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Characteristics of Ginsenoside Rg₃-Mediated Brain Na⁺ Current Inhibition

Research Laboratory for the Study of Ginseng Signal Transduction and Department of Physiology, College of Veterinary Medicine, Konkuk University, Seoul, Korea (Ju.-H.L., S.M.J., J.-H.K., B.-H.L., I-S.Y., Jo.-H.L., S.-H.C., S.-Y.N); College of Pharmacy, KyungHee University, Seoul, Korea (D.-H.K.); Biomedical Research Center, Korean Institute of Science and Technology, Seoul, Korea (H.R.); Korea Food Research Institute, Gyeonggi-do, Korea (S.S.K.); Department of Life Science, Kwangju Institute of Science & Technology, Kwangju, Korea (J.-I.K.); Department of Pharmacology, College of Pharmacy Sungkyunkwan University, Seoul, Korea (C.-G.J.); and Department of Pharmacology, College of Medicine, Chung-Ang University, Seoul, Korea (J.-H.S.)

Received May 24, 2005; accepted July 13, 2005

	Abstract

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We demonstrated previously that ginsenoside Rg₃ (Rg₃), an active ingredient of Panax ginseng, inhibits brain-type Na⁺ channel activity. In this study, we sought to elucidate the molecular mechanisms underlying Rg₃-induced Na⁺ channel inhibition. We used the two-microelectrode voltage-clamp technique to investigate the effect of Rg₃ on Na⁺ currents (I_Na) in Xenopus laevis oocytes expressing wild-type rat brain Na_V1.2 and 1 subunits, or mutants in the channel entrance, the pore region, the lidocaine/tetrodotoxin (TTX) binding sites, the S4 voltage sensor segments of domains I to IV, and the Ile-Phe-Met inactivation cluster. In oocytes expressing wild-type Na⁺ channels, Rg₃ induced tonic and use-dependent inhibitions of peak I_Na. The Rg₃-induced tonic inhibition of I_Na was voltage-dependent, dose-dependent, and reversible, with an IC₅₀ value of 32 ± 6 µM. Rg₃ treatment produced a 11.2 ± 3.5 mV depolarizing shift in the activation voltage but did not alter the steady-state inactivation voltage. Mutations in the channel entrance, pore region, lidocaine/TTX binding sites, or voltage sensor segments did not affect Rg₃-induced tonic blockade of peak I_Na. However, Rg₃ treatment inhibited the peak and plateau I_Na in the IFMQ3 mutant, indicating that Rg₃ inhibits both the resting and open states of Na⁺ channel. Neutralization of the positive charge at position 859 of voltage sensor segment domain II abolished the Rg₃-induced activation voltage shift and use-dependent inhibition. These results reveal that Rg₃ is a novel Na⁺ channel inhibitor capable of acting on the resting and open states of Na⁺ channel via interactions with the S4 voltage-sensor segment of domain II.

Ginseng, the root of Panax ginseng C.A. Meyer, is well known in herbal medicine as a tonic and restorative agent. The main molecular ingredients responsible for the actions of ginseng are the ginsenosides (also called ginseng saponins), which are amphiphilic molecules comprising a hydrophobic backbone of aglycone (a hydrophobic, four-ring, steroid-like structure) linked to hydrophilic carbohydrate side chains consisting of monomers, dimers, or tetramers (Fig. 1). The ginsenosides are classified as protopanaxadiol or protopanaxatriol, according to the positions of the carbohydrate moieties at carbons -3, -6, and -20, which can be either free or connected to sugar rings (Nah, 1997). We recently demonstrated that ginsenoside Rg₃ (20-S-protopanaxadiol-3-[O--D-glucopyranosyl (12)--glucopyranoside]) (Rg₃), one of the active ingredients in Panax ginseng, inhibits voltage-dependent brain Na⁺ channel activity expressed in Xenopus laevis oocytes (Jeong et al., 2004; Kim et al., 2005). However, no previous work has examined the underlying mechanisms by which Rg₃ regulates Na⁺ channel currents.

View larger version (13K): [in this window] [in a new window]   Fig. 1. Structure of the 20(S)-ginsenoside Rg3 (Rg3). Glc, glucopyranoside. Subscripts indicate the carbon in the glucose ring that links the two carbohydrates.

	Experimental Procedures

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Materials. The 20(S)-ginsenoside Rg₃ (Fig. 1) was kindly provided by the Korean Ginseng Cooperation (Taejon, Korea). The cDNA for the rat brain Na⁺ channel Na_V1.2 subunit was kindly provided by Dr. A. L. Goldin (University of California, Irvine, CA) and that for the Na⁺ channel 1 subunit was kindly provided by Dr. T. Zimmer (Friedrich Schiller University, Jena, Germany). Other agents were purchased from Sigma (St. Louis, MO).

Preparation of X. laevis Oocytes and Microinjection. X. laevis frogs were purchased from Xenopus I (Ann Arbor, MI). Their care and handling was in accordance with the highest standards of institutional guidelines. For isolation of oocytes, frogs were anesthetized with an aerated solution of 3-amino benzoic acid ethyl ester, and ovarian follicles were removed. The oocytes were separated by treatment with collagenase and agitation for 2 h in Ca²⁺-free medium containing 82.5 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 5 mM 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 (96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, and 5 mM HEPES, pH 7.5) supplemented with 0.5 mM theophylline and 50 µg/ml gentamicin. This oocyte-containing solution was maintained at 18°C with continuous gentle shaking and was renewed everyday. Electrophysiological experiments were performed within 5 to 6 days of oocyte isolation, with chemicals applied to the bath. For Na⁺ channel experiments, 40 nl of cRNAs encoding the Na_V1.2 and 1 subunits was injected into the animal or vegetal pole of each oocyte one day after isolation, using a 10-µl VWR microdispenser (VWR Scientific, San Francisco, CA) fitted with a tapered glass pipette tip (15–20 µm in diameter; Choi et al., 2003).

Site-Directed Mutagenesis of Na_V1.2 and in Vitro Transcription of Na⁺ Channel Subunit cDNAs. The substitution mutations of single or triplet amino acids were performed using the QuikChange XL site-directed mutagenesis kit (Stratagene, La jolla, CA), along with Pfu DNA polymerase and mutated sense and antisense primers. Overlap extension of the target domain by sequential polymerase chain reactions was carried out according to the manufacturer's recommended protocol. The final PCR products were transformed to Escherichia coli strain DH5, screened by PCR and confirmed by DNA sequencing of the target region. The mutant DNA constructs were linearized at the 3' end by NotI digestion, and run-off transcripts were prepared using methylated cap analog m⁷G(5')ppp(5')G. The cRNAs were prepared using the mMessage mMachine transcription kit (Ambion, Austin, TX) with T7 RNA polymerase. The absence of degraded RNA was determined by denaturing agarose gel electrophoresis followed by ethidium bromide staining. Likewise, recombinant plasmids containing wild-type Na_V1.2 or 1 subunit cDNA inserts were linearized by digestion with the appropriate restriction enzymes, and cRNAs were obtained using the mMessage mMachine in vitro transcription kit (Ambion) with SP6 RNA or T7 polymerases. The final cRNA products were resuspended at a concentration of 1 µg/µl in RNase-free water and stored at -80°C until use (Choi et al., 2003).

Data Recording. A custom-made Plexiglas net chamber was used for two-electrode voltage-clamp recordings. The chamber was constructed by milling two concentric wells into the chamber bottom (the diameter and height of the upper well were 8 and 3 mm, respectively, and those of the lower well were 6 and 5 mm, respectively) and gluing plastic meshes (0.4-mm grid diameter) onto the bottom of the upper well. A perfusion inlet (1 mm in diameter) was drilled through the wall of the lower well, and a suction tube was placed on the edge of the upper well. For experiments, a single oocyte was placed on the net separating the upper and lower wells. The net grids helped anchor the oocyte in place during the electrophysiological recordings. The oocyte was then impaled with two microelectrodes filled with 3 M KCl (0.2–0.7 M) and electrophysiological experiments were carried out at room temperature using an oocyte clamp (Warner Instruments, Hamden, CT). Stimulation and data acquisition were controlled with a pClamp 8 (Axon Instruments, Union City, CA) (Choi et al., 2003). For most of the electrophysiological experiments on Na⁺ channel activity, oocytes were clamped at a holding potential of -100 mV and the membrane potential was depolarized to -10 mV for 100 ms every 5 s. Linear leak currents were corrected by means of the leak subtraction procedure (Jeong et al., 2005).

The voltage-dependence of Na⁺ channel activation was calculated by measuring the peak current at test potentials ranging from -50 mV to +50 mV evoked in 5-mV increments. The conductance (g_Na) was calculated according to the equation, g_Na = I_Na/(V_g - V_r), where I_Na is the peak amplitude of the Na⁺ current, V_g is the test potential, and V_r is the reversal potential for Na⁺. The conductance-voltage curves were drawn according to the equation g_Na/g_{Na max} = 1/{1+ exp [(V_g0.5 - V_g)/kg]}, where g_{Na max} is the maximum value for g_Na, V_g0.5 is the potential at which g_Na is 0.5 g_{Na max}, and kg is the slope factor (potential required for an e-fold change). The voltage-dependence of Na⁺ channel inactivation was determined using 200 ms conditioning prepulses ranging from -60 mV to +20 mV from a holding potential of -100 mV in 5-mV increments, followed by a test pulse to -10 mV for 5 ms. The peak I_Na was normalized to its respective maximum value (I_{Na max}) and plotted as a function of the prepulse potential. The steady-state inactivation curves were drawn according to the equation I_Na/I_{Na max} = 1/{1 + exp [(V_h - V_h0.5)/kh]}, where V_h is the prepulse potential, V_h0.5 is the potential at which I_Na is 0.5 I_{Na max}, and kh is the slope factor.

The frequency-dependent effect of Rg₃ was examined using a protocol in which 50 depolarizing pulses of 10-ms duration and 10 Hz frequency were applied to -10 mV from a holding potential of -100 mV. The protocol was run in the absence (control) and presence of 10 or 100 µM Rg₃. The current amplitude of each pulse was normalized to the peak maximal current (pulse number 1) and plotted as a function of pulse number.

The kinetics of the Rg₃ blockade of IFMQ3, which is the fastinactivation–deficient mutant of Na_V1.2, were examined by clamping oocytes at -100 mV in ND96 solution. A single 500-ms depolarizing pulse to -10 mV was applied, and the I_Na was recorded. Different concentrations of Rg₃ (10, 30, 100, or 300 µM) were perfused into the bath for 1 min, and a second, single depolarizing pulse from -100 to -10 mV was given. The data were individually fit to either a single [1 - A_slow x exp(-t/_slow)] or double [1 - A_fast x exp(-t/_fast)] + [1 - A_slow x exp(-t/_slow)] exponential equation, in which A_fast and A_slow represent the proportion of current decaying with time constants _fast and _slow, respectively, and t is the time interval. If we assume that a first-order relationship describes the dependence of the blocking rate on the concentration of the blocking chemical, the apparent rate constant for binding (k_on) can be obtained by fitting the _fast values with the equation: 1/_fast = k_on x [Rg₃] + l (Lansman et al., 1986). Recovery from open channel blockade was measured at a holding potential of -100 mV with a 10-ms depolarizing prepulse to -10 mV (P1) followed by a variable recovery period from 10 to 500 ms, subsequently followed by a 10-ms test pulse to -10 mV (P2).

Data Analysis. To obtain the concentration-response curve of the effect of Rg₃ on I_Na, the peak amplitudes at different concentrations of Rg₃ were plotted and then fitted to the following Hill equation using the Origin software (OriginLab Corp, Northampton, MA): y/y_max = [A]^nH/([A]^nH + [EC₅₀]^nH), where y is the peak I_Na at given concentration of Rg₃, y_max is the maximal peak I_Na, EC₅₀ is the concentration of Rg₃ producing a half-maximum effect, [A] is the concentration of Rg₃, and n_H is the interaction coefficient. All values are presented as means ± S.E.M. The differences between the means of control and treatment values were determined using an unpaired Student's t test. A value of P < 0.05 was considered statistically significant.

	Results

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The Effect of Rg₃ on Peak I_Na in Oocytes Expressing Na_V1.2. The effect of Rg₃ on currents from brain Na⁺ channels expressed in X. laevis oocytes was examined. I_Na was recorded by two-electrode voltage clamping of oocytes injected with cRNAs encoding Na_V1.2 and 1 subunits. Oocytes were held at -100 mV, and I_Na was elicited by depolarization to -10 mV at a low frequency (0.2 Hz). This procedure minimized the use-dependent blockade and allowed evaluation of whether Rg₃ produced a tonic blockade of peak I_Na (Pugsley and Goldin, 1998; Pugsley et al., 2000). For comparison, we also examined the effect of lidocaine on the peak I_Na. As shown in Fig. 2A, the depolarizing voltage step induced a large inward I_Na with rapid inactivation. Application of Rg₃ (100 µM) or lidocaine (1000 µM) inhibited the peak I_Na by 64 ± 7 and 41 ± 10%, respectively (Fig. 2A, inset), indicating that both agents induced a tonic inhibition of the Na⁺ current.

View larger version (26K): [in this window] [in a new window]   Fig. 2. Tonic inhibition of INa by Rg3 and lidocaine. A, oocytes were injected with wild-type NaV1.2 and 1 subunit cRNAs and maintained for 2 to 4 days before Na+ currents (INa) were recorded in ND96 using the two-electrode voltage clamp technique. , peak inward current amplitudes elicited by 100-ms depolarizations to -10 mV from a holding potential of -100 mV, evoked every 5 s. Rg3 (100 µM) or lidocaine (1000 µM) were applied during the period indicated by the solid bars. Inset, left, traces are representatives of six separate oocytes from three different frogs. Inset, right, the histograms show the percentage blockade of peak INa by Rg3 or lidocaine (Lido). Data represent the means ± S.E.M. (n = 10–13/group). B, the current-voltage relationship was obtained using voltage steps between -50 and +50 mV taken in 5-mV increments. Voltage steps were applied in the absence () and presence () of 100 µM Rg3 or after washout of Rg3 (). Data represent the means ± S.E.M. (n = 5–6/group). C and D, concentration-response curves for the Rg3- (C) or lidocaine- (D) induced inhibition of peak INa. Inset, traces are representatives of six separate oocytes from three different frogs. The solid lines were fit by the Hill equation as described under Experimental Procedures. Data represent the means ± S.E.M. (n = 10–13/group).

The Effects of Rg₃ on the Activation and Inactivation of Na_V1.2. We next examined the effects of Rg₃ on the voltage-dependence of Na⁺ channel steady-state activation and inactivation. First, the effect of Rg₃ on Na⁺ channel activation was determined by a conductance transformation of the peak current-voltage relationship (Fig. 3, A and B), with the curves representing the best data fit using the Boltzmann function. There was a significant depolarizing shift of the half-maximal activation voltage (V_g0.5). The V_g0.5 was -28.9 ± 0.57 mV in control experiments and -17.8 ± 0.21 mV in Rg₃-treated oocytes (P < 0.01, compared with control, n = 10). However, the slope factor (kg) was not significantly different, yielding values of 4.6 ± 0.5 mV under control conditions and 4.8 ± 0.2 mV after Rg₃ treatment. We then investigated the effect of Rg₃ on voltage-dependent Na⁺ channel inactivation by plotting the normalized peak I_Na against the conditioning prepulse voltage (Fig. 3, C and D) and then fitting the data to the Boltzmann function. There was no significant difference in the half-maximal inactivation voltage (V_h0.5) and the slope factor (kh) between control and Rg₃ treatment groups; V_h0.5 was -28.9 ± 0.57 and -26.9 ± 0.68 mV, respectively, and kh was 7.2 ± 0.4 and 8.1 ± 0.68 mV, respectively (n = 10). These findings indicate that Rg₃ affects the steady-state activation but not inactivation of the Na⁺ channel.

View larger version (32K): [in this window] [in a new window]   Fig. 3. The effect of Rg3 on steady-state activation and inactivation of INa in wild-type and K859Q mutant channels. A, effect of Rg3 on steady-state activation and inactivation of INa in wild-type channels. The voltage dependence of conductance was compared in the absence () and presence () of 100 µM Rg3. Inactivation was measured using a two-pulse protocol in which oocytes were held at -100 mV and depolarized to potentials from -60 to +20 mV for 200 ms, followed by a test-pulse to -10 mV for 10 ms to determine channel availability. Inactivation curves are shown in the absence () and presence of 100 µM Rg3 (). B, representative current traces for the activation and inactivation of wild-type channels were obtained as described under Experimental Procedures. The indicated traces are presented for clarity. C, the effect of Rg3 on the steady-state activation and inactivation of INa in the K859Q mutant. D, representative current traces for the activation and inactivation of the K859Q mutant channel were obtained as described. The indicated traces are represented for clarity. Data represent the means ± S.E.M. (n = 10–11/group). The curves represent a two-state Boltzmann function.

View larger version (41K): [in this window] [in a new window]   Fig. 4. Use-dependent block of Na+ channels by Rg3 or lidocaine. Fifty 20-ms depolarizing pulses to -10 mV were applied from a holding potential of -100 mV at 10 Hz in the absence () or presence of 10 µM () or 100 µM () Rg3 in wild-type (A), IFMQ3 (B), K859Q (C), and IFMQ3-K859Q (D) mutants. Lidocaine (1000 µM) () was also applied in wild-type (A) and the K859Q mutant (C). The current amplitude during each pulse was normalized to the peak current of the first pulse and plotted as a function of the pulse number. Inset, only sweeps 1 and 50 are shown for both control and Rg3 treatment groups in the wild-type and mutants (IFMQ3, K859Q, and IFMQ3-K859Q). The other sweeps were omitted for clarity. Data represent the means ± S.E.M. (n = 9–10/group).

The Effect of Rg₃ on Peak I_Na at Different Holding Potentials. The effect of Rg₃ on peak I_Na at different holding potentials was examined. Diphenylhydantoin (DPH), which is a well known anticonvulsant that preferentially binds to the inactivated Na⁺ channel (Valenzuela et al., 1996), did not significantly affect the peak I_Na evoked at -10 mV from a holding potential of -110 mV (6 ± 1% inhibition by 100 µM, n = 10) (Fig. 5A). However, at a more depolarized holding potential of -50 mV, DPH dramatically reduced the peak I_Na (86 ± 7% inhibition by 100 µM, n = 10). This indicates that the blockade of peak I_Na by DPH is highly sensitive to the membrane potential and that DPH has a much higher affinity to the inactivated state than to the resting state of the Na⁺ channel as shown by Kuo and Bean (1994) (Fig. 5, A and C, left). We then tested the effect of Rg₃ on the peak I_Na at different holding potentials. In contrast to the action of DPH, the inhibitory effect of 100 µM Rg₃ was not significantly affected by changes in the holding potential (78 ± 14% and 69 ± 10% inhibition at -110 and -50 mV, respectively; n = 15 each), indicating that the inhibitory effect of Rg₃ on the peak I_Na is independent of the membrane holding potential (Fig. 5, B and C, right).

View larger version (23K): [in this window] [in a new window]   Fig. 5. Rg3-induced INa inhibition is independent of the holding potential. A, representative traces of INa in the absence or presence of 30 or 100 µM phenytoin (DPH). Each oocyte was held at -50 or -100 mV and stepped to -10 mV for 100 ms every 5 s. B, representative traces of INa in control oocytes or those treated with 30 or 100 µM Rg3. Each oocyte was held at -50 or -100 mV and stepped to -10 mV for 100 ms every 5 s. C, left, histograms for the inhibition of INa by 30 or 100 µM DPH at different holding potentials. C, right, histograms for the inhibition of INa by 30 or 100 µM Rg3 at different holding potentials. Data represent the means ± S.E.M. (n = 10–11/group).

View larger version (25K): [in this window] [in a new window]   Fig. 6. Lidocaine and TTX do not interfere with the action of Rg3. A, , peak inward current amplitudes elicited by 100-ms depolarizations to -10 mV from a holding potential of -100 mV, evoked every 5 s. Rg3 (30 µM) was first applied during the period indicated by the solid bars, and then lidocaine (1000 µM) was applied in absence or presence of Rg3 (30 µM) as indicated by the solid bars. B, traces are representatives of six separate oocytes from three different frogs. Inset, the histograms show the percentage blockade of INa by Rg3, lidocaine (Lido), or Rg3 + lidocaine (Lido). Data represent the means ± S.E.M. (n = 10–13/group). C and D, experiments were performed as above with TTX (1 nM) used in place of lidocaine. Data represent the means ± S.E.M. (n = 10–12/group).

View larger version (26K): [in this window] [in a new window]   Fig. 7. Effect of Rg3 on various mutant Na+ channels. The site-directed mutants were generated at the channel pore, the lidocaine or TTX binding sites, and the S4 voltage sensor segment of domains I and II, as described under Experimental Procedures. A, traces are representative of six separate oocytes from three different frogs for analysis of channels with mutations in the channel pore, TTX interaction site, and S4 voltage sensor segment of domain II. B, representative concentration-response curves for the effect of Rg3 on various mutants. The solid lines were fit by the Hill equation. Additional IC50, Hill coefficient, and Vmax values for the various mutants are presented in Table 1.

View larger version (27K): [in this window] [in a new window]   Fig. 8. The effect of Rg3 or lidocaine on F1764A, Y1771A, and F1764A-Y1771A mutant Na+ channels. The site-directed mutants F1764A, Y1771A, and F1764A-Y1771A were constructed as described under Experimental Procedures. A, C, and E, traces are representatives of six separate oocytes from three different frogs for the F1764A (A), Y1771A (C), and F1764A-Y1771A (E) mutants, respectively. B, D, and F, Rg3 but not lidocaine inhibited peak INa in a use-dependent manner in the F1764A (B), Y1771A (D), and F1764A-Y1771A (F) mutants. Insets, only sweeps 1 and 50 are shown for control (), 1000 µM lidocaine (), and 100 µM Rg3 (). The other sweeps were omitted for clarity. Data represent the means ± S.E.M. (n = 9–10/group).

View larger version (26K): [in this window] [in a new window]   Fig. 9. The effects of Rg3 on IFMQ3 mutant Na+ channels. The IFMQ3 mutant was constructed as described under Experimental Procedures. A, INa was elicited in IFMQ3 mutant by a 500-ms depolarization to -10 mV from a holding potential of -100 mV, evoked every 5 s. The evoked current exhibited a slower decay and sizable persistent noninactivating or plateau current. Rg3 treatment dose dependently inhibited the peak and plateau INa values. B, concentration-response curves for the effect of Rg3 on the IFMQ3 peak () and plateau () INa values. The solid lines were fit by the Hill equation. C, current traces of the IFMQ3-K859Q mutant in the absence or presence of Rg3 were obtained from the procedures described in A. D, concentration-response curves for the effect of Rg3 on IFMQ3-K859Q peak () and plateau () INa values. The IC50, Hill coefficient, and Vmax values for Rg3 in these mutants are presented in Table 1. Data represent the means ± S.E.M. (n = 10–11/group).

A Point Mutation in the S4 Voltage-Sensor Segment of Domain II of Na_V1.2 Abolishes the Rg₃-Induced Voltage Shift of Na⁺ Channel Activation. As shown in Fig. 3, A and B, Rg₃ treatment strongly depolarized the Na⁺ channel activation voltage, suggesting that Rg₃ might modify its activation gating. The S4 segments comprise four homologous domains (I–IV) of the Na⁺ channel and are believed to act as the voltage-sensing apparatus (Kontis et al., 1997). To investigate whether mutations in the voltage-sensor segments of the Na⁺ channel affect the Rg₃-induced depolarization of the Na⁺ channel activation voltage, we constructed four different mutants in the S4 segments of domain I to IV (Table 1) (Kontis et al., 1997) and examined their influences on the Rg₃-induced voltage shift of the Na⁺ channel activation curve. Our results revealed that replacing Lys859 (domain II) with glutamine (K859Q) abolished the Rg₃-induced voltage shift, although the mutation itself resulted in a depolarizing shift of the activation curve by 10 mV compared with wild-type channel (Fig. 3, C and D). The half-maximal activation voltage (Vg_0.5) was -10.9 ± 0.4 mV and the slope factor (kg) was 5.1 ± 0.3 mV in the Rg₃ control, but only -9.8 ± 0.4 mV and 5.3 ± 0.4 mV in the mutant (Fig. 3, C and D). The other tested mutants did not show significant alterations of the activation curve (data not shown), indicating that the Lys859 residue of domain II may play an important role in the Rg₃-induced modification of voltage-dependent Na⁺ channel activation.

A Single Point Mutation in the S4 Voltage-Sensor Segment of Domain II of Na_V1.2 in Both Wild-Type and IFMQ3 Mutant Channels Abolishes Rg₃-Induced Use-Dependent Inhibition. We next examined whether the K859Q mutation affects the Rg₃-induced use-dependent inhibition of Na⁺ channels, because this mutation abolished Rg₃-induced modification of Na⁺ channel activation gating. Rg₃ treatment did not produce use-dependent inhibition in either K859Q or IFMQ3-K859Q mutants (Fig. 4, C and D), suggesting that the Lys859 residue of the S4 voltage sensor segment of domain II might play an important role in Rg₃-induced use-dependent inhibition of the IFMQ3 mutant. These mutations also significantly increased the IC₅₀ values by 1.5-fold compared with wild-type in terms of tonic inhibition (^*, P < 0.01, compared with wild-type; Table 1). It is noteworthy that lidocaine treatment produced wild-type level use-dependent inhibition in the K859Q mutant (Fig. 4C), suggesting that the Rg₃ site is not related with the action of lidocaine. In contrast, when we examined whether the mutation in the S4 voltage-sensor segment of domain II affected Rg₃-induced open-channel blocking pattern, we found that Rg₃ treatment inhibited both peak and plateau I_Na levels to comparable degrees in the IFMQ3-K859Q and IFMQ3 mutants (compare Fig. 9, C and A). The IC₅₀ values of the IFMQ3-K859Q mutant were 56 ± 6 and 20 ± 1 µM for the peak and plateau I_Na levels, respectively (Fig. 9D and Table 1), which was significantly higher than that for the IFMQ3 mutant in terms of peak (1.5-fold) but not plateau I_Na inhibition (^**, P < 0.01, compared with IFMQ3 mutant, Table 1). These results indicate that the K859Q mutant might decrease the affinity of Rg₃ for the IFMQ3 mutant to a comparable degree as that seen in the wild-type but that the K859Q mutation did not affect the Rg₃-induced open channel blocking pattern.

Developmental Rate of Rg₃-Induced Open Channel Blockade. We examined the ability of various concentrations of Rg₃ to block IFMQ3 (Fig. 10A). Untreated control traces showed that during a depolarizing pulse to -10 mV, the IMFQ3 current decayed exponentially with a single, slow time constant (_slow) of 585.4 ± 2.7 ms (n = 9). Currents elicited after Rg₃ treatment decayed exponentially with two distinct time constants. In the presence of low concentration of Rg₃ (10 µM), the currents decayed with a slow time constant that was similar to that of the control, whereas treatment with higher concentrations of Rg₃ such as 30 to 300 µM induced a concentration-dependent fast exponential component to the curves (_fast), which represented the Rg₃ block (_B). The time constants of the fast components were 464.5 ± 6.4, 235.1 ± 7.8, 64.1 ± 14.2, and 51.8 ± 9.4 ms in samples treated with 10, 30, 100, and 300 µM Rg₃, respectively. This result indicates that Rg₃ also interacts with the open state of the Na⁺ channel. A plot of the reciprocal of _B (1/_B) against drug concentration (Lansman et al., 1986) was used to approximate the drug-channel interaction kinetics. The relationship between 1/_B and the concentration of Rg₃ was determined with a straight line representing the best fit to the equation 1/_B = k_on[Rg₃] + l. (Fig. 10B). The slope of the line is the apparent binding constant (k_on), which equals 0.062 ± 0.009 x 10⁶ M^-1s^-1, and the intercept l equals 1.89 ± 0.78 s^-1. The latter value is close to the reciprocal of _slow (1.71 ± 0.41 s^-1), indicating that in the IFMQ3 mutant, a residual inactivation determines the intercept in the absence of Rg₃. This suggests that the unbinding constant (k_off) is likely to be very small.

View larger version (23K): [in this window] [in a new window]   Fig. 10. Kinetics of open channel blockade by Rg3 and recovery delay in IFMQ3 mutant Na+ channels. A, traces evoked from -100 mV to -10 mV in the absence (Con) and presence of various concentrations of Rg3 showing open channel blockade of IFMQ3. B, the rate of Rg3 block as a function of Rg3 concentration. A detailed description of the determination of tB can be found in the text. Data represent the means ± S.E.M. (n = 10–12/group). C, delayed recovery of IFMQ3 channels from open channel blockade by Rg3. Shown is a representative pair of current traces recorded in the absence (Con, top) and presence (Rg3, bottom) of 100 µM Rg3 with a recovery time interval of 10 ms between pulses. Channels recovered almost fully at this recovery interval in the absence of Rg3. In the presence of 100 µM Rg3, the test current measured during the second pulse (P2) was significantly reduced. D, recovery from inactivation or open channel blockade was assessed in detail using the paired-pulse voltage-clamp protocol shown in the inset. After oocytes were depolarized for 10 ms (P1), fractional recovery during a subsequent test pulse (P2) was assessed after an intervening recovery interval. Rg3 ()-free channels recovered rapidly (<50 ms), whereas complete recovery from inactivation was delayed for 260 ms in the presence of Rg3 (). Data represent the means ± S.E.M. (n = 9–10/group).

Rg₃ Treatment Slows Recovery from Inactivation. Because Rg₃ blocked the open state of the IFMQ3 channel (Fig. 9) and exhibited an additional use-dependent block compared with the wild-type channel (Fig. 4B), we examined whether the inhibitory effect of Rg₃ on I_Na was derived from a delayed recovery of the channel from the open channel block. Current traces were recorded in the absence (Fig. 10C, control, top) and presence (Fig. 10C, bottom) of 100 µM Rg₃ with a recovery time interval of 10 ms between pulses. Recovery from open channel block was analyzed as shown in Fig. 10D. After a 10-ms prepulse (P1), recovery from open channel block was assessed using a test pulse (P2) after increasing recovery intervals (Fig. 10C and D, inset). The IFMQ3 Na⁺ channels were found to recover rapidly, probably because of a slow inactivation associated with the mutation (Fig. 10D, < 50 ms, ). In contrast, Rg₃ treated channels (Fig. 10D, ) showed a delayed recovery from open channel block (up to 260 ms). This slow recovery seems to underlie the enhanced use-dependent inhibition produced by Rg₃.

	Discussion

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Ginseng has long been used as a treatment for a wide variety of ailments, and some of the purported effects of this root have been documented in laboratory studies (Nah, 1997). Although the beneficial effects and functional mechanisms of ginsenosides have not been fully elucidated, accumulating evidence suggests that they may target the ion channels involved in neuronal excitability. Ginsenosides have been shown to affect several ion channels found at pre- and postsynaptic sites in the nervous system (Nah and McCleskey, 1994; Nah et al., 1995; Kim et al., 1998, 2002; Choi et al., 2002, 2003; Sala et al., 2002), and their effects are closely coupled to the inhibition of neurotransmitter release (Tachikawa et al., 1995; Kudo et al., 1998). We demonstrated recently that Rg₃ stereospecifically inhibits voltage-dependent brain Na⁺ currents and that the carbohydrate portion of Rg₃ plays a key role in the inhibition of Na⁺ currents (Kim et al., 2005). However, very little is known about the molecular mechanism(s) underlying Rg₃-induced Na⁺ channel modulation.

Herein, we characterized Rg₃-induced channel regulation of brain Na⁺ channels (Na_V1.2) expressed in X. laevis oocytes. Our results revealed four major findings. First, Rg₃ produced a tonic inhibition of the peak I_Na via an interaction with the resting state of the Na⁺ channel (Fig. 2). Second, Rg₃ induced a large depolarizing shift in the steady-state activation of the Na⁺ channel (Fig. 3). Third, Rg₃ produced a use-dependent block of the Na⁺ channel after high-frequency stimulation, indicating that Rg₃ could exert an inhibitory effect on the open state of the Na⁺ channel (Fig. 4). Fourth, the inhibitory effect of Rg₃ on the peak I_Na was independent of the holding potential, indicating that Rg₃ might have a lower affinity for the inactivated state of the Na⁺ channel (Fig. 5).

To examine the molecular mechanism by which Rg₃ regulates brain Na⁺ channel activity, we used site-directed mutagenic methods similar to those previously used to identify drug- or toxin-Na⁺ channel interaction site(s) (Cestele and Catterall, 2000). We used six different types of Na_V1.2 mutants to assess the sites at which Rg₃ interacts with Na⁺ channels: 1) mutations in the channel pore entrance of the S6 segment of domain I; 2) mutations in the pore region of domain II; 3) mutations in the lidocaine binding sites; 4) mutations in the TTX binding sites; 5) mutations in the S4 voltage-sensor segments of domains I, II, III, and IV; and 6) mutations in the inactivation cluster. The pore site(s) are unlikely to be the target for Rg₃, because the inhibitory potency of Rg₃ on Na⁺ channel activity was not altered in cells injected with pore site mutants (Fig. 7 and Table 1). In addition, it does not seem as though Rg₃ interacts with the binding sites for lidocaine or TTX (Ragsdale et al., 1994, 1996), because the inhibitory potency of Rg₃ on Na⁺ channel activity was not altered in cells injected with the F385C, F385S, F385Y, F385T, F385M, E387G, E387T, E387Q, F1764A, Y1771A, or F1764A-Y1771A mutants, which harbor changes in the lidocaine or TTX binding sites (Fig. 7 and Table 1). In addition, the inhibitory effect of Rg₃ on the peak I_Na seemed additive in the presence of lidocaine or TTX in occlusion experiments (Fig. 6, C and D), providing further evidence that Rg₃ uses a separate binding site. Finally, we tested the possibility that the hydrophobic cluster related with Na⁺ channel inactivation might be involved in the Rg₃-induced inhibition of I_Na. In the IFMQ3 mutant channel, which lacks fast inactivation (West et al., 1992), Rg₃ treatment inhibited the noninactivating plateau I_Na to a greater degree than the peak I_Na, providing further evidences that Rg₃ inhibits I_Na in both the open and resting states of the Na⁺ channel.

Two other lines of evidence that support the possibility that Rg₃ also inhibits open Na⁺ channels are the observations that 1) the inhibitory effect of Rg₃ on peak I_Na is not dependent on membrane holding potentials and 2) Rg₃ does not shift the steady-state inactivation curve in wild-type Na⁺ channels (Figs. 3 and 5), which does occur in many drugs (e.g., antiarrhythmic agents and anticonvulsants) that act on inactivated Na⁺ channels (Willow et al., 1985). Moreover, Rg₃ treatment induced an additional use-dependent block of I_Na in the IFMQ3 mutant compared with wild-type channel, indicating that Rg₃ might prefer to bind and block the open state of the Na⁺ channel. Similar use-dependent open channel blockades have been observed in the case of disopyramide, RSD921, tetracaine, and flecainide (Cahalan, 1978; Grant et al., 1996; Pugsley and Goldin, 1999; Wang et al., 2003; Ramos and O'Leary, 2004).

We next used S4 voltage-sensor segment mutants (K226Q, K859Q, R1312Q, and R1368Q) to examine whether the voltage-sensor segment of the Na⁺ channel was involved in Rg₃-induced Na⁺ channel regulation (Noda et al., 1989). The K859Q mutation in domain II, but not K226Q, R1312Q, or R1638Q, significantly increased the IC₅₀ values by 1.5-fold compared with wild-type (^*, P < 0.01, compared with wild-type; Table 1), indicating that this mutation might decrease the affinity of Rg₃ to Na⁺ channels. In addition, K859Q alone abolished the Rg₃-induced voltage shift in Na⁺ channel activation (Fig. 3C) and Rg₃-induced use-dependent but not tonic inhibition of I_Na (Figs. 4C and 7). Although these results do not allow precise elucidation of the interaction between Rg₃ and Na⁺ channels, our data seem to indicate that the voltage-sensor S4 segment of domain II might be an important portion for Rg₃-induced Na⁺ channel regulation (Figs. 3C and 4C). It is unlikely that Rg₃ interacts nonspecifically with Na⁺ channels, because the 20(S)-ginsenoside Rg₃ used in the present study inhibits I_Na but 20(R)-ginsenoside Rg₃ does not (Jeong et al., 2004) (Fig. 1). Moreover, modifications of the hydrophilic portion of Rg₃ by opening the cyclic glucoses or conjugating the glucoses with other hydrophobic molecule abolished the inhibitory effect of Rg₃ on peak I_Na (Kim et al., 2005). These findings indicate that Rg₃ specifically modulates Na⁺ channel by interaction with unidentified site of Na⁺ channel. Further works will be necessary to determine specific interaction site(s) of Rg₃ with Na⁺ channel.

Based on our findings, it seems reasonable to speculate as to whether the in vitro Rg₃-induced Na⁺ channel blockade could translate to in vivo pharmacological effects. Qian et al. (2005) and Xie et al. (2005) determined plasma concentration of Rg₃ after administration of Rg₃ via intravenous (5 mg/kg) or intragastric (10 mg/kg) routes in rats and found that plasma Rg₃ peaked at 1 µg/ml 10 min after intravenous administration and 4 h after intragastric administration. These findings suggest that plasma-borne Rg₃ might exert in vivo pharmacological effects. In addition, in rats, Bae et al. (2004) and Tian et al. (2005) showed that oral (100 mg/kg) or intravenous administration (5 and 10 mg/kg) of Rg₃ exerted significant neuroprotective effects against focal cerebral ischemic injury by decreasing neurological deficit scores and reducing the infarct area compared with the control group. Rg₃ also significantly improved mitochondrial energy metabolism, antagonized decreases in superoxide dismutase and glutathione-peroxidase activities, and increased malondialdehyde levels in a cerebral ischemia model (Tian et al., 2005). Na⁺ channel-blocking drugs such as local anesthetics, antiarrhythmics, and anticonvulsants have been shown to exhibit significant neuroprotective effects against cerebral ischemia-, hypoxia-, and head trauma-induced neuronal damages (Taylor and Meldrum, 1995). Thus, it is possible that Rg₃-mediated brain I_Na inhibition could be an underlying mechanism for neuroprotection against in vivo ischemic brain injury.

We demonstrated previously that Rg₃ and other ginsenosides inhibit voltage-dependent Ca²⁺ and K⁺ channels as well as ligand-gated ion channels such as 5-HT₃ and some subsets of nicotinic acetylcholine receptors (Nah et al., 1995; Choi et al., 2002, 2003; Jeong et al., 2004). We are currently examining the detailed characteristics of Rg₃-mediated regulation of Ca²⁺, K⁺, and ligand-gated ion channels. The elucidation of the regulatory modes and identification of interaction site(s) with those channels or receptors by Rg₃ and other ginsenosides will provide important insights into the molecular basis by which ginsenosides interact with membrane proteins such as ion channels and receptors.

In this study, we used brain Na⁺ channel gene expression in a X. laevis oocyte model system to show that Rg₃ blocks the resting and open state of brain Na⁺ channels. This novel work provides new insights into one possible mechanism underlying the effects of ginsenosides in the nervous system.

	Footnotes

 
This work was supported in part by a grant from the Korean Food Research Institute (2003), the Neurobiology Research Program from the Korean Ministry of Science and Technology (to S.Y.N.), and two Brain Research Center of the 21st Century Frontier Research Program grants funded by the Korea Ministry of Science and Technology (M103KV010008-05K2201-00830 and M103KV010004-03K2201-00420).

Ju.-H.L. and S.M.J. contributed equally to this work.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.105.015115.

ABBREVIATIONS: Rg₃, 20-S-protopanaxadiol-3-[O--D-glucopyranosyl(12)--glucopyranoside]; TTX, tetrodotoxin; IFMQ3, Ile1488-Phe1489-Met1490 mutated to I1488Q-F1489Q-M1490Q; DPH, diphenylhydantoin.

Address correspondence to: Prof. Seung-Yeol Nah, Research Laboratory for the Study of Ginseng Signal Transduction and Dept. of Physiology, College of Veterinary Medicine, Konkuk University, Seoul 143-701, Korea. E-mail: synah{at}konkuk.ac.kr

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