Allocryptopine and benzyltetrahydropalmatine block hERG potassium channels expressed in HEK293 cells

Abstract

Aim: Allocryptopine (ALL) is an alkaloid extracted from Corydalis decumbens (Thunb) Pers. Papaveraceae, whereas benzyltetrahydropalmatine (BTHP) is a derivative of tetrahydropalmatine extracted from Corydalis ambigua (Pall) Cham et Schlecht. The aim of this study was to investigate the effects of ALL and BTHP on the human ether-a-go-go related gene (hERG) current expressed in HEK293 cells.

Methods: Cultured HEK293 cells were transiently transfected with hERG channel cDNA plasmid pcDNA3.1 using Lipofectamine. The whole-cell current IHERG was evoked and recorded using Axon MultiClamp 700B amplifier. The drugs were applied via supserfusion.

Results: Both ALL and BTHP reversibly suppressed the amplitude and density of IHERG in concentration- and voltage-dependent manners (the respective IC50 value was 49.65 and 22.38 μmol/L). BTHP (30 μmol/L) caused a significant negative shift of the steady-state inactivation curve of IHERG, while ALL (30 μmol/L) did not affect the steady-state inactivation of IHERG. Furthermore, BTHP, but not ALL, shortened the time constants of fast inactivation and slow time constants of deactivation of IHERG. But both the drugs markedly lengthened the time constants for recovery of IHERG from inactivation. Using action potential waveform pulses, it was found that both the drugs at 30 μmol/L significantly suppressed the current densities in the late phase of action potential, but did not significantly affect the current densities in the early phase of action potential.

Conclusion: Both ALL and BTHP derived from Chinese herbs potently block hERG current.

Figure 1

Chemical formula of ALL (A) and BTHP (B). ALL, an alkaloid, is extracted from Corydalis decumbens (Thunb) Pers. Papaveraceae and BTHP is a derivative of tetrahydropalmatine which is extracted from Corydalis ambigua (Pall) Cham et Schlecht.

Figure 2

Inhibition of hERG channels by ALL and BTHP. (A and B) Representative current traces recorded from the same cell under control conditions and after superfusion with ALL and BTHP (10, 30, and 100 μmol/L). (C and D) Concentration-response relationship of the effects of ALL and BTHP on hERG peak tail currents (n=20). The IC₅₀ of ALL was 49.65 μmol/L (95% CI: 34.75–64.54 μmol/L) with a Hill coefficient of 1.21 and the IC₅₀ of BTHP was 22.38 μmol/L (95% CI: 13.56–31.20 μmol/L) with a Hill coefficient of 1.34. (E and F) Time course of hERG tail current inhibition by 30 μmol/L ALL and BTHP and washout effect (n=15). (G) The hERG current amplitude was decreased by dofetilide 10 nmol/L.

Figure 3

Inhibition effect of ALL and BTHP on step and tail currents by repolarizing pulse of −40 mV. (A) Representative current traces recorded under control conditions and after superfusion with ALL and BTHP (30 μmol/L). (B) The inhibitory effect of drugs (30 μmol/L) on repolarizing pulse of -40 mV is shown and the peak amplitude of step current was reduced. At test potential of +60 mV, the step current was reduced from 51.2±3.2 pA/pF to 34.6±2.9 pA/pF by ALL and to 24.5±2.4 pA/pF by BTHP and the magnitude of I_hERG tail was from 143.5±7.8 to 97.1±5.6 pA/pF by ALL and to 65.3±4.2 pA/pF by BTHP. (C and D) Summary of ALL and BTHP on current density-voltage relationship of step and tail currents. ^bP<0.05, ^cP<0.01, n=15.

Figure 4

Inhibitory effect of ALL and BTHP on step and tail currents by reploarizing pulse of −110 mV. (A) Representative current traces recorded under control conditions and after superfusion with ALL and BTHP (30 μmol/L). (B) The inhibitory effect of drugs (30 μmol/L) on reploarizing pulse of −110 mV is shown and the magnitude of I_hERG tail was reduced from −190.3±11.7 to −137.4±9.9 pA/pF by ALL and to −105.4±7.8 pA/pF by BTHP. (C) Summary of ALL and BTHP on current density-voltage relationship of tail currents. ^bP<0.05, ^cP<0.01, n=15.

Figure 5

Effects of ALL and BTHP on rectification characteristics of hERG current. (A and B) Representative current traces before and after exposure to two drugs are shown. (C and D) I–V relationship for steady-state exposure to two drugs exhibits similar pattern to inward rectification characteristics of control between −70 mV to +20 mV, but rectification curve by 30 μmol/L BTHP diverges slightly lower than that of control.

Figure 6

Effects of ALL and BTHP on the activation and deactivation of hERG current. Normalized tail currents were displayed as a function of the preceding test pulse voltages and fitted with a Boltzmann function. Steady-state activation curves are shown differently in the absence of and presence of 30 μmol/L ALL and 30 μmol/L BTHP (n=17, A and B). Time constants of activation were not markedly different before and after exposure to two drugs (C and D). Fast time constants of deactivation were similar before and after exposure to two drugs. The slow time constants of deactivation were shortened by 30 μmol/L BTHP, but not affected by 30 μmol/L ALL (E and F). The fast and slow time constant proportion under each test potential is not changed before and after exposure to two drugs, respectively (G and H).

Figure 7

Effects of ALL and BTHP on steady-state inactivation and fast inactivation of hERG current. V_1/2 of the steady-state inactivation curve of hERG current showed more negative shift by 30 μmol/L BTHP (from −54.13±2.42 mV to −78.89±2.38 mV) (P<0.01, n=17), while showed only slight change by 30 μmol/L ALL (from −55.33±3.14 mV to −57.38±2.52 mV) (A–D). Inactivating currents were fitted with single exponential functions to obtain time constants. Voltage dependence of time course of fast inactivation was investigated. Compared with control, time constant of fast inactivation was significantly shortened by 30 μmol/L BTHP (P<0.01, n=17) but was only slightly shortened by 30 μmol/L ALL over +10 mV (E–H).

Figure 8

Effects of ALL and BTHP on voltage dependence of recovery of inactivation of hERG currents. Recovery from inactivation traces was observed as the time-dependent initial increase in current amplitude at potentials from −120 mV to −50 mV for 3000 ms after condition pulse at +50 mV for 1500 ms (A and B). Tail currents were fitted by a single-exponential function. For two drugs, the time constants for recovery from inactivation were significantly lengthened over -80 mV test potentials compared to control with more significant acceleration of prolongation by 30 μmol/L BTHP (C and D). ^bP<0.05, ^cP<0.01 vs control, n=17.

Figure 9

Effects of ALL and BTHP on characteristics of hERG current with action potential waveform pulse. Digitized action potential waveform was used as command potential. (A) Representative current tracings before and after exposure to two drugs. (B) The current densities by two drugs, as a function of the voltage applied, showed marked decrease compared to control during late phase of action potential, especially for peak current. The peak current densities, as a function of the voltage applied, showed a decrease of 43.22%±5.3% of ALL and 56.78%±2.7% of BTHP during the late phase of action potential (n=6, B), while they were not significantly different during early phase of action potential. ^cP<0.01 vs control.