Ginseng ginsenoside pharmacology in the nervous system: involvement in the regulation of ion channels and receptors

Ginseng, the root of Panax ginseng C.A. Meyer, is one of the oldest traditional medicines and is thought to be a tonic. It has been claimed that ginseng may improve vitality and health. Recent studies have advanced ginseng pharmacology and shown that ginseng has various pharmacological effects in the nervous system. Ginsenosides, steroid glycosides extracted from ginseng, were one of the first class of biologically active plant glycosides identified. The diverse pharmacological effects of ginsenosides have been investigated through the regulation of various types of ion channels and receptors in neuronal cells and heterologous expression systems. Ginsenoside Rg3 regulates voltage-gated ion channels such as Ca2+, K+, and Na+ channels, and ligand-gated ion channels such as GABAA, 5-HT3, nicotinic acetylcholine, and N-methyl-D-aspartate (NMDA) receptors through interactions with various sites including channel blocker binding sites, toxin-binding sites, channel gating regions, and allosteric channel regulator binding sites when the respective ion channels or receptors are stimulated with depolarization or ligand treatment. Treatment with ginsenoside Rg3 has been found to stabilize excitable cells by blocking influxes of cations such as Ca2+ and Na+, or by enhancing Cl− influx. The aim of this review is to present recent findings on the pharmacological functions of the ginsenosides through the interactions with ion channels and receptors. This review will detail the pharmacological applications of ginsenosides as neuroprotective drugs that target ion channels and ligand-gated ion channels.


INTRODUCTION
Ginseng, the root of Panax ginseng C.A. Meyer, contains a variety of ingredients useful in herbal medicines (Tyler, 1995). Ginseng glycosides, also called ginsenosides or ginseng saponin, are derivatives of triterpenoid dammarane, which consists of 30 carbon atoms. Each ginsenoside has a common hydrophobic four ring steroid-like structure with carbohydrate moieties attached (Nah et al., 2007). Several types of ginsenosides have been isolated and identified from the roots of various ginseng species from America, China, and Korea. They are mainly classified as protopanaxadiol (PD), protopanaxatriol (PT), oleanolic ginsenosides, and ginsenoside metabolites according to the position of different carbohydrate moieties at the carbon-3 and carbon-6 positions, as well as the aliphatic side chain (Figure 1). Recent studies have demonstrated that ginsenosides exhibit a variety of pharmacological effects in nervous and non-nervous systems. A line of accumulating evidence shows that ginsenoside Rg 3 , the most active ginsenoside, interacts and regulates voltage-gated ion channels and ligand-gated ion channel activity through interaction with specific amino acid(s) at channel entryways or channel pore regions that are associated with ion influx or efflux (Lee et al., 2004b). This review will describe the physiology and pharmacology of ginseng ginsenoside in the regulation of voltage-gated ion and ligand-gated ion channel activities through interactions with specific amino acids of channel proteins and receptors.

REGULATION OF VOLTAGE-GATED Ca 2+ CHANNELS BY GINSENOSIDES
Ginseng extract and ginsenosides inhibit Ca 2+ channel currents in sensory neurons. Among the various ginsenosides such as Rb 1 , Rc, Re, Rf, and Rg 1 , ginsenoside Rf was found to be the most effective in inhibiting Ca 2+ channel activities (Nah et al., 1995). Ginsenosides also inhibit Ca 2+ channels in rat chromaffin cells, which are one of the representative neurosecretory cells involved in catecholamine release under various stress conditions (Kim et al., 1998a). The ginsenosides listed in order of inhibitory potency on Ca 2+ channels in rat chromaffin cells are: ginsenoside Rc > Re > Rf > Rg 1 > Rb 1 . Ginsenosides showed a selectivity in Ca 2+ channel regulation, inhibiting N-, P-, and Q/R-, but not L-type Ca 2+ channels in bovine chromaffin cells (Choi et al., 2001). Ginsenoside Rg 3 more potently inhibits L-, N-, and P-type Ca 2+ channels than other ginsenosides tested in rat sensory neurons . Ginsenoside Rb1 selectively inhibits L-type Ca 2+ channel activity in cultured rat hippocampal neurons (Lin et al., 2012). Besides neuronal Ca 2+ channels, ginsenoside Re also selectively inhibits L-type Ca 2+ channel activity in guinea pig cardiomyocytes (Bai et al., 2003(Bai et al., , 2004. In addition, ginsenosides attenuated the stimulation of membrane capacitance increase ( C m ) in rat chromaffin cells. The ginsenosides listed in order of inhibitory potency on C m are: ginsenoside Rf > Rc > Re > Rg 1 > Rb 1 (Kim et al., 1998a). The regulation of Ca 2+ channel activity and membrane capacitance by ginsenosides indicates that ginsenosides are closely involved in the regulation of neurotransmitter release from presynaptic nerve terminal(s) (Duan and Nicholson, 2008).

IDENTIFICATION OF GINSENOSIDE INTERACTION SITES IN VOLTAGE-GATED Ca 2+ CHANNEL REGULATION
Among the various domains of the L-type Ca 2+ channel protein, mutations in L427R, N428R, and L431K in transmembrane domain-I-segment 6 (IS6) of the channel significantly attenuated the action of ginsenoside Rg 3 , resulting in a shift to the right in dose-response curves, although the inhibitory effects of ginsenoside Rg 3 on Ca 2+ channel currents was not completely abolished ). In addition, while ginsenoside Rg 3 treatment produced a negative shift in the inactivation voltage, it did not alter the steady-state activation voltage, and none of the mutant channels affected the ginsenoside Rg 3 -induced negative shift in inactivation voltage. Ginsenoside Rg 3 had no effect on the inactivation time constant in wild-type and mutant channels. Thus, mutations in L427R, N428R, and L431K in transmembrane domain-I-segment 6 (IS6) of the channel partially attenuated ginsenoside Rg 3 action. The Leu427, Asn428, and Leu431 residues of the transmembrane domain-I-segment 6 of L-type Ca 2+ channels play important roles in the regulation of L-type Ca 2+ channels by ginsenoside Rg 3   (Figure 2).

REGULATION OF VOLTAGE-GATED K + CHANNELS BY GINSENOSIDES
Ginseng total saponins and ginsenoside Rg 3 have been shown to activate Ca 2+ -activated K + and ATP-sensitive K + channels in rabbit coronary artery smooth muscle cells (Chung and Kim, 1999;Chung and Lee, 1999). Ginsenosides activate Ca 2+activated K + channels in vascular smooth muscle cells . In addition, the external application of ginseng total saponin fraction and ginsenoside Rg 3 to rabbit coronary artery smooth muscle cells has been shown to increase the amplitude of whole-cell BK Ca currents (Chung and Kim, 1999). Treatment with ginseng total extract relaxed rabbit vaginal tissue via hyperpolarization of BK Ca channel activation (Kim et al., 2008a). In guinea pig cardiomyocytes, ginsenoside Re enhanced the delayed rectifier K + channel (I Ks ) (Bai et al., 2003(Bai et al., , 2004. Ginsenosideinduced relaxation of blood vessels and other smooth muscles may be achieved via activation of K + channels . By contrast, ginsenoside Rg 3 inhibits voltage-dependent Kv1.4 and Kv4.2 in human cells, but not Kv1.3, Kv1.5, and Kv2.1 expressed in Xenopus laevis oocytes (Jeong et al., 2004;Lee et al., 2013c), indicating that ginsenoside Rg 3 differentially regulates Kv channel subtypes. The regulatory effect of ginsenoside Rg 3 on Kv1.4 channel activity has been found to be strongly dependent on the extracellular K + concentration, by shifting the ginsenoside Rg 3 concentration-response curve to the right, indicating that ginsenoside Rg 3 competes with extracellular [K + ] for the same interaction site(s) . Further study showed that the inhibitory effects of ginsenoside Rg 3 on Kv1.4 channel currents were abolished by K + activation, which is induced by increasing extracellular K + concentrations. In addition, some subsets of Kv channel currents are also affected by extracellular and intracellular tetraethylammonium (TEA), which is a well-known K + channel blocker. The wild-type Kv1.4 channel, however, is nearly insensitive to TEA (Pardo et al., 1992;Lee et al., 2008a). Thus, although extracellular TEA treatment did not inhibit the wild-type Kv1.4 channel, it appeared that extracellular TEA competed with ginsenoside Rg 3 to inhibit Kv1.4 channel currents by shifting the ginsenoside Rg 3 concentration-response curve to the right . Based on these results, ginsenoside Rg 3 may have specific interaction site(s) for Kv1.4 channel activity regulation.

GINSENOSIDE Rg 3 INTERACTS WITH THE EXTRACELLULAR TEA BINDING SITE TO REGULATE Kv1.4 CHANNEL ACTIVITY
The K + activation site of the Kv1.4 channel, which is located at the outer pore entry, consists of several amino acids including lysine 531 (K531) (Claydon et al., 2004). In addition, one of the extracellular TEA binding sites also contains the K531 residue. Mutations in this K531 residue to tyrosine (i.e., K531Y) increased the sensitivity of the Kv1.4 channel to extracellular TEA, abolished K + activation, and also abolished the effect of ginsenoside Rg 3 on the Kv1.4 channel. Thus, ginsenoside Rg 3mediated action of Kv1.4 channel activity may occur through common interaction site(s) for K + activation and TEA binding sites. Alternatively, the ginsenoside Rg 3 interaction site(s) may overlap or share the K + activation site or the TEA binding FIGURE 2 | Topology and sequence alignment of Cav1.2, Nav1.2, and Nav1.4 channels. Ca 2+ and Na + channels consist of the main α subunit and other auxiliary subunits. α subunit has 4 domains and each domains contain 6 segments. Each segment 4 plays a membrane voltage sensor and segments 5 and 6 form channel pore. The mutated amino acid residues are underlined in the pore-forming segment 6. The underlined specific amino acid residues in these channels are thought to interact with 20(S)-ginsenoside Rg 3 (Rg 3 ) (Lee et al., 2008b;Choi et al., 2009). site, as shown by Lee et al. (2008a) using various Kv1.4 channel mutations, including the K531 residue. Mutations have been generated in channel pore sites (S510K, D513Q, V525L, and V535Q) and outer pore sites (K531A, P532A, I533A, T534A, and V535A) (Watanabe et al., 2004). Kv1.4 channel mutations have also been generated in the N-glycosylation site (N353Q) (Judge et al., 1999), the voltage sensor site (R447C and R450C) (Claydon et al., 2004), the voltage shift sites (L478F and G548P) (Bett and Rasmusson, 2004), the pH sensitive site (H507Q), and the C-type inactivation site (V560A) (Claydon et al., 2004). The K531A mutant, located in one of the outer pores, significantly attenuated ginsenoside Rg 3 inhibition of Kv1.4 channel currents, while the other mutations had no significant effect. Thus, ginsenoside Rg 3 regulates Kv1.4 channel activity by interacting with Lys531, which is also known to be one of the K + activation sites and one of the extracellular TEA binding sites. Other mutant channels at the K531 residue, such as K531Y, I533M, and K531Y-I533M, showed that the K531Y substitution, but not I533M, and the K531Y-I533M double substitution mostly abolished ginsenoside Rg 3 inhibition of Kv1.4 channel currents . Ginsenoside Rg 3 -mediated action of Kv1.4 channel activity is closely associated with the Lys531 residue (Figure 3).
Using homology and virtual docking model methods, which give three-dimensional configurations, Lee et al. (2008a) demonstrated that ginsenoside Rg 3 could bind to the Kv1.4 channel protein through various interactions such as hydrogen bonds or hydrophobic interactions. Therefore, the carbohydrate portion of ginsenoside Rg 3 plays an important role in its interaction with the Kv1.4 channel. The second, but not the first, carbohydrate attached at carbon-3 of the ginsenoside Rg 3 backbone forms six FIGURE 3 | Topology and sequence alignment of BK Ca , hERG, KCNQ1, and Kv1.4 K + channels. BK Ca , hERG, KCNQ1, and Kv1.4 K + consist of the main α subunit and auxiliary β subunits. Tetramer of α subunit forms a functional K + channel. α subunit contains 6 segments. Each segment 4 plays a membrane voltage sensor and segments 5 and 6 form channel pore. The mutated amino acid residues are underlined in the pore-forming segment 5 and 6. The underlined specific amino acid residues in those K + channels are thought to interact with 20(S)-ginsenoside Rg 3 near the K + channel "signature sequence" of -TXGYGD- Choi et al., 2010Choi et al., , 2011a.
hydrogen bonds with amino acids in the pore entryway of the Kv1.4 channel. Among the amino acids forming hydrogen bonds with ginsenoside Rg 3 , K531 forms three bonds, and threonine and histidine form the other hydrogen bonds. In addition, the ginsenoside Rg 3 backbone is located in the pore portion of the Kv1.4 channel, enabling it to block the pore and interrupt K + efflux when the channel is stimulated by depolarization. The backbone of ginsenoside Rg 3 may act as a physical plug or wedge in ginsenoside Rg 3 -mediated Kv1.4 channel regulation. However, the K531Y mutant in the Kv1.4 channel forms only two hydrogen bonds with ginsenoside Rg 3 . Thus, the use of site-directed mutagenesis, as well as homology and virtual docking model methods, are useful tools to confirm and identify ginsenoside Rg 3 interaction sites on the Kv1.4 channel (Figure 3).

GINSENOSIDE Rg 3 REGULATES BK Ca CHANNEL ACTIVITY THROUGH INTERACTION WITH AMINO ACID RESIDUES HOMOLOGOUS TO THE Kv1.4 CHANNEL
Ginsenoside Rg 3 enhanced BK Ca channel currents, in contrast to its inhibitory effect on Kv1.4 channel currents (Choi et al., 2011a). Interestingly, BAPTA, a Ca 2+ chelator, did not block ginsenoside Rg 3 -induced enhancement of BK Ca channel currents, indicating that ginsenoside Rg 3 -enhancement of BK Ca channel currents was independent of intracellular Ca 2+ . The BK Ca channel was sensitive to TEA, with a significant shift in the concentration response curve of ginsenoside Rg 3 to the right in the presence of TEA, and vice versa in the wild-type channel, indicating that ginsenoside Rg 3 competes for extracellular TEA binding site(s), similarly to mutant Kv1.4 channel interaction . In addition, Choi et al. (2011a) reported that ginsenoside Rg 3 -induced enhancement of the BK Ca channel current was independent of the β subunit, suggesting that ginsenoside directly interacts with the BK Ca channel α subunit. At the channel pore entry of the α subunit in mutant BK Ca channels, the effect of ginsenoside Rg 3 on BK Ca channel current enhancements was also almost abolished, for instance, in Y360I mutant channels, in which the residue is located at near K + channel signature sequence, which is also known as the extracellular TEA binding site of the BK Ca channel (Choi et al., 2011a) (Figure 3).

GINSENOSIDE Rg 3 ALSO REGULATES hERG K + (I Kr ) AND KCNQ K + CHANNEL (I Ks ) ACTIVITY THROUGH INTERACTION WITH AMINO ACID RESIDUES HOMOLOGOUS TO THE Kv1.4 CHANNEL
Ginsenosides have been shown to exhibit anti-hypertension and cardio-protective effects (Attele et al., 1999). Ginseng extract shortens action potential duration, whereas ginsenoside Re has been shown to regulate the I Kr and I Ks channel currents of guinea pig cardiomyocytes (Bai et al., 2003;Furukawa et al., 2006). However, relatively little is known at the molecular level about how ginseng extract and ginsenosides shorten action potential duration through the activation of hERG (I Kr ) and KCNQ (I Ks ) K + channels. Ginsenoside Rg 3 has been shown to regulate hERG (I Kr ) K + channel by enhancing I hERG and I tail . Ginsenoside Rg 3 not only caused a persistent I deactivating−tail without decay but also decelerated deactivating time constants. The mutation of S631 to S631C in the hERG α subunit has been reported to abolish ginsenoside Rg 3 -mediated action of hERG K + channels. Thus, ginsenoside Rg 3 enhanced I hERG and I tail , and induced a persistent I deactivating−tail with delayed deactivation of the hERG K + channel through interaction with the S631 residue (Choi et al., 2011b) (Figure 3).
Ginsenoside Rg 3 enhances I Ks currents, indicating that it regulates the KCNQ (I Ks ) K + channel, which consists of KCNQ1 and KCNE1 subunits (Choi et al., 2010). Site-directed mutagenesis has shown that the K318 and V319 residues of the KCNQ1 or KCNQ1 plus KCNE1 channels are involved in ginsenoside Rg 3 -mediated action of KCNQ1 plus KCNE1 channel activity (Figure 3). Homology docking models show that the K318 residue plays an important role in the interaction of ginsenoside Rg 3 in the closed or open state of the channels (Choi et al., 2010). Ginsenoside Rg 3 interacts with the S631 residue of the hERG K + channel and the K318 and V319 residues of the KCNQ1 plus KCNE1 channel, respectively.

REGULATION OF VOLTAGE-GATED Na + CHANNEL BY GINSENOSIDES
Ginsenosides also regulate Na + channel activities, for instance, inhibiting neuronal Na + channels expressed in tsA201 cells and Xenopus laevis oocytes. Higher concentrations of ginseng extract and ginsenoside Rb 1 than those used in other channel interaction experiments were required to inhibit Na + channel currents (Liu et al., 2001). Ginsenoside Rg 3 was highly potent in inhibiting Na + channel currents compared to ginseng extract and the other ginsenosides tested (Jeong et al., 2004). In structure-activity relationships of the ginsenoside Rg 3 stereoisomers, 20(S)-ginsenoside Rg 3 but not 20(R)ginsenoside Rg 3 inhibited the neuronal Na + channel currents in a dose-and voltage-dependent manner. The hydroxyl group at carbon-20 of 20(S)-ginsenoside Rg 3 may be geometrically better aligned with the hydroxyl acceptor group in the ion channels than that of the 20(R)-ginsenoside Rg 3 (Jeong et al., 2004).
A structure-activity relationship study of ginsenoside Rg 3 investigated the role of the aliphatic side chain, [-CH 2 CH 2 CH = C(CH 3 ) 2 ], which is coupled to carbon-20 of the 20(S)ginsenoside Rg 3 backbone in Na + channel interaction (Lee et al., 2008c). The reduction of the double bond in the aliphatic side chain of 20(S)-ginsenoside Rg 3 strengthens the inhibitory effect on Na + channel activity, shifting the concentration-response curve significantly to the left. However, deletion, hydroxylation, or oxygenation of the aliphatic side chain caused an attenuation or loss of Na + channel current inhibition. The aliphatic side chain of 20(S)-ginsenoside Rg 3 , as well as the hydroxyl group of carbon-20 of ginsenoside Rg 3 stereoisomers, plays an important role in Na + channel regulation. Thus, the aliphatic side chain of ginsenoside Rg 3 may be a future target for chemical modifications to tune regulation of Na+ channels by Rg 3 .
In further studies on ginsenoside Rg 3 -mediated voltage-gated neuronal Na + channel interaction, two main characteristics have been identified. One is that ginsenoside Rg 3 treatment causes a depolarizing shift in the activation voltage step in wild-type Na + channels, indicating that ginsenoside Rg 3 binding to the Na + channel does not allow the Na + channel to easily open at a given voltage step, requiring greater depolarizing stimulation compared to untreated channels . The other characteristic is that ginsenoside Rg 3 induces use-dependent inhibition, meaning that the channel pore-blocking actions of ginsenoside Rg 3 are enhanced by rapid, repeated stimulation over a very short time period, indicating that ginsenoside Rg 3 may be a kind of open channel blocker.
The idea that ginsenoside Rg 3 is a kind of open channel blocker of Na + channel is supported by experiments using inactivationdeficient Na + channel mutants, in which the inactivation gate has been mutated from IFM to Q3, and transient inward currents are converted into long-lasting inward currents . Ginsenoside Rg 3 more potently inhibited the plateau than peak I Na , and facilitated channel closing in inactivation-deficient channel mutants. Interestingly, mutations in one amino acid (Lys859 to Glu859 in brain Na V 1.2 channel) in the voltage-sensor domain in the S4 helix abolished the ginsenoside Rg 3 -mediated depolarizing shift without affecting the ginsenoside Rg 3 -mediated inhibition of peak current (I Na ). These results indicate that ginsenoside Rg 3 may have interaction sites for the brain Nav1.2 channel regulation.

IDENTIFICATION OF GINSENOSIDE INTERACTION SITES IN VOLTAGE-GATED Na + CHANNEL REGULATION
Although ginsenoside Rg 3 regulates neuronal Na + channels as a kind of open channel blocker, showing use-dependent inhibition and a depolarizing shift in the activation curve of wild-type Na + channels, the exact interaction sites in the Na + channel proteins have not been identified. Batrachotoxin (BTX) is a neurotoxin that acts on Na + channels. BTX toxin was first found in the skin of the South American frog Phyllobates terribilis, and persistently activates brain Na V 1.2 and skeletal muscle Na V 1.4 channels, rather than inhibiting Na + currents as lidocaine and tetrodotoxin do (Wang and Wang, 1998). In addition, BTX is a steroidal alkaloid toxin, with a backbone structure similar to that of ginsenoside Rg 3 . Interestingly, ginsenoside Rh 2 inhibited [ 3 H]BTX-B binding in rat brain membrane fractions and attenuated glutamate release (Duan et al., 2006;Duan and Nicholson, 2008), showing that the ginsenoside Rg 3 -induced interaction with rat brain Na V 1.2 channel activity may involve the BTX binding sites, and that the interference by ginsenoside of [ 3 H]BTX-B binding in rat brain membrane fractions is relevant to ginsenoside Rg 3 -induced Na + channel regulation. BTX interaction sites are located at the I433, N434, and L437 residues of the Na V 1.4 channel and equivalent residues such as I417, N418, and L421 of brain Na V 1.2 channels in domain-I segment 6 (IS6).
Channel mutations in BTX binding sites, such as N418K and L421K in rat brain Na V 1.2, and L437K in the mouse skeletal muscle Na V 1.4 channel, have been shown to attenuate or abolish ginsenoside Rg 3 inhibition of Na + currents (Figure 2). In addition, channel mutations in BTX binding sites also greatly attenuate the ginsenoside Rg 3 -mediated depolarizing shift in the activation voltage observed in wild-type channels. Moreover, ginsenoside Rg 3 -mediated use-dependent inhibition was almost abolished in these mutant channels. BTX binding sites in brainand muscle-type Na + channels play important roles in ginsenoside Rg 3 -mediated Na + channel interaction at cellular and molecular levels (Lee et al., 2008b).

REGULATION OF GABA A AND GLYCINE RECEPTOR CHANNEL ACTIVITY BY GINSENOSIDES
Ginseng has been shown to have an anxiolytic-like effect in animal model studies . Recent studies have shown that ginsenosides interact with the GABA A receptor, and may regulate the binding of the ligand with the GABA A receptor. Ginsenosides differentially regulate the binding of [ 3 H]-flunitrazepam or [ 3 H]muscimol to the GABA A receptor in a rat brain membrane fraction (Kimura et al., 1994). On the other hand, prolonged infusion with ginsenoside Rc in the rat brain elevates [ 3 H]-muscimol binding to the GABA A receptor in a brain region-specific manner, while ginsenoside Rg 1 has no effect . Thus, ginsenosides may regulate the GABA A receptor by affecting the binding affinities of its ligands.
In a GABA A receptor channel activity study, ginsenosides were also shown to regulate GABA A receptor channel activity by enhancing GABA-mediated channel activity (Choi et al., 2003a). Thus, in studies using Xenopus oocytes expressing human recombinant GABA A receptor, ginsenosides Rb 1 , Rb 2 , Rc, Rd, Re, Rf, Rg 1 , and Rg 2 affected GABA A receptor channel activity, and ginsenoside Rc most potently enhanced the GABA-induced inward peak current (I GABA ). Bicuculline, a GABA A receptor antagonist, and picrotoxin, a GABA A channel blocker, blocked the stimulatory effect of ginsenoside Rc on I GABA . Niflumic acid (NFA) and 4,4 -diisothiocyanostilbene-2,2 -disulfonic acid, both Cl − channel blockers, attenuated the effect of ginsenoside Rc on GABA-induced inward peak current.
Compared to the GABA A receptor, few investigations have been carried out into the ginsenoside-mediated action on glycine receptor channel activity. In a study using human glycine α1 receptor channel expressed in Xenopus oocytes, Noh et al. (2003) demonstrated that although treatment with ginsenoside Rf enhanced the glycine-induced inward peak current in a dosedependent and reversible manner, ginsenoside Rf itself did not elicit membrane currents. The effect of ginsenoside Rf action on glycine receptor channel activity was blocked by strychnine, a glycine receptor antagonist, and 4,4 -disothiocyanostilbene-2,2 -disulfonic acid (DIDS), a Cl − channel blocker. The various ginsenosides, listed in order of potency for the enhancement of the glycine-induced inward Cl − current, were ginsenoside Rb 1 > >Rb 2 > Rg 2 = Rc > Rf > Rg 1 > Re. Further study will be helpful for elucidation of the interaction of ginsenosides with glycine receptor proteins to enhance the glycine-induced inward Cl − current.

GINSENOSIDE Rg 3 INTERACTS WITH THE γ 2 SUBUNIT TO REGULATE GABA A RECEPTOR CHANNEL ACTIVITY
Two characteristics of ginsenoside Rg 3 -induced GABA A receptor interaction have been described. First, ginsenoside Rg 3 itself evoked an inward current in a concentration-dependent manner in Xenopus oocytes expressing human recombinant GABA A receptor (α 1 β 1 γ 2S ) in the absence of GABA. Ginsenoside Rg 3elicited inward currents were blocked by a GABA A receptor antagonist, indicating that ginsenoside Rg 3 itself activates GABA A receptors (Lee et al., 2013b). Ginsenoside Rg 3 -elicited inward currents were not observed in the absence of the γ 2S or γ 2L subunits. The magnitude of the ginsenoside Rg 3 -elicited inward current was dependent on the expression ratio of the γ 2S subunit. However, ginsenoside Rg 3 exerted no effects in oocytes expressing other subunits, such as γ 1 , γ 3 , δ, and ε, with α 1 β 1 .
The γ 2 subunit of the GABA A receptor plays an important role in the action of human epilepsy (Bowser et al., 2002). The γ 2 subunit of the GABA A receptor is also essential for the formation of high-affinity benzodiazepine-binding sites, and mutations in benzodiazepine-binding sites greatly attenuated the benzodiazepine-induced potentiation of I GABA (Buhr et al., 1997). In addition, increased expression of the γ 2 subunit compared to other subunits further potentiated benzodiazepineinduced I GABA (Boileau et al., 2003). However, GABA A receptors with mutant γ 2 subunit benzodiazepine-binding sites did not affect the action of ginsenoside Rg 3 (Lee et al., 2013b). Thus, it is unlikely that the ginsenoside Rg 3 -induced activation of α 1 β 1 γ 2 GABA receptors is achieved through interaction with benzodiazepine-binding sites.
Second, co-treatment of ginsenoside Rg 3 with GABA further potentiated I GABA in oocytes expressing GABA A receptor (α 1 β 1 γ 2S ) (Lee et al., 2013b). However, the potentiating effect of ginsenoside Rg 3 on I GABA was not specific to the γ 2 subunit, and co-expression of other subunits such as γ 1 , γ 3 , δ, and ε also enhanced I GABA . The degree of potentiation of I GABA by ginsenoside Rg 3 was not significantly different in the presence of different subunits. Even ginsenoside Rg 3 enhanced I GABA in oocytes expressing α 1 β 1 subunits and ginsenoside Rg 3 itself did not elicit inward currents in oocytes expressing α 1 β 1 GABA A receptor in the absence of GABA. Thus, ginsenoside Rg 3 may have dual binding sites for GABA A receptor modifications, one for GABA binding sites in the presence of GABA and the other one for γ 2 subunit in the absence of GABA. However, ginsenoside Rg 3 had no effects on GABAc receptor channel activity (Lee et al., 2013b).

GINSENOSIDE Rg 3 REGULATES 5-HT 3 RECEPTOR CHANNEL ACTIVITY THROUGH INTERACTION WITH AMINO ACIDS AT THE CHANNEL GATING REGION
Mutations in V291A, F292A, and I295A in the transmembrane domain 2 (TM2) greatly attenuated or abolished ginsenoside Rg 3induced inhibition of peak I 5−HT . Thus, ginsenoside Rg 3 acts through the 5-HT 3 receptor protein, and alterations in TM2 of the 5-HT 3 receptor could affect the action of ginsenoside Rg 3 . Interestingly, the V291A mutation, although not the F292A or I295A mutations, induced constitutively active ion currents, with a decreased current decay rate. Ginsenoside Rg 3 treatment of this mutant receptor accelerated the rate of current decay in the presence of 5-HT, suggesting that the presence of ginsenoside Rg 3 caused channel closure rather than opening. Thus, ginsenoside Rg 3 and TMB-8, an open channel blocker, inhibited the constitutively active ion currents. Diltiazem, another open channel blocker, did not prevent ginsenoside Rg 3 -induced inhibition of the constitutively active ion currents in occlusion experiments . This report provides the following insight: first, ginsenoside Rg 3 inhibits 5-HT 3A receptor channel activity through interaction with residues V291, F292, and I295 in the channel gating region of TM2, and second, ginsenoside Rg 3 regulates 5-HT 3A receptor channel activity in the open state at different site(s) from those used by TMB-8 and diltiazem. Thus, ginsenoside Rg 3 inhibits the 5-HT 3 receptor in the open state through interactions with V291, F292, and I295. The identification of ginsenoside Rg 3 interacting sites in the 5-HT 3 receptor indicates that ginsenoside Rg 3 achieves its pharmacological actions via a specific interaction with the 5-HT 3 receptor (Figure 4).

REGULATION OF NICOTINIC ACETYLCHOLINE RECEPTOR CHANNEL ACTIVITY BY GINSENOSIDES
Ginsenosides inhibit acetylcholine-stimulated catecholamine release from chromaffin cells, which mainly contain α3β4 nicotinic acetylcholine receptors involved in catecholamine release FIGURE 4 | Topology and sequence alignment of α7 nicotinic acetylcholine (nACh) and 5-HT 3A receptors at transmembrane domain 2 (TM2). α7 nACh and 5-HT 3A receptors consist homomeric pentamer. TM2 of each segment forms a channel pore. The underlined specific amino acid residues in those ligand-gated channels are thought to interact with 20(S)-ginsenoside Rg 3 ). (Tachikawa et al., 2001). Furthermore, ginsenosides also inhibited acetylcholine-induced inward currents in oocytes expressing nicotinic receptor α1β1δε or α3β4 subunits, suggesting that ginsenosides directly regulate nicotinic acetylcholine receptor channel activities . However, ginsenosides themselves had no effect on basal currents in oocytes expressing nicotinic acetylcholine receptor αβδε or α3β4 subunits. The inhibition of acetylcholine-induced inward currents by ginsenosides in oocytes expressing nicotinic acetylcholine receptor αβδε or α3β4 subunits was reversible, voltage-independent, and noncompetitive, indicating that ginsenosides may bind to the nicotinic acetylcholine receptor when channels are open, but do not compete with acetylcholine to regulate these receptors . Interestingly, it appears that protopanaxatriol ginsenosides such as Re, Rf, Rg 1 , or Rg 2 were more potent than protopanaxadiol ginsenosides such as Rb 1 , Rb 2 , Rc, and Rd in inhibiting acetylcholine-induced inward currents . On the other hand, ginsenoside Rg 2 reduced the peak current and increased the desensitization of acetylcholine-induced inward currents in oocytes expressing human neuronal nicotinic acetylcholine receptors such as α3β4, α3β2, α4β4, and α4β2 . Thus, the inhibitory effects of ginsenosides on acetylcholine-stimulated attenuation of catecholamine release are achieved through nicotinic acetylcholine receptor channel activity.
In Xenopus oocytes heterologously expressing the α9α10 nicotinic acetylcholine receptor, ginsenosides blocked acetylcholineinduced inward currents (I ACh ), listed in order of potency, with ginsenoside Rg 3 > Rb2 > CK > Re = Rg2 > Rf > Rc > Rb1 > Rg1 in a reversible manner, and the blocking effect of ginsenoside Rg 3 on I ACh was the same after pre-application compared to co-application of ginsenoside Rg 3 . Ginsenoside Rg 3 -induced I ACh inhibition was not affected by acetylcholine concentration and was independent of membrane holding potential. The inhibitory effect of ginsenoside Rg 3 on I ACh was not observed in oocytes expressing the α9 subunit alone, indicating that the presence of the α10 subunit is required for ginsenoside Rg 3 -induced regulation of α9α10 nicotinic acetylcholine receptor channel activity (Lee et al., 2013a). The α10 subunit of the α9α10 nicotinic acetylcholine receptor may play an important role in ginsenoside Rg 3 -induced interaction with the α9α10 nicotinic acetylcholine receptor.

IDENTIFICATION OF GINSENOSIDE INTERACTION SITES IN THE REGULATION OF THE NICOTINIC ACETYLCHOLINE RECEPTOR
In contrast to heteromeric acetylcholine receptors, ginsenosides had no effect on wild-type homomeric α7 nicotinic acetylcholine receptor-mediated ion currents . The homomeric α7 nicotinic acetylcholine receptors, which are predominantly expressed in the cortical and limbic areas, are the major binding sites for α-bungarotoxin in the mammalian central nervous system and are Ca 2+ permeable (Gotti et al., 2009). The α7 nicotinic acetylcholine receptor and the 5-HT 3A receptors are both homomeric ligand-gated ion channels. Interestingly, single point mutations of Leu247 to Thr247 in the highly conserved TM2, which forms the channel pore region, creates gain-offunction alterations, including slower desensitization, increased acetylcholine affinity, and a linear current-voltage relationship, as well as altering pharmacological properties such as the conversion of various α7 nicotinic acetylcholine receptor antagonists into agonists (Revah et al., 1991;Bertrand et al., 1992). Thus, the L247 residue of the α7 nicotinic acetylcholine receptor could be a useful target for drug development and a focal point in the investigation of channel gating of the acetylcholine receptor (Palma et al., 2006). In addition, the Leu residue corresponding to position 247 of the chick α7 nicotinic acetylcholine receptor channel is highly conserved in all nicotinic, GABA A , 5-HT 3 , and glycine receptors, and is believed to be positioned at the gate (Lester et al., 2004). Moreover, recently acquired high resolution structures of the nicotinic acetylcholine receptor channel show that the conserved Leu is located at the narrowest part of the channel, and that the side chain of the amino acid head points toward the lumen of the pore (Miyazawa et al., 2003). The importance of position 247 for gating and conductance has also been demonstrated in a functional study (Bertrand et al., 1992).
The mutation of Leu247 to various other amino acid residues induces changes in the receptor sensitivity to ginsenoside Rg 3 . Interestingly, mutations in L247 to L247A, L247D, L247E, L247I, L247S, and L247T, although not L247K, rendered mutant receptors sensitive to ginsenoside Rg 3 ). Ginsenoside Rg 3 inhibition of the mutant α7 nicotinic acetylcholine receptor channel currents was reversible and concentration-dependent. In addition, ginsenoside Rg 3 inhibition of the mutant α7 nicotinic acetylcholine receptor was strongly voltage-dependent and non-competitive. The homology docking model between ginsenoside Rg 3 and the mutant receptor revealed that ginsenoside Rg 3 forms hydrogen bonds with amino acids, such as Ser240 of subunit I and Thr244 of subunits II and V at the channel pore, whereas the wild-type receptor ginsenoside Rg 3 localizes at the interface of the two wild-type receptor subunits. Thus, the mutation of Leu247 to Thr247 may induce conformational changes in the wild-type receptor, creating a binding pocket for ginsenoside Rg 3 at the channel pore (Figure 4).

REGULATION OF N-METHYL-D-ASPARTATE (NMDA) RECEPTOR CHANNEL ACTIVITY BY GINSENOSIDES
Ginsenosides Rb 1 and Rg 3 have been shown to attenuate glutamate-and NMDA-induced neurotoxicity by inhibiting the overproduction of nitric oxide, the formation of malondialdehyde, and the influx of Ca 2+ in rat cortical cultures (Kim et al., 1998b). In addition, in rat hippocampal cultures, ginsenosides and ginsenoside Rg 3 attenuated high K + -, glutamate-, and NMDA-induced Ca 2+ influx . Seong et al. (1995) showed that ginsenosides attenuated glutamate-induced swelling of cultured rat astrocytes. Notoginsenoside R1 prevents glutamate-mediated neurotoxicity in cultured mouse cortical neurons (Gu et al., 2009). On the other hand, in an in vivo study using anesthetized rats, intracerebroventricular administration of ginsenoside Rb 1 , and not Rg 1 ,significantly inhibited the magnitude of long term potentiation induced by strong tetanus in the dentate gyrus, although ginsenoside Rb 1 did not affect the basal synaptic responses evoked by low-frequency tests (Abe et al., 1994). Pretreatment with ginsenosides via the intrathecal route attenuated NMDA-or substance P-induced nociceptive behavior, but had no effect on glutamate-induced behavior (Yoon et al., 1998;Shin et al., 1999). Pretreatment of ginsenosides via the intraperitoneal route also attenuated cell death of hippocampal neurons induced by kainate . Ginsenosides Rh2 and Rg 3 inhibit NMDA receptor channel currents in cultured rat hippocampal neurons (Lee et al., 2006a). Regarding the effects of ginsenoside metabolites such as compound K (CK), protopanaxadiol (PPD), and protopanaxatriol (PPT) on NMDA receptor channel activity, PPT, unlike CK and PPD, reversibly inhibited NMDA-mediated inward currents (I NMDA ) in a concentration-dependent manner. PPT inhibition of I NMDA was non-competitive with NMDA, and was independent of the membrane holding potential (Shin et al., 2012), even though ginsenoside Rh2, Rg 3 , and PPT interact with the NMDA receptor.

IDENTIFICATION OF GINSENOSIDE INTERACTION SITES IN THE REGULATION OF THE NMDA RECEPTOR
Ginsenoside Rg 3 does not compete with NMDA in NMDA receptor-mediated ion current inhibition in rat hippocampal neuron cultures Lee et al., 2006a). In addition, ginsenoside Rg 3 does not interact with Mg 2+ or phencyclidine (PCP, MK-801, ketamine) binding sites. However, the inhibitory effect of ginsenoside Rg 3 on NMDA receptor-mediated ion currents was attenuated by increasing glycine concentrations, whereas ginsenoside Rh2-mediated inhibition of NMDA receptor-mediated ion currents were attenuated by increasing spermine concentrations. Thus, ginsenoside Rg 3 and ginsenoside Rh2 selectively targeted NMDA receptors with different NMDA receptor regulatory sites (Lee et al., 2006a). These reports show the possibility that ginsenoside Rg 3 is a competitive antagonist at the glycine-and polyamine-binding site, although the specific amino acid(s) involved in ginsenoside Rg 3 binding have not been identified. Site-directed mutagenesis may provide further information about the interaction of ginsenoside Rg 3 with glycine binding site(s).

CHACTERIZATIONS OF GINSENOSIDE Rg 3 -MEDIATED ACTONS ON ION CHANNELS AND RECEPTORS
Ginsenosides exhibit several principles in their actions of a variety of ion channels and receptors. Firstly, ginsenosides do not affect most of ion channel and receptor activities, when they are at rest or without ligand stimulations (Nah et al., 2007). But, when voltage-gated ion channels are depolarized or receptors are activated by the relevant receptor agonists, ginsenosides affect ion channel or ligand-gated ion channel activities. Thus, conformational changes to ion channels or receptor proteins induced by depolarization or receptor ligand binding may provide an opportunity for ginsenoside Rg 3 to bind ion channels or receptors to exert its action (i.e., Na + channels and 5-HT 3 receptor) (Lee et al., , 2008c. Secondly, in ginsenoside Rg 3 -induced voltage-gated ion channel regulations, the ginsenoside Rg 3 effects on voltage-gated Ca 2+ , K + , and Na + channel currents were achieved via interaction with transmembrane domain-I-segment 6, which is known to form a channel pore or pore entryway. Ginsenoside Rg 3 -induced inhibition of L-type Ca 2+ channel currents was attenuated by mutations of Leu427, Asn428, and Leu431 in transmembrane domain-I-segment 6 residues . Similarly, N418K and L421K mutant rat brain Na V 1.2, and L437K mutant mouse skeletal muscle Na V 1.4 channel attenuated or abolished ginsenoside Rg 3 inhibition of Na + currents. Another interesting observation was the consistent pattern exhibited in ginsenoside Rg 3 -mediated action on various K + channel subtypes. As shown in Figure 3, K + channels have a common feature, in that they all have a pore-lining P-loop with a consensus amino acid sequence -TXGYGD-, which is called the K + channel "signature sequence" (Heginbotham et al., 1994). These residues, repeated in each of the 4 α subunits, form the K + selectivity filter. Ginsenoside Rg 3 -mediated human Kv1.4, KCNQ (I Ks ), hERG, and BK Ca K + channels are regulated through interaction with K531 and K318, V319, S631, and Y360 residues respectively, all of which are the first or second amino acid after -TXGYGD-. They are all located at the channel pore entry Choi et al., 2010Choi et al., , 2011a (Figure 2). Thus, ginsenoside Rg 3 has a common interaction site near the "signature sequence" in the subsets of the K + channels examined. However, the interaction of ginsenoside Rg 3 with these K + channel subtypes exhibits differential effects (i.e., activation of BK Ca , I Kr , I Ks or inhibition of Kv1.2 and Kv4.2). Another important characteristic in Kv1.4 and BK Ca channel interaction with ginsenoside Rg 3 is the sharing of other K + channel regulator binding sites such as the extracellular TEA binding site, although TEA structure is different from that of ginsenoside .
Thirdly, ginsenoside Rg 3 exhibits its effects via differential interactions with ligand-gated ion channels. Ginsenoside Rg 3induced actions of 5-HT 3 and mutant α7 nicotinic acetylcholine receptor are achieved via interaction with amino acids in TM2, which forms channel pore . Ginsenoside Rg 3 regulates GABA A receptor channel activity via γ 2 subunit, whereas ginsenoside Rg 3 regulate NMDA receptor channel activity via allosteric interaction sites such as co-agonist, glycine or polyamines, binding site.
Finally, there might be a compensatory action by ginsenoside action in ion channel regulations. For example, ginsenoside Rg 3 stabilizes membrane potentials through the inhibition of both Ca 2+ and Na + channels, but ginsenoside Rg 3 might induce a depolarization of neuronal cells by inhibiting Kv1.4 and Kv4.2 channel activity. Thus, ginsenoside Rg 3 action is interesting because the same inhibitory effects on Ca 2+ , Kv1.4 and Kv4.2, and Na + channels could result in opposite effects in neurons. One possible speculation on the actions of ginsenoside Rg 3 on nervous system is that suppression of neuronal excitability by ginsenoside Rg 3 -mediated Ca 2+ and Na + channel inhibition could be to some extent compensated by inhibition of Kv1.4 and Kv4.2 channel activity. On the other hand, there is no evidence that ginsenoside directly binds to ion channels or receptors, since most of studies are indirectly obtained from mutation experiments. In future studies, it will be needed to obtain direct evidence(s) to confirm ginsenoside binding to ion channel or ligand-gated ion channel proteins.

A LINKAGE OF THE GINSENOSIDE-MEDIATED ION CHANNELS AND RECEPTOR INTERACTIONS BETWEEN GINSENG PHARMACOLOGY
In traditional medicine, ginseng was utilized as a tonic to invigorate body (Nah et al., 2007). Ginsenoside-mediated actions of ion channels and receptors could underlie molecular bases for the explanations of traditional ginseng pharmacology. Here, this review illustrates representative examples how ginsenoside-mediated actions of ion channels and receptors link to ginseng pharmacology. In normal Ca 2+ homeostatic state, cytosolic calcium plays a key role for learning, memory and other many cellular events (Berridge et al., 1998). In contrast, Ca 2+ dyshomeostasis is one of important events in brain ischemic and traumatic brain injury (Wojda et al., 2008). Ginsenoside might be helpful for neuroprotection by negative coupling of [Ca 2+ ] i in Ca 2+ dyshomeostasis of nervous systems.

FIGURE 5 | A schematic illustration on cytosolic Ca 2+ overload signalings and ginsenoside-mediated attenuation against cytosolic Ca 2+
overload. This schematic drawing shows that cytosolic Ca 2+ levels could be elevated in ischemic or traumatic brain injury. The elevation of cytosolic Ca 2+ levels occurs either via direct activation of voltage-gated Ca 2+ channels or via depolarization caused by voltage-dependent Na + channel activation and other excitatory ligand-gated ion channels at pre-and post-synaptic sites. The overload of cytosolic Ca 2+ levels caused by excitatory neurotransmitters or neurotoxins may initiate the persistent activation of Ca 2+ -dependent signaling, resulting in damage, for instance through apoptosis or necrosis. Although ginsenoside has no effects on ion channels and ligand-gated ion channels at rest state of neurons, ginsenoside might exhibit its effects by attenuation of cytosolic Ca 2+ elevation by abnormal conditions by inhibiting ion channels and receptors (as indicated with "x" in arrow). The detailed explanations were described in text. Ginsenoside exhibits differential regulations on subsets of K + channels. So, one possible hypothesis is that K + channels regulated by ginsenoside might play a balancing role on Ca 2+ and Na + channel inhibitions by ginsenoside.
Brain injury will cause ATP exhaustion by mitochondrial dysfunction and also subsequently couples to the slow secondary excitotoxicity by glutamate (Pang and Geddes, 1997). The secondary glutamate-induced excitotoxicity in ATP deficient neurons is initiated by voltage-dependent Na + channel activation, which is coupled to membrane depolarization, Ca 2+ channel activation, and subsequent NMDA receptor activation (Alzheimer, 2002). Moreover, pathologically elevated Na + and Ca 2+ levels in the cytosol are likely to trigger a cascade of molecular events that eventually lead to neuronal death (e.g., formation of reactive oxygen species, lipid peroxidation, mitochondrial dysfunction, activation of calpain and caspases, etc.). As described above, ginsenosides inhibit cytosolic Ca 2+ and Na + elevation through these channel activations as well as NMDA receptor inhibition (Lee et al., 2006a). Thus, negative couplings of ginsenosides to Ca 2+ and Na + channels and NMDA receptors might be helpful for the attenuation of excitotoxity under the brain ischemic and traumatic brain injury. Figure 5 illustrate a schematic example based on reports how ginsenoside-mediated interactions with Ca 2+ and Na + channels and NMDA receptors may be coupled to neuroprotective effects against Ca 2+ dyshomeostasis induced by uncontrolled Ca 2+ and Na + channels or NMDA receptors (Lee et al., , 2006aKim and Rhim, 2004;Park et al., 2004;Kim et al., 2005c;Lian et al., 2005;Tian et al., 2005;Kim et al., 2007Kim et al., , 2008bZhang et al., 2008;Cheon et al., 2013). Ginseng and ginsenosides have also been reported to alleviate stress-induced symptoms and lesions (Attele et al., 1999). Stress is one of main causes for development of almost all types of diseases. The adrenal gland is one of the peripheral organs responding to stress. In stress situation, the adrenal gland secrets catecholamines from the medulla and renders the organs to cope with stress. α3β4 nicotinic acetylcholine receptors in adrenal medulla play a key role in catecholamine secretion. Ginsenosides and their metabolites inhibit α3β4 nicotinic acetylcholine receptor channel activity and catecholamine release (Tachikawa et al., 2001;Choi et al., 2002). Thus, ginsenosides might be helpful for alleviation of stress by controlling catecholamine secretion during over-stress situation.
On the other hand, traditional medicine also showed that ginseng is utilized for the alleviation of emesis, which includes nausea and vomiting. Nausea and vomiting are significant adverse effects of anti-cancer agents such as cisplatin, and cause significant patient morbidity. 5-HT 3 receptors are involved in vomiting and irritable bowel syndrome (Kim et al., 2005a,b). Ginsenosidemediated action of 5-HT 3A receptor channel activity may be the molecular basis of the anti-vomiting action of ginseng. Ginsenoside might be used for attenuation of anticancer agentinduced side effects of vomiting and nausea.
There are multiple cardiovascular effects attributed to ginseng, including cardioprotection, antihypertensive effects, and attenuation of myocardial hypertrophy, heart failure, and the ischemic and reperfused heart in a variety of experimental models (Attele et al., 1999). Ginseng total saponins and ginsenoside Rg 3 regulate L-type Ca 2+ and various K + channel activities. Especially, ginsenoside Re enhances I Kr and I Ks channel currents in guinea myocytes (Bai et al., 2003(Bai et al., , 2004. Ginsenoside Rg 3 also delays deactivation of I Ks and enhances I Kr . In heart, I Kr and I Ks channels are clinically important and are target for drug development, since dysfunction of these channels is associated with sudden death in human (Bai et al., 2004). Thus, ginsenosides-mediated actions of I Kr and I Ks channels as well as K + channels might contribute to cardioprotective effects of ginseng.

SUMMARY
The pharmacological behaviors of ginseng are diverse rather than unique, since ginsenosides, as one of active ingredients of ginseng, exhibit differential pharmacological actions in ion channels or ligand-gated ion channel regulations. Therefore, it is unlikely that ginseng ginsenoside achieves its diverse effects on ion channels and receptors via mediation of its own receptor activations on plasma membrane for the following reasons. Firstly, ginsenoside affinity for ion channels and receptors is very low compared to other receptor-specific ligands or toxins (Table 1). Rather, ginsenosides directly interact with various ion channel proteins and receptors without selectivity (Table 1). Secondly, since ginsenoside does not have its receptor on plasma membrane, ginsenoside does not induce spontaneous cellular responses in ion channel or receptor activity without stimuli such as depolarization or receptor ligands. Thus, diverse actions of ginsenoside on ion channels and receptors without its specific receptor mediations could be a limitation on its therapeutic use. Nevertheless, since ginseng influences various body functions as a traditional medicine, a pharmacological role of ginsenosides through the interactions with diverse ion channels and receptors might have potential as a prevention or treatment for a variety of nervous system disorders.