Functional Expression of a Novel Ginsenoside Rf Binding Protein from Rat Brain mRNA inXenopus laevisOocytes

Abstract

We have shown that ginsenoside Rf (Rf) regulates voltagedependent Ca 2ϩ channels through pertussis toxin (PTX)-sensitive G proteins in rat sensory neurons. These results suggest that Rf can act through a novel G protein-linked receptor in the nervous system. In the present study, we further examined the effect of Rf on G protein-coupled inwardly rectifying K ϩ (GIRK) channels after coexpression with size-fractionated rat brain mRNA and GIRK1 and GIRK4 (GIRK1/4) channel cRNAs in Xenopus laevis oocytes using two-electrode voltage-clamp techniques. We found that Rf activated GIRK channel in a dose-dependent and reversible manner after coexpression with subfractions of rat brain mRNA and GIRK1/4 channel cRNAs. This Rf-evoked current was blocked by Ba 2ϩ , a potassium channel blocker. The size of rat brain mRNA responding to Rf was about 6 to 7 kilobases. However, Rf did not evoke GIRK current after injection with this subfraction of rat brain mRNA or GIRK1/4 channel cRNAs alone. Other ginsenosides, such as Rb 1 and Rg 1 , evoked only slight induction of GIRK currents after coexpression with the subfraction of rat brain mRNA and GIRK1/4 channel cRNAs. Acetylcholine and serotonin almost did not induce GIRK currents after coexpression with the subfraction of rat brain mRNA and GIRK1/4 channel cRNAs. Rf-evoked GIRK currents were not altered by PTX pretreatment but were suppressed by intracellularly injected guanosine-5Ј-(2-O-thio) diphosphate, a nonhydrolyzable GDP analog. These results indicate that Rf activates GIRK channel through an unidentified G protein-coupled receptor in rat brain and that this receptor can be cloned by the expression method demonstrated here.

Ginseng pharmacology: a new paradigm based on gintonin-lysophosphatidic acid receptor interactions

“…To observe the pharmacological effects of ginsenosides, cells must be pre-stimulated by electrical currents, excitatory ligands, or other treatments or subjected to injuries like hypoxia or ischemia, in the case of organs ( Nah, 2014 ). Second, ginsenosides must be applied at high micromolar concentrations (≈ 30–97 μM in EC 50 or IC 50 ) to elicit any physiological or pharmacological effects compared to other endogenous or exogenous ligands ( Choi et al, 2002 Nah et al, 2014). Third, the effects of ginsenosides are miscellaneous, non-selective, and receptor-independent (Figure 4 ).…”

Ginsenosides: Are Any of them Candidates for Drugs Acting on the Central Nervous System?

“…In the heart, acetylcholine released from the vagus nerve binds to M 2 receptors in the heart and activates GIRK channels, slowing the heart rate (Dascal 1997). One study showed that ginsenoside Rf activates GIRK channels when GIRK channel genes are co‐expressed in Xenopus oocytes with rat brain mRNA (Choi et al 2002a). The effect of ginsenoside Rf on GIRK current was concentration dependent and reversible; the EC 50 was 34 ± 3 μM, and the maximal effect was obtained at about 100 μM.…”

“…Ginsenoside Rf‐induced GIRK current enhancement was blocked by Ba 2+ , a K + channel blocker. Intracellular injection of GDPβS, but not pretreatment with PTX, attenuated ginsenoside Rf‐induced GIRK currents (Choi et al 2002a). These results provide evidence that ginsenoside Rf interacts with unidentified ginsenoside Rf‐binding protein(s) in the brain, and the activation of unidentified ginsenoside Rf‐binding protein(s) could be coupled to GIRK channels.…”

Modulating effect of ginseng saponins on heterologously expressed HERG currents in Xenopus oocytes

“…In our experiments, the effects of GS on HERG potassium currents were not blocked by intracellular Ca 2+ chelation and intraoocyte injection of BAPTA nor pretreatment with PTX (2 mg/L, 16 h) ( Figure 6A, 6B). Therefore, it should be noted that ginsenoside Rf could regulate GIRK channels with unidentified proteins derived from the rat brain through PTXinsensitive G proteins [30] . However, we still cannot exclude the possibility of direct interaction between ginsenosides and HERG channel proteins.…”