Ginsenosides are the active ingredients of Panax ginseng. Ginsenoside Rg(3) exists as two stereoisomers of carbon-20: 20-S-protopanaxatriol-3-[O-beta-D-glucopyranosyl (1 -> 2)-beta-glucopyranoside] (20(S)-Rg(3)) and 20-R-protopanaxatriol-3-[O-beta-D-glucopyranosyl (1 -> 2)-beta-glucopyranoside] (20(R)-Rg(3)). Recently, we reported that 20(S)-Rg(3) regulates voltage-dependent Ca2+ channel activity and several types of ligand-gated ion channels, whereas 20(R)-Rg(3) 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 (Na(v)1.5). We found that 20(S)-Rg(3) but not 20(R)-Rg3 inhibited Na+ channel current in a dose- and voltage-dependent manner. The difference between Rg(3) epimers in voltage-dependent ion channel regulation indicates that the structure of 20(S)-Rg(3) may be geometrically better aligned than that of 20(R)-Rg(3) for interaction with receptor regions in Na+ channels. The H-1 and C-13 NMR chemical shifts, including all hydroxyl protons of 20(S)-Rg(3) and 20(R)-Rg(3), were completely assigned, and their tertiary structures were determined. 20(S)-Rg(3) has more tight hydrophobic packing near the chiral center than 20(R)-Rg(3). Tertiary structures and activities of 20(S)-Rg(3) and 20(R)-Rg(3) indicate that 20(S)-Rg(3) may have stronger interactions with the receptor region in ion channels than 20(R)-Rg(3). This may result in different stereo selectivity of Rg(3) stereoisomers in the regulation of voltage-dependent Na+ channel activity. This is the first structural approach to ginsenoside action on ion channel. (c) 2005 Elsevier Inc. All rights reserved.