Effects of Ginsenoside Rg₃ on α9α10 Nicotinic Acetylcholine Receptor-Mediated Ion Currents

Abstract

Ginsenosides is a low molecular weight substance found in ginseng as one of the active ingredients. Ginsenosides, like other herbal medicines, has a wide range of neuropharmacological actions including neuroprotective effects. The α9α10 nicotinic acetylcholine receptor is one of numerous nicotinic acetylcholine receptors that exists as a heteropentameric form in auditory hair cells of the cochlea. In this study, we report the effects of ginsenosides on rat α9α10 nicotinic acetylcholine receptor-mediated ion currents using the two-electrode voltage clamp technique. Treatment with acetylcholine evoked inward currents (I_ACh) in oocytes heterologously expressing the α9α10 nicotinic acetylcholine receptor. Ginsenosides blocked I_ACh in order of potency of Rg₃> Rb₂> CK>Re=Rg₂> Rf>Rc> Rb₁> Rg₁ with reversible manners, and the blocking effect of Rg₃ on I_ACh was same after pre-application than co-application of Rg₃. The half maximal inhibitory concentration (IC₅₀) of Rg₃ was 39.6±4.9 µm. Rg₃-induced I_ACh inhibition was not affected by acetylcholine concentration and was independent of membrane holding potential. Although the inhibitory effect of Rg₃ on I_ACh was abolished in oocytes expressing α9 subunit alone, indicating that the presence of α10 subunit might be required for Rg₃-induced regulations of α9α10 nicotinic acetylcholine receptor channel activity. These results indicate that α10 subunit of α9α10 nicotinic acetylcholine receptor might play an important role in Rg₃-induced regulation of the α9α10 nicotinic acetylcholine receptor.

Nicotinic acetylcholine receptors are members of the Cys-loop family of ligand-gated ion channels, which also includes 5-hydroxytryptamine 3, γ-aminobutyric acid A, and glycine receptors.¹⁾ Sixteen different nicotinic acetylcholine receptor subunits are currently known, and subunits of nicotinic acetylcholine receptor α (α1–7, α9 and α10), β (β1–4), γ, δ and ε have been identified.²⁾ Neuronal nicotinic acetylcholine receptors contain α2–6 subunits that are usually expressed as heteropentamers in combination with β2–4 subunits^3–5) and are found in the central and peripheral nervous systems.⁶⁾ In contrast, the α7 and α9 subunits can form homomeric receptors.^5,7,8) In particular, the α9 subunits can form heteropentameric receptors in combination with the α10 subunits.⁹⁾ Although many nicotinic acetylcholine receptor subunits are expressed in the central and peripheral nervous systems, the distributions of α9α10 nicotinic acetylcholine receptor are restricted to certain cell populations, such as leukocyte, pituitary, skin keratinocyte, sperm, and dorsal root ganglion.^9–14) The α9α10 nicotinic acetylcholine receptor is also expressed in mammalian vestibular and cochlear mechanosensory hair cells but has not been detected in the brain.^9,10) The α9α10 nicotinic acetylcholine receptor displays a biphasic response to concentrations of extracellular calcium and exists as a heteropentamer with a stoichiometry of (α9)₂(α10)₃.^9,15) The α9α10 nicotinic acetylcholine receptor is also related to various diseases such as tinnitus, hearing loss and auditory processing disorders.¹⁶⁾ It is known that blockage of α9α10 nicotinic acetylcholine receptor reduces inflammation-related nerve injury.¹⁷⁾

Ginsenosides, one of ginseng components, is a substance of low molecular weight uniquely found in ginseng¹⁸⁾ (Fig. 1A). Ginsenosides exhibits diverse biological activities in nervous systems,¹⁸⁾ with neuropharmacological actions such as analgesia, neuroprotection against neurotoxins or excitatory amino acids.¹⁹⁾ However, the cellular mechanisms of ginsenoside activity are relatively unknown, especially with regards to possible regulation of receptors or ion channels involved in synaptic transmission in nervous system.

Fig. 1. Chemical Structure of Ginsenosides

Glc=β-d-glucose, Rha=α-l-rhamnose, Ara=α-l-arabinose, Pyr=pyranose, Fur=furanose, Xyl=β-d-xylose.

In previous reports, we have shown that ginsenosides or ginsenoside metabolites regulates the Cys-loop family of ligand-gated ion channels, such as 5-hydroxytryptamine 3A, human glycine α1 and nicotinic acetylcholine receptors. For example, the application of ginsenoside Rg₂ inhibits 5-hydroxytryptamine- and glycine-induced peak inward currents (I_5-HT and I_Gly) of mouse 5-hydroxytryptamine 3A and human glycine α1 receptor channels expressed in Xenopus laevis oocytes. Ginsenoside Rg₃ inhibits I_5-HT in a competitive and voltage-independent manner through interaction with amino acids locate in channel pore region, whereas ginsenoside Rg₃ had no effects on wild-type α7 nicotinic acetylcholine receptors but inhibited mutant α7 nicotinic acetylcholine receptor at channel pore region.^20,21) As noted above, the α9α10 nicotinic acetylcholine receptor plays an important role in auditory systems and contains the same Cys-loop as 5-hydroxytryptamine 3A and glycine receptors, which are all pentameric ligand-gated ion channels. However, relatively little is known about the effects of ginsenosides on α9α10 nicotinic acetylcholine receptor channel activity.

In this study, we investigated the effects of ginsenosides on the α9α10 nicotinic acetylcholine receptor channel activity regulation in Xenopus oocytes. We initially expressed rat α9 and α10 nicotinic acetylcholine receptor cRNAs in Xenopus oocytes. We examined the effect of ginsenosides on acetylcholine evoked inward currents (I_ACh) and found that ginsenoside Rg₃ (Rg₃) was most potent for the inhibition of I_ACh in oocytes expressing α9α10 nicotinic acetylcholine receptor. Inhibition of I_ACh by Rg₃ was concentration dependent, reversible and voltage independent. Moreover, inhibition of Rg₃ on I_ACh was non-competitive with acetylcholine. Rg₃-induced inhibition of I_ACh was abolished in oocytes expressing α9 subunit alone. In the present study, we demonstrate that Rg₃ is a novel agent that regulates the α9α10 nicotinic acetylcholine receptor through interaction with α10 subunit.

Materials and Methods

Materials

Ginsenosides (Fig. 1) and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Ginsenosides was dissolved in dimethyl sulfoxide (DMSO) as a stock solution and was diluted with bath medium before use. The cDNAs for the rat α9 and α10 nicotinic acetylcholine receptors (Gene bank ID: NM_022930 and NM_022639) were used.⁹⁾

Preparation of Xenopus laevis Oocytes and Microinjection

Xenopus laevis frogs were purchased from Xenopus I (Ann Arbor, MI, U.S.A.). Animal care and handling were in accordance with the highest standards of Konkuk University guidelines. To isolate oocytes, frogs were anesthetized with an aerated solution of 3-amino benzoic acid ethyl ester, and the ovarian follicles were removed. The oocytes were separated with collagenase followed by agitation for 2 h in a Ca²⁺-free medium containing 82.5 mm NaCl, 2 mm KCl, 1 mm MgCl₂, 5 mm N-(2-hydroxyethyl)piperazine-N′-2-ethansulfonic acid (HEPES), 2.5 mm sodium pyruvate, 100 units/mL penicillin, and 100 µg/mL streptomycin. Stage V–VI oocytes were collected and stored in ND96 medium (96 mm NaCl, 2 mm KCl, 1 mm MgCl₂, 1.8 mm CaCl₂, and 5 mm HEPES, pH 7.5) supplemented with 50 µg/mL gentamicin. The solution containing the oocytes was maintained at 18°C with continuous gentle shaking and was replaced daily. Electrophysiological experiments were performed 3 to 6 d after oocyte isolation. For α9α10 nicotinic acetylcholine receptor experiments, oocytes were injected with both α9 and α10 nicotinic acetylcholine receptor-encoding cRNAs (40 nL, a 1 : 1 molar ratio) into the animal or vegetal pole of each oocyte one day after isolation, using a 10-µL microdispenser (VWR Scientific, West Chester, PA, U.S.A.) fitted with a tapered glass pipette tip (15 to 20 µm in diameter).²⁰⁾

cRNA Preparation of the Rat α9α10 Nicotinic Acetylcholine Receptor

The cDNA constructs were linearized at the 3′ ends by digestion with NotI, and run-off transcripts were prepared using the methylated cap analogue m⁷G(5′)ppp(5′)G. The cRNAs were prepared using a mMessage mMachine transcription kit (Ambion, Austin, TX, U.S.A.) with T7 RNA polymerase. The absence of degraded RNA was confirmed by denaturing agarose gel electrophoresis followed by ethidium bromide staining. The final cRNA products were re-suspended at a concentration of 1 µg/µL in RNase-free water and stored at –80°C.²⁰⁾

Data Recording

A custom-made Plexiglas net chamber was used for two-electrode voltage-clamp recordings, as previously reported.²⁰⁾ A single oocyte was superfused continuously with ND96 medium (96 mm NaCl, 2 mm KCl, 1 mm MgCl₂, 1.8 mm CaCl₂, and 5 mm HEPES, pH 7.5) in the absence or presence of acetylcholine or ginsenosides during recording. Both voltage and current microelectrodes were filled with 3 m KCl and had a resistance of 0.2 to 0.7 MΩ. Two-electrode voltage-clamp recordings were obtained at room temperature using an Oocyte Clamp (OC-725C, Warner Instruments) and were digitized using Digidata 1200A (Molecular Devices, Sunnyvale, CA, U.S.A.). Stimulation and data acquisition were controlled using pClamp 8 software (Molecular Devices). For electrophysiological experiments, the oocytes were clamped at a holding potential of –80 mV, and 1.5 s voltage steps were applied from –120 to +50 mV to assess the relationship between current and voltage. Linear leak and capacitance currents were corrected by means of the leak subtraction procedure. In all experiments, we incubated oocytes with the Ca²⁺-chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM; 100 µm) for 3 to 4 h prior to electrophysiological recording to avoid activation of the endogenous Ca²⁺-sensitive Cl^－ currents.⁹⁾

Data Analysis

To obtain the concentration-response curve for the effect of Rg₃ on the inward I_ACh mediated by α9α10 nicotinic acetylcholine receptor, I_ACh was plotted as a function of different concentrations of Rg₃. Origin software (OriginLab Corp., Northampton, MA, U.S.A.) was used to fit the plot to the Hill equation: I/I_max=1/[1 + (IC₅₀/[A])^nH], where I_max was the maximal current obtained from each IC₅₀ value of acetylcholine in receptors, IC₅₀ was the concentration of Rg₃ required to decrease the response by 50%, [A] was the concentration of Rg₃, and nH was the Hill coefficient. All values were presented as the mean±S.E.M. The differences between the means of the control and treatment data were determined using the unpaired Student’s t-test or one-way ANOVA. A value of p<0.05 was considered to be statistically significant.

Results

Effect of Rg₃ on I_ACh in Oocytes Expressing α9α10 Nicotinic Acetylcholine Receptor

The addition of acetylcholine (10 µm) to the bathing solution induced a large I_ACh in oocytes injected with rat α9α10 nicotinic acetylcholine receptor cRNAs (Fig. 2A). In H₂O-injected control oocytes, the application of acetylcholine did not induce any inward currents (data not shown). Ginsenosides (100 µm each) itself also had no effect in oocytes expressing the α9α10 nicotinic acetylcholine receptor at a holding potential of –80 mV (data not shown). However, the co-application of ginsenosides (100 µm each) with acetylcholine (10 µm) for 30 s inhibited I_ACh in oocytes expressing the α9α10 nicotinic acetylcholine receptor (Fig. 2A, n=10–14 from three different frogs). Thus, the co-application of ginsenoside Rb₁, Rb₂, Rc, Re, Rf, Rg₁, Rg₂, Rg₃ or ginsenoside metabolite CK with acetylcholine inhibited I_ACh by 22.9±3.0, 56.4±4.8, 27.9±3.2, 43.1±5.6, 35.5±4.4, 5.5±1.5, 44.1±3.0, 70.6±6.6, or 58.5±5.1% (Fig. 2B). Interestingly, the pre-application of Rg₃ (100 µm) alone for 30 s before co-application with acetylcholine (10 µm) or co-application of Rg₃ with acetylcholine induced almost the same inhibition of I_ACh (70.6±6.6 and 74.6±4.6%) (Figs. 3A, B). To determine the concentration-dependent effect of Rg₃, we experimented with different concentrations of Rg₃. Co-application with Rg₃ for 30 s inhibited I_ACh by 0.5±1.2, 3.7±1.6, 15.8±2.6, 34.7±6.0, 69.0±5.1, and 80.9±4.8% at 1, 3, 10, 30, 100, and 300 µm, respectively, in oocytes expressing the α9α10 nicotinic acetylcholine receptor (Figs. 3C, D). The IC₅₀ of I_ACh was 39.6±4.9 µm for co-application in oocytes expressing the α9α10 nicotinic acetylcholine receptor (n=10 or 11, with samples taken from three different frogs for each point).

Fig. 2. Effect of Ginsenosides on I_ACh in Oocytes Expressing α9α10 Nicotinic Acetylcholine Receptors

A. Acetylcholine (ACh; 10 µm) was applied first, followed by co-application of various individual ginsenosides and acetylcholine. The trace in (A) represents seven separate oocytes from three different batches of frogs. B. Summary histograms of I_ACh inhibition by co-application of ginsenosides. Each point represents the mean±S.E.M. (n=9 to 12 per group).

Fig. 3. Effects of Co- or Pre-application of 100 µm Rg₃ with Acetylcholine on I_ACh and Concentration-Dependent Inhibition of I_ACh by Rg₃ in α9α10 Nicotinic Acetylcholine Receptors

A and B. I_ACh in oocytes expressing the α9α10 nicotinic acetylcholine receptors was elicited at a holding potential of –80 mV for 30 s in the presence of 10 µm acetylcholine with co- or pre-application of 100 µm Rg₃. C and D. Concentration-dependent effects on I_ACh following co-application of Rg₃. Rg₃ inhibited I_ACh in a concentration-dependent manner. Each point represents the mean±S.E.M. (n=8 to 12 per group).

Current–Voltage Relationship in Rg₃-Mediated α9α10 Nicotinic Acetylcholine Receptor Regulation

In experiments examining the current–voltage (I–V) relationship, the membrane potential was held at –80 mV, and a voltage ramp was applied from –120 to +50 mV for 1.5 s. Leakage correction was executed by subtraction of the I–V curve obtained by the same voltage protocol before the application of acetylcholine. The application of acetylcholine to the bathing medium induced a mainly inward current at negative voltages and an outward current at positive voltages (Fig. 4A). Co-application of Rg₃ with acetylcholine decreased both inward and outward currents. The reversal potentials were –8.8±1.6 mV and –10.5±1.9 mV with application of acetylcholine alone and co-application of Rg₃ with acetylcholine in oocytes expressing the α9α10 nicotinic acetylcholine receptor. The co-application of Rg₃ with acetylcholine did not affect α9α10 nicotinic acetylcholine receptor channel properties; Rg₃ did not alter the reversal potential of the α9α10 nicotinic acetylcholine receptor (Fig. 4A). In addition, the inhibitory effect of Rg₃ (40 and 100 µm) on I_ACh was independent of the membrane-holding potential (Fig. 4B). Forty and one hundred micromole Rg₃ inhibited I_ACh by 41.9±3.8, 43.2±5.6, 40.5±2.8, 41.8±4.5 and 69.1±7.0, 69.3±4.7, 73.7±5.9, 62.4±8.9% at membrane-holding potentials of −120, −90, −60, −30 mV, respectively, in oocytes expressing the α9α10 nicotinic acetylcholine receptor (n=8 to 11, from three different frogs).

Fig. 4. Current–Voltage Relationship and Voltage-Independent Inhibition of I_ACh by Rg₃

A. Current–voltage relationships of I_ACh inhibition by Rg₃ in α9α10 nicotinic acetylcholine receptors. Representative current–voltage relationships were obtained using voltage ramps of –120 to +50 mV for 1.5 s at a holding potential of –80 mV. Voltage steps were applied before and after application of 10 µm acetylcholine in the absence or presence of 100 µm Rg₃. B. Voltage-independent inhibition of I_ACh in the α9α10 nicotinic acetylcholine receptors by Rg₃. The values were obtained from the receptors in the absence or presence of 40 or 100 µm Rg₃ at the indicated membrane-holding potentials. Each point represents the mean±S.E.M. (n=7 to 9 per group).

Non-competitive Inhibition of α9α10 Nicotinic Acetylcholine Receptor Channel Currents by Rg₃

To further study the mechanism by which the co-application of Rg₃ inhibits I_ACh in oocytes expressing the α9α10 nicotinic acetylcholine receptor, we analyzed the effect of Rg₃ on I_ACh evoked by different acetylcholine concentrations (Figs. 5A, B). Co-application of Rg₃ of 100 µm for 30 s with various concentrations of acetylcholine did not significantly shift the concentration–response curve of acetylcholine to the right (EC₅₀ values were changed from 10.9±0.6 to 14.9±1.5 µm, * p<0.08, while the Hill coefficient changed from 1.5 to 1.7) in oocytes expressing the α9α10 nicotinic acetylcholine receptor. Thus, the inhibitory effect of Rg₃ on I_ACh was not affected by increasing concentrations of acetylcholine in the range of 1 to 300 µm acetylcholine (Fig. 5B). These results indicate that Rg₃ inhibited I_ACh in a non-competitive manner and inhibition of I_ACh by Rg₃ was not related to the acetylcholine-binding site.

Fig. 5. Concentration-Dependent Effects of Acetylcholine on Rg₃-Mediated Inhibition of I_ACh

A. The representative traces were obtained from α9α10 nicotinic acetylcholine receptor-expressing oocytes. The I_ACh of the upper and lower panels were elicited by concentrations of 10 µm acetylcholine and 300 µm acetylcholine at a holding potential of –80 mV, respectively. B. Concentration–response relationships for acetylcholine (ACh) in the α9α10 nicotinic acetylcholine receptors treated with acetylcholine (1 to 300 µm) alone or with acetylcholine plus pre-application of 100 µm Rg₃. The I_ACh of oocytes expressing the α9α10 nicotinic acetylcholine receptors was measured using the indicated concentration of acetylcholine in the absence (□) or presence (○) of 100 µm Rg₃. Oocytes were exposed to acetylcholine alone or to acetylcholine with Rg₃ for 30 s. Oocytes were voltage-clamped at a holding potential of –80 mV. Each point represents the mean±S.E.M. (n=8 to 11 per group).

Effects of Rg₃ on I_ACh in Oocytes Expressing α9 Subunit Alone

Since α9 subunit can form homomeric receptors,^5,8) we next examined the effects of Rg₃ on I_ACh in oocytes expressing α9 subunit alone. Interestingly, as shown in Fig. 6A, Rg₃ had no effect on I_ACh in oocytes expressing α9 subunit alone even with high concentration of Rg₃ compared to α9α10 nicotinic acetylcholine receptors. However, we could not observe any acetylcholine-induced inward currents in oocytes expressing α10 subunit (data not shown). These results show that co-expressions of α9 and α10 subunits of nicotinic acetylcholine receptors are required for Rg₃-induced regulation of α9α10 nicotinic acetylcholine receptors. Furthermore, the present study shows that α10 subunit of nicotinic acetylcholine receptor might play an important role in Rg₃-induced α9α10 nicotinic acetylcholine receptor regulation.

Fig. 6. Effects of Rg₃ on I_ACh in Oocytes Expressing α9 Subunit Alone

A and B. The representative traces show effects of Rg₃ (100 and 300 µm) on I_ACh in oocytes expressing α9 subunit alone or α9α10 nicotinic acetylcholine receptors. Traces in A and B represent eight separate oocytes from three different frogs. C. Concentration-dependent effects of Rg₃ on I_ACh in two different concentration of Rg₃. Each point represents the mean±S.E.M. (* p<0.001, compared to α9 subunit alone; n=8 to 12 per group).

Effects of Extracellular Ca²⁺ on Rg₃-Mediated Inhibition of I_Ach

Channels of the α9α10 nicotinic acetylcholine receptor are known to be permeable and have a biphasic response to extracellular Ca²⁺, in contrast to other ligand-gated ion channels.^9,22) We examined whether Rg₃-mediated inhibition of I_ACh was related to extracellular Ca²⁺ concentration. As shown in Figs. 7A and 7B, I_ACh was potentiated with extracellular Ca²⁺. Next we examined the effects of various concentrations of Rg₃ on I_ACh in the absence of extracellular Ca²⁺. As shown in Fig. 7C, the removal of extracellular Ca²⁺ from ND96 in the presence of 0.1 mm EGTA did not decrease the inhibitory effects of Rg₃ on I_ACh. Thus, the inhibitory effects of Rg₃ (100 µm) on I_ACh did not change in ND96 and Ca²⁺ free ND96. Next, we examined whether the inhibitory effects of Rg₃ on I_ACh are affected by various concentrations of extracellular Ca²⁺. We found that the inhibitory effects of Rg₃ (100 µm) on I_ACh were not affected by varying concentrations of extracellular Ca²⁺ (Fig. 7C). These results show that the presence of extracellular Ca²⁺ may not relate Rg₃-mediated inhibition of I_ACh and that extracellular Ca²⁺ does not play a role in Rg₃-mediated regulation of the α9α10 nicotinic acetylcholine receptor.

Fig. 7. Effects of Extracellular Ca²⁺ on Rg₃-Mediated Inhibition of I_ACh

A and B. The representative traces show effects of Rg₃ (100 µm) on I_ACh in the absence or presence of extracellular Ca²⁺. Traces in A and B represent eight separate oocytes from three different frogs. C. The summary histograms of I_ACh inhibition by 100 µm Rg₃ in Ca²⁺-free ND96 medium or with various concentrations of extracellular Ca²⁺. Each point represents the mean±S.E.M. (n=7 to 10 per group).

Discussion

α9α10 Nicotinic acetylcholine receptors are abundantly expressed in auditory system and is related to various auditory-related diseases such as tinnitus, hearing loss and auditory processing disorders.¹⁶⁾ Inhibitions of α9α10 nicotinic acetylcholine receptor attenuates inflammation-related nerve injury in auditory systems.¹⁷⁾ Accumulating evidences have shown that the ginsenosides protects the central nervous system against excitatory amino acids- or neurotoxins-induced brain damage through regulations of various ion channels or ligand-gated ion channels.¹⁹⁾ However, the effects of ginsenosides in the nervous system are not fully understood. Furthermore, ginsenosides’ molecular mechanisms and ability to exhibit various beneficial effects are relatively unknown at the cellular level. In previous studies, we have demonstrated that ginsenosides regulates subsets of nicotinic acetylcholine receptor channel activity such as α3β4 and other heteromeric nicotinic acetylcholine receptors.^23,24) Interestingly, Rg₃ had no effects on homomeric α7 nicotinic acetylcholine receptor. Although expression of the α9α10 nicotinic acetylcholine receptor is limited in several tissues,^9–14) little is known about the effects of Rg₃ on the α9α10 nicotinic acetylcholine receptor.

In the present study, we examined the effects of Rg₃ on the α9α10 nicotinic acetylcholine receptor heterologously expressed in Xenopus oocytes. We found that: (1) pre- or co-application of Rg₃ with acetylcholine inhibited I_ACh in a reversible and concentration-dependent manner; (2) inhibition of I_ACh by Rg₃ pre-application with acetylcholine was independent of the concentration of acetylcholine or membrane-holding potential; (3) I_ACh inhibition by Rg₃ was non-competitive and Rg₃ had no effects on I_ACh in oocytes expressing α9 subunit alone; and (4) extracellular Ca²⁺ did not play a role in inhibitory effect of Rg₃ on I_ACh. These results show the possibility that α10 subunit might play a role in Rg₃-induced regulation of α9α10 nicotinic acetylcholine receptor channel activity.

Previous reports have shown that ginsenosides regulates heteromeric nicotinic acetylcholine receptors expressed in oocytes.^23,24) However, ginsenosides Rg₃ had no effects on homomeric α7 nicotinic acetylcholine receptor.²¹⁾ Instead, Rg₃ inhibited mutant homomeric α7 nicotinic acetylcholine receptor, which was mutated at channel pore regions.²¹⁾ These studies showed that ginsenosides regulate nicotinic acetylcholine receptor channel activity through interaction with amino acids in channel pore region. Similarly, we have reported that Rg₃ inhibits 5-hydroxytryptamine 3A receptor-gated ion currents through interactions with amino acids at channel pore region with both non-competitive and voltage-independent manners.²⁰⁾ Thus, the main target of ginsenosides in regulation of ligand-gated ion channels might be channel pore region rather than ligand binding site(s).

In the present study examining how Rg₃ regulates α9α10 nicotinic acetylcholine receptor-gated ion currents, we found that Rg₃ inhibited I_ACh in Xenopus oocytes expressing the α9α10 nicotinic acetylcholine receptor. We could observe I_ACh in oocytes expressing α9 but not α10 subunit alone, indicating that α9 but not α10 subunit alone could form homomeric α9 nicotinic acetylcholine receptor channels^8,9) but the currents were not large as α9α10 nicotinic acetylcholine receptor co-expression.⁸⁾ As shown in Fig. 6, Rg₃ had no effect on I_ACh in oocytes expressing α9 subunit alone, showing that Rg₃ could not exert its effect on homomeric nicotinic acetylcholine receptors such as α7 and α9 nicotinic acetylcholine receptors. Interestingly, when α10 nicotinic acetylcholine subunit was co-expressed with α9 nicotinic acetylcholine receptor, Rg₃ exhibited inhibitory effects on I_ACh. These results show the possibility that α10 nicotinic acetylcholine subunit might play an important role in Rg₃-induced α9α10 nicotinic acetylcholine receptor regulation and that co-expressions of both subunits might provide Rg₃ binding site(s) through the induction of conformational changes of receptor proteins. However, further studies will be required to elucidate the role of α9 or α10 subunit in Rg₃-induced α9α10 nicotinic acetylcholine receptor regulation.

In conclusion, we found that Rg₃ inhibited I_ACh of α9α10 nicotinic acetylcholine receptor in a concentration-dependent, non-competitive, and voltage-independent manner. Moreover, inhibition of Rg₃ on I_ACh was not observed in homomeric α9 nicotinic acetylcholine receptor. These results indicate that ginsenosides are a novel agent acting on the α9α10 nicotinic acetylcholine receptor and that ginsenosides-mediated I_ACh regulation of the α9α10 nicotinic acetylcholine receptor could provide a molecular basis for the pharmacological actions of ginseng in the nervous system.

Acknowledgment

This work was supported by the Basic Science Research Program (2011-0021144) and the Priority Research Centers Program through the National Research Foundation of Korea (NRF), which is funded by the Ministry of Education, Science, and Technology (2012-0006686) and by the BK21 project fund to S.-Y. Nah.

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