreased interaction between hERG and Hsp70 or Hsp90 respectively after treatment with 10 μM DHB. *P < 0.05 vs. control, n = 3.

Fig 8. Unfolded protein response is activated by DHB.

(A) DHB incubation for 24 h significantly decreased ATF-6 (~90 kDa) expression and proportionally increased cleaved ATF-6 (~50 kDa) expression. *P < 0.05 and #P < 0.05 vs. control, n = 6. (B) Western blot analysis shows DHB markedly increased the expression of both chaperones, calnexin and calreticulin. *P < 0.05, **P < 0.01, #P < 0.05, and ##P < 0.01 vs. control, n = 6.

Fig 9. Western blot analysis suggests that DHB accelerated mature 155-kDa hERG degradation.

hERG-HEK cells were treated with or without 10 μM DHB in the presence of BFA, which inhibits hERG protein transportation from the ER to the Golgi body. Cells were harvested and assayed using western blot at the designated time points. Intensities of the mature 155-kDa hERG bands were normalized to the time 0 h value. *P < 0.05 vs. control, n = 4.