Abstract

We demonstrated previously that ginsenoside Rg₃ (Rg₃), an active ingredient of Panax ginseng, inhibits brain-type Na⁺ channel activity. In this study, we sought to elucidate the molecular mechanisms underlying Rg₃-induced Na⁺ channel inhibition. We used the two-microelectrode voltage-clamp technique to investigate the effect of Rg₃ on Na⁺ currents (I_Na) in Xenopus laevis oocytes expressing wild-type rat brain Na_V1.2 α and β1 subunits, or mutants in the channel entrance, the pore region, the lidocaine/tetrodotoxin (TTX) binding sites, the S4 voltage sensor segments of domains I to IV, and the Ile-Phe-Met inactivation cluster. In oocytes expressing wild-type Na⁺ channels, Rg₃ induced tonic and use-dependent inhibitions of peak I_Na. The Rg₃-induced tonic inhibition of I_Na was voltage-dependent, dose-dependent, and reversible, with an IC₅₀ value of 32 ± 6 μM. Rg₃ treatment produced a 11.2 ± 3.5 mV depolarizing shift in the activation voltage but did not alter the steady-state inactivation voltage. Mutations in the channel entrance, pore region, lidocaine/TTX binding sites, or voltage sensor segments did not affect Rg₃-induced tonic blockade of peak I_Na. However, Rg₃ treatment inhibited the peak and plateau I_Na in the IFMQ3 mutant, indicating that Rg₃ inhibits both the resting and open states of Na⁺ channel. Neutralization of the positive charge at position 859 of voltage sensor segment domain II abolished the Rg₃-induced activation voltage shift and use-dependent inhibition. These results reveal that Rg₃ is a novel Na⁺ channel inhibitor capable of acting on the resting and open states of Na⁺ channel via interactions with the S4 voltage-sensor segment of domain II.