18β-Glycyrrhetinic acid preferentially blocks late Na current generated by ΔKPQ Nav1.5 channels

Abstract

Aim: To compare the effects of two stereoisomeric forms of glycyrrhetinic acid on different components of Na(+) current, HERG and Kv1.5 channel currents.

Methods: Wild-type (WT) and long QT syndrome type 3 (LQT-3) mutant ΔKPQ Nav1.5 channels, as well as HERG and Kv1.5 channels were expressed in Xenopus oocytes. In addition, isolated human atrial myocytes were used. Two-microelectrode voltage-clamp technique was used to record the voltage-activated currents.

Results: Superfusion of 18β-glycyrrhetinic acid (18β-GA, 1-100 μmol/L) blocked both the peak current (I(Na,P)) and late current (I(Na,L)) generated by WT and ΔKPQ Nav1.5 channels in a concentration-dependent manner, while 18α-glycyrrhetinic acid (18α-GA) at the same concentrations had no effects. 18β-GA preferentially blocked I(Na,L) (IC(50)=37.2 ± 14.4 μmol/L) to I(Na,P) (IC(50)=100.4 ± 11.2 μmol/L) generated by ΔKPQ Nav1.5 channels. In human atrial myocytes, 18β-GA (30 μmol/L) inhibited 47% of I(Na,P) and 87% of I(Na,L) induced by Anemonia sulcata toxin (ATX-II, 30 nmol/L). Superfusion of 18β-GA (100 μmol/L) had no effects on HERG and Kv1.5 channel currents.

Conclusion: 18β-GA preferentially blocked the late Na current without affecting HERG and Kv1.5 channels.

Figure 1

Effects of 18α-GA and 18β-GA on I_Na,P mediated by the WT Nav1.5 channel. (A) and (C) Representative current traces elicited by 500-ms test pulses from -70 mV to 30 mV with 10 mV increments at 0.1 Hz in typical oocytes perfused before and after in 100 μmol/L 18α-GA (A) or 30 μmol/L 18β-GA (C). The membrane potential was held at -120 mV. (B) and (D) Averaged current-voltage relationships of I_Na,P before and after perfusion with 100 μmol/L 18α-GA (B) or 30 μmol/L 18β-GA (D); n=6 per group; ^bP<0.05.

Figure 2

Effects of 18α-GA and 18β-GA on ΔKPQ Nav1.5 channels. (A) and (C) Original current recordings in the absence and presence of 100 μmol/L 18α-GA (A) or 18β-GA (C). Current was elicited by a depolarizing pulse from a holding potential of -120 mV to -30 mV for 500 ms. Insets show magnified current traces for comparison of the late component. (B) and (D) Averaged current-voltage relationships of I_Na,P before and after perfusion with 100 μmol/L 18α-GA (B) or 18β-GA (D); n=6 per group; ^bP<0.05.

Figure 3

Concentration-dependent block by 18β-GA of WT I_Na,P (A), ΔKPQ I_Na,P (B), and I_Na,L (C). I_Na was elicited by a depolarizing pulse from a holding potential of -120 mV to -30 mV for 500 ms at 0.1 Hz. Representative traces (upper panels) were superimposed before (control) and during perfusion of 18β-GA (1–100 μmol/L). (B, C) are traces from the same oocyte on an expanded scale. Summarized dose-response data (lower panels) fitted with the Hill equation. IC₅₀ values and the Hill coefficient are provided in the figure. n=6–27 oocytes/point.

Figure 4

Rate-dependent blockade of WT and ΔKPQ Nav1.5 channels. I_Na was elicited by a series of 30 depolarizing pulses of -120 mV to -20 mV at different stimulation frequencies. The relative current amplitude elicited by each pulse was normalized to the respective amplitudes the currents elicited by the first pulse and plotted against each pulse number: for WT I_Na,P, 30 μmol/L 18β-GA at 4 Hz (A) (n=6) and 100 μmol/L lidocaine at 1, 2, and 4 Hz (B) (n=5); for ΔKPQ, 100 μmol/L 18β-GA of I_Na,P (C) and I_Na,L (D) at 4 Hz (n=6). ^bP<0.05, ^cP<0.01.

Figure 5

Effects of 18β-GA on HERG (A) and Kv1.5 potassium channels expressed in Xenopus oocytes. The voltage protocols are shown in the upper panels. 18β-GA at 100 μmol/L had no significant effects on HERG (n=6) or Kv1.5 channels (n=7).

Figure 6

Effects of 18β-GA on I_Na,P and I_Na,L induced by ATX-II in isolated human atrial myocytes. (A) Original I_Na,P recoding in control oocytes and in the presence of 30 μmol/L 18β-GA. Current was elicited by a depolarizing pulse from a holding potential of -80 mV to -30 mV for 100 ms. (B) Representative I_Na,L induced by ATX-II (30 nmol/L) in the absence and in the presence of 30 μmol/L 18β-GA. Current was elicited by a depolarizing pulse from a holding potential of -80 mV to -30 mV for 500 ms. (C) The bar graph shows the percentage of inhibition of I_Na,P and I_Na,L induced by ATX-II mediated by 30 μmol/L 18β-GA. The numbers of cells are indicated in parentheses. ^bP<0.05.