Evidence that the tertiary structure of 20(S)-ginsenoside Rg3 with tight hydrophobic packing near the chiral center is important for Na+ channel regulation

Ginsenosides are the active ingredients of Panax ginseng. Ginsenoside Rg3 exists as two stereoisomers of carbon-20: 20-S-protopanaxatriol-3-[O-β-d-glucopyranosyl (1 → 2)-β-glucopyranoside] (20(S)-Rg3) and 20-R-protopanaxatriol-3-[O-β-d-glucopyranosyl (1 → 2)-β-glucopyranoside] (20(R)-Rg3). Recently, we reported that 20(S)-Rg3 regulates voltage-dependent Ca2+ channel activity and several types of ligand-gated ion channels, whereas 20(R)-Rg3 does not have this activity. In this study, we investigated the structure-activity relationship of these two stereoisomers by NMR spectroscopy and by measurement of the current in Xenopus oocytes expressing the mouse cardiac voltage-dependent Na+ channel (Nav1.5). We found that 20(S)-Rg3 but not 20(R)-Rg3 inhibited Na+ channel current in a dose- and voltage-dependent manner. The difference between Rg3 epimers in voltage-dependent ion channel regulation indicates that the structure of 20(S)-Rg3 may be geometrically better aligned than that of 20(R)-Rg3 for interaction with receptor regions in Na+ channels. The 1H and 13C NMR chemical shifts, including all hydroxyl protons of 20(S)-Rg3 and 20(R)-Rg3, were completely assigned, and their tertiary structures were determined. 20(S)-Rg3 has more tight hydrophobic packing near the chiral center than 20(R)-Rg3. Tertiary structures and activities of 20(S)-Rg3 and 20(R)-Rg3 indicate that 20(S)-Rg3 may have stronger interactions with the receptor region in ion channels than 20(R)-Rg3. This may result in different stereoselectivity of Rg3 stereoisomers in the regulation of voltage-dependent Na+ channel activity. This is the first structural approach to ginsenoside action on ion channel.