Ginsenoside Rg₃Inhibits Human Kv1.4 Channel Currents by Interacting with the Lys531 Residue

Abstract

We have demonstrated previously that the 20(S) but not the 20(R) form of ginsenoside Rg 3 inhibited K ϩ currents flowing through Kv1.4 (hKv1.4) channels expressed in Xenopus laevis oocytes, pointing to the presence of specific interaction site(s) for Rg 3 in the hKv1.4 channel. In the current study, we sought to identify this site(s). To this end, we first assessed how point mutations of various amino acid residues of the hKv1.4 channel affected inhibition by 20(S)-ginsenoside Rg 3 (Rg 3 ). Lys531 residue is known to be a key site for K ϩ activation and to be part of the extracellular tetraethylammonium (TEA) binding site; the mutation K531Y abolished the Rg 3 effect and made the Kv1.4 channel sensitive to TEA applied to the extracellular side of the membrane. Mutations of many other residues, including the pH sensitive-site (H507Q), were without any significant effect. We next examined whether K ϩ and TEA could alter the effect of Rg 3 and vice versa. We found that 1) raising [K ϩ ] o reduced the inhibitory effect of Rg 3 on hKv1.4 channel currents, whereas Rg 3 shifted the K ϩ activation curve to the right, and 2) TEA caused a rightward shift of the Rg 3 concentration-response curve of wild-type hKv1.4 channel currents, whereas Rg 3 caused a rightward shift of the TEA concentration-response curve of K531Y mutant channel currents. The docked modeling revealed that Lys531 plays a key role in forming hydrogen bonds between Rg 3 and hKv1.4 channels. These results indicate that Rg 3 inhibits the hKv1.4 channel current by interacting with residue Lys531.

Ginseng Gintonin Activates the Human Cardiac Delayed Rectifier K+ Channel: Involvement of Ca2+/Calmodulin Binding Sites

“…Similarly, recombinant plasmids containing human KCNQ cDNA inserts were linearized by digestion with the appropriate restriction enzymes, and cRNAs were obtained using the mMessagemMachine transcription kit with SP6 RNA polymerase or T7 polymerase. The final cRNA products were resuspended at a concentration of 1 μg/μl in RNase-free water and stored at −80°C (Lee et al, 2008). …”

Molecular Mechanisms of Large-Conductance Ca2+-Activated Potassium Channel Activation by Ginseng Gintonin

“…Electrophysiological experiments were performed within 5-6 days of oocyte isolation, with gintonin or ginsenoside applied to the bath. For BK Ca channel experiments, BK Ca channel-encoding cRNAs (40 nl) were injected into the animal or vegetal pole of each oocyte one day after isolation, using a 10- μ L microdispenser (VWR Scientific, San Francisco, CA, USA) fitted with a tapered glass pipette tip (diameter, 15–20  μ m) [25]. …”

“…Similarly, recombinant plasmids containing rat BK Ca channel cDNA inserts were linearized by digestion with the appropriate restriction enzymes, and cRNAs were obtained using the mMessage mMachine in vitro transcription kit (Ambion, Austin, TX, USA) with SP6 RNA polymerase or T7 polymerase. The final cRNA products were resuspended at a concentration of 1  μ g/ μ L in RNase-free water and stored at −80°C [25]. …”

“…Therefore, a custom-made Plexiglas net chamber was used for two-electrode voltage-clamp recordings, as previously reported [25]. The oocytes were impaled with two microelectrodes filled with 3 M KCl (0.2–0.7 MΩ), and electrophysiological experiments were carried out at room temperature using an Oocyte Clamp (OC-725C, Warner Instruments, Hamsden, CT, USA).…”

Yin and Yang of ginseng pharmacology: ginsenosides vs gintonin

“…For example, the EC 50 values for the ginsenoside Rg 3 are approximately 0.41–97.3 μmol/L 4,14,15,16,17,18,19,20 . The ginsenoside Rg 3 not only inhibits voltage-gated Ca 2+ , K + , and Na + channels, ligand-gated 5-HT 3 , α7 nicotinic acetylcholine, and NMDA receptors but also activates K + channels such as KCNQ K + , BK Ca , and hERG K + 4,14,15,16,17,18,19,20,21 . Other ginsenosides enhance the activation of anion GABA A and glycine receptors 22,23 (Table 1).…”