Ginsenoside Rg₃ Enhances Large Conductance Ca²⁺-Activated Potassium Channel Currents: A Role of Tyr360 Residue

Abstract

Ginsenosides, active ingredients of Panax ginseng, are known to exhibit neuroprotective effects. Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels are key modulators of cellular excitability of neurons and vascular smooth muscle cells. In the present study, we examined the effects of ginsenosides on rat brain BK_Ca (rSlo) channel activity heterologously expressed in Xenopus oocytes to elucidate the molecular mechanisms how ginsenoside regulates the BK_Ca channel activity. Ginsenoside Rg₃ (Rg₃) enhanced outward BK_Ca channel currents. The Rg₃-enhancement of outward BK_Ca channel currents was concentration-dependent, voltage-dependent, and reversible. The EC₅₀ was 15.1 ± 3.1 μM. Rg₃ actions were not desensitized by repeated treatment. Tetraetylammonium (TEA), a K⁺ channel blocker, inhibited BK_Ca channel currents. We examined whether extracellular TEA treatment could alter the Rg₃ action and vice versa. TEA caused a rightward shift of the Rg₃ concentration-response curve (i.e., much higher concentration of Rg₃ is required for the activation of BK_Ca channel compared to the absence of TEA), while Rg₃ caused a rightward shift of the TEA concentration-response curve in wild-type channels. Mutation of the extracellular TEA binding site Y360 to Y360I caused a rightward shift of the TEA concentration-response curve and almost abolished both the Rg₃ action and Rg₃-induced rightward shift of TEA concentration-response curve. These results indicate that Tyr360 residue of BK_Ca channel plays an important role in the Rg₃-enhancement of BK_Ca channel currents.

INTRODUCTION

Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels are a family of potassium-selective ion channels activated in response to elevation of intracellular free Ca²⁺ level following mem-brane depolarization (Hille, 2001). BK_Ca channels are composed of two subunits: the α (also called Slo) subunit, which forms the channel pore (Ghatta et al., 2006; Salkoff et al., 2006), and the β subunit (Wanner et al., 1999), which modifies the voltage and calcium sensitivity of the pore-forming subunit (Qian et al., 2002). The α subunit has large cytoplasmic C terminus, is responsible for the calcium-dependent activation of the channel (Fig. 1B) (Schreiber and Salkoff, 1997; Schreiber et al., 1999; Wei et al., 1994), and is activated by increased intracellular Ca²⁺ and/or Ca²⁺-dependent kinases including CaM kinases, PKA, and PKC (Toro et al., 1998; Weiger et al., 2002). BK_Ca channels play key roles in a variety of neuronal and non-neuronal cell functions. For example, in neurons, BK_Ca channels regulate frequency of firing, action potential afterhyperpolarization and neurotransmitter release. BK_Ca channels are also one of the main ion channels that contribute action potential repolarization and afterhyperpolarization during excitation-contraction coupling in vascular smooth muscle cells (Ohi et al., 2001).

Fig. 1. Chemical structures of ginsenosides and the primary amino acid sequence of mutated BK_Ca subunit. (A) Structures of ginsenosides. Abbreviations for carbohydrates are as follows: Glc, glucopyranoside; Ara (pyr), arabinopyranoside; Rha, rhamnopyrano-side. The subscript indicates the carbon in the glu-cose ring that links the two carbohydrates. (B) The brief sequence alignment of BK_Ca channel and the mutated amino acid residues (underlined) in the pore helix. Amino acid residues in BK_Ca, KCNQ1, and Kv1.4 channels that were proposed to interact with 20(S)-ginsenoside Rg₃ (Rg₃) are marked (*) amino acid.

Ginseng, the root of Panax ginseng C.A. Meyer, has been used as a representative tonic or an adaptogen to promote longevity and enhance bodily functions against hypertension and various ailments for several hundred years in Far Eastern countries. Recent reports have shown that ginsenosides, one of the active ingredients in ginseng, inhibit ion channels that are involved in neuronal excitabilities such as voltage-dependent Ca²⁺ and Na⁺ channels (Fig. 1A) (Choi et al., 2009; Lee et al., 2005). BK_Ca channels are usually co-localized with voltage-dependent Ca²⁺ channels (Berkefeld et al., 2006). Little is known about ginsenoside effects on BK_Ca channel activities.

In the present study, we examined ginsenoside effects on rat brain BK_Ca (rSlo) channel activities expressed in Xenopus oocytes using the two-electrode voltage clamp and outside-out patch clamp techniques (Ha et al., 2006; Lu et al., 1990). We demonstrated that among various ginsenosides Rg₃ most potently enhanced BK_Ca channel currents and that this action was concentration- and voltage-dependent and reversible. However, site-directed mutagenesis of the channel pore entry site greatly attenuated Rg₃ action. In this communication, we present results suggesting that the Y360 residue in the channel pore entry site is involved in Rg₃-mediated enhancement of the BK_Ca channel currents.

MATERIALS AND METHODS

Materials

Ginsenosides were provided by AMBO Institute (Korea) (Fig. 1A). We used the cDNAs for rat brain BK_Ca channel (GenBank No. NM_000218) that were previously reported (Ha et al., 2006). All other reagents were purchased from Sigma-Aldrich (USA).

Preparation of Xenopus oocytes and microinjection

Xenopus laevis frogs were purchased from Xenopus I (Ann Arbor, USA). Their care and handling were in accordance with the highest standards of institutional guidelines of Konkuk University. For isolation of oocytes, frogs were anesthetized with an aerated solution of 3-amino benzoic acid ethyl ester followed by removal of ovarian follicles. The oocytes were treated with col-lagenase and then agitated for 2 h in Ca²⁺-free OR2 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 medium (in mM: 96 NaCl, 2 KCl, 1 MgCl₂, 1.8 CaCl₂, and 5 HEPES, pH 7.5) supplemented with 50 μg/ml gentamicin. The oocyte-containing solution was maintained at 18℃ with continuous gentle shaking and renewed daily. Electrophysiological experiments were performed within 5-6 days of oocyte isolation, with ginsenoside applied to the bath. For BK_Ca channel experiments, BK_Ca channel-encoding cRNAs (40 nl) were injected into the animal or vegetal pole of each oocyte one day after isolation, using a 10 μl microdispenser (VWR Scien-tific, USA) fitted with a tapered glass pipette tip (15-20 μm in diameter) (Lee et al., 2008).

Site-directed mutagenesis of the BK_Ca α and in vitro transcription of BK_Ca channel cDNAs

Single amino acid substitutions of BK_Ca channel (Fig. 1B) were made using a QuikChange™ XL Site-Directed Mutagenesis Kit (Stratagene, USA), along with Pfu DNA polymerase, in addition to sense and antisense primers encoding the desired mutations. Overlap extension of the target domain by sequential polymerase chain reaction (PCR) was carried out according to the manufacturer’s protocol. The final PCR products were trans-formed into E. coli strain DH5α, screened by PCR and confirmed by sequencing of the target regions. The mutant DNA constructs were linearized at their 3′ ends by digestion with NotI, and run-off transcripts were prepared using the methylated cap analog, m⁷G(5′)ppp(5′)G. The cRNAs were pre-pared using a mMessage mMachine transcription kit (Ambion, USA) with T7 RNA polymerase. The absence of degraded RNA was con-firmed by denaturing agarose gel electrophoresis followed by ethidium bromide staining. Similarly, recombinant plasmids containing rat BK_Ca channel cDNA inserts were linearized by digestion with the appropriate restriction enzymes, and cRNAs were obtained using the mMessage mMachine in vitro transcription kit (Ambion, USA) with SP6 RNA polymerase or T7 polymerase. The final cRNA products were resuspended at a concentration of 1 μg/μl in RNase-free water, and stored at -80℃ (Lee et al., 2008).

Data recording

Data recording for BK_Ca channel currents were performed as described by Lu et al. (1990). A custom-made Plexiglas net chamber was used for two-electrode voltage-clamp recordings as previously reported. The oocytes were impaled with two microelectrodes filled with 3 M KCl (0.2-0.7 MΩ), and electro-physiological experiments were carried out at room temperature using an Oocyte Clamp (OC-725C, Warner Instruments, USA). Stimulation and data acquisition were controlled with a pClamp 8 (Axon Instruments, USA). For most electrophysio-logical experiments, oocytes were perfused initially with Cl^-- and Ca²⁺-free solution (in mM: 96 NaOH, 2 KOH, 8 Mg-gluconate, 5 HEPES, 5 EGTA, pH 7.4 with methanesulfonic acid) in the presence of Cl^- channel blocker (500 μM anthracene-9-carboxylic acid) (Lu et al., 1990) to inhibit endogenous Cl^- channels. The oocytes were then clamped at a holding potential of -80 mV, membrane potential was depolarized to +40 mV for 400 ms at 10 s intervals, and currents were recorded as otherwise indicated.

All macroscopic current recordings were performed using the gigaohm-seal patch-clamp method in an outside-out configuration (Hamil et al., 1981). Patch pipettes were fabricated from borosilicate glass (WPI, USA) and were fire-polished to a resistance of 5-7 MΩ. The channel currents were amplified using an Axopatch 200B amplifier (Axon Instruments, USA), low-pass filtered at 1 or 2 kHz using a four-pole Bessel filter, and digitized at a rate of 10 or 20 points/ms using a Digidata 1200A apparatus (Axon Instruments). The macroscopic currents of expressed channels were activated by voltage-clamp pulses delivered from a holding potential of -100 mV to membrane poten-tials ranging from -80 to 200 mV in 10 mV increments. The intracellular and extracellular solutions contained the following components, unless otherwise specified: 116 mM KOH, 4 mM KCl, 10 mM HEPES, and 5 mM EGTA at pH 7.2.

To provide the precise free concentration of intracellular Ca²⁺ ([Ca²⁺]_i), the appropriate amount of total Ca²⁺ to be added to the intracellular solution was calculated using the MaxChelator software (Patton et al., 2004; http://www.standford.edu/~cpatton/maxc.html). The pH was adjusted to 7.0 with 2-[N-morpholino]ethanesulfonic acid. To compare the channel characteri-stics accurately, an identical set of intracellular solutions was used throughout the experiments. Commercial software packages, which included Clampex 8.0 or 8.1 (Axon Instruments) and Origin 6.1 (OriginLab Corp., USA), were used for the acquisition and analysis of macroscopic recording data.

Data analysis

To obtain the concentration-response curve of the effect of Rg₃ or TEA on the K⁺ currents from the BK_Ca channel, the peak amplitudes at different concentrations of Rg₃ or TEA were plot-ted. The current enhancements or inhibitions evoked by drug treatment were analyzed after subtraction of currents elicited by H₂O injection. Origin software (Origin, USA) was used to fit the plot to the Hill equation: y/y_max = [A]^nH/([A]^nH + [EC₅₀]^nH), where y represented the peak current at a given concentration of Rg₃, y_max was the maximal peak current, EC₅₀ was the concentration of Rg₃ producing a half-maximal effect, [A] was the concentra-tion of Rg₃, and nH was the Hill coefficient. All values were pre-sented as mean ± S.E.M. The significance of differences be-tween mean control and treatment values was determined using Student’s t-test, where P < 0.05 was considered statisti-cally significant.

RESULTS

Treatment of Rg₃ enhances BK_Ca channel currents in Xenopus oocytes expressing rat brain BK_Ca channels

Application of a voltage step from -80 to +40 mV with duration of 400 ms at 10-s intervals to oocytes injected with cRNA encoding the BK α subunit gene elicited a large outward current in the absence of Rg₃ (Fig. 2A, inset). Charybdotoxin and Iberio-toxin, highly specific peptidyl inhibitors of maxi-K channels (Candia et al., 1992; Gao et al., 2003), blocked the outward currents (98.2 ± 1.2 and 95.5 ± 2.1% inhibition by 100 nM charybdotoxin and iberiotoxin, respectively, n = 5), indicating that the BK_Ca channel is functionally working (Kaczorowski et al., 1996). Next, we examined the effects of various individual gin-senosides on the BK_Ca channel currents in Xenopus oocytes expressing BK_Ca channel α subunits (Fig. 1A). Rg₃ (100 μM) activated the BK_Ca channel currents by an average of 340%, and other ginsenosides (Rb₁, Rc, Rf, Rh₁, and Rg₁) were much less effective (Fig. 2A). As shown in Fig. 2B, application of Rg₃ to oocytes injected with rSlo cRNAs resulted in a rapid stimula-tion of outward BK_Ca channel currents in a concentration-dependent manner. However, Rg₃ had no effect on H₂O-injected control oocytes that were not injected with cRNA en-coding the BK α subunit gene. Thus, the outward currents in H₂O-injected control oocytes were 130 ± 25 and 131 ± 10.2 nA in the absence and presence of Rg₃, respectively (n = 4). In addition, in oocytes expressing α subunit alone, Rg₃ (100 μM) enhanced BK_Ca channel currents to a degree similar to that observed in oocytes co-expressing α and β subunits (350 ± 80.6% for α subunit alone; 367 ± 83.1% for α + β subunits, n = 4), indicating that Rg₃-mediated enhancement of BK_Ca channel currents is independent of β subunit. These results indicated that Rg₃ is most potent among various ginsenosides in enhancing BK_Ca channel currents.

Fig. 2. Effects of Rg₃ on wild-type BK_Ca channel activities. (A) Effects of various ginsenosides (100 μM each) on wild-type BK_Ca channel currents. Inset, the representative traces elicited by ginse-noside Rb₁, Rg₁ and Rg₃. (B) The representative traces on BK_Ca channel current enhancements by different concentrations of Rg₃ (0, 10, 30 or 100 μM). Rg₃ enhanced BK_Ca channel current in a con-centration-dependent manner. Currents were in response to 400 ms voltage steps to +40 mV from a holding potential of -80 mV. Data represented the mean ± S.E.M. (n = 6-7).

Rg₃-enhancement of BK_Ca channel currents was not desensitized by repeated application and was independent of intracellular Ca²⁺

We next examined the possible changes of Rg₃ effect on BK_Ca channel currents by repeated application of Rg₃ to oocytes expressing BK_Ca channel. As shown in Fig. 3A, treatment with Rg₃ induced an enhancement of BK_Ca channel currents. Oocytes stimulated with Rg₃ were first washed with recording buffer for 3 min until the basal current was recovered, and then restimulated with Rg₃. The second, third, or fourth BK_Ca current responses for Rg₃ were not significantly diminished and the magnitudes of BK_Ca currents were 94.8 ± 10.3, 78.7 ± 15.4 and 81.0 ± 9.3 % respectively, of the first responses of Rg₃ (n = 15, oocytes from three different batches of donors) (Fig. 3B). In addition, we also examined the effect of BAPTA-AM, a cell permeable intracellular Ca²⁺ chelator, on Rg₃ enhancement of BK_Ca channel currents to investigate whether Rg₃ enhancement of BK_Ca channel currents was due to the increase of intracellular free Ca²⁺ levels. As shown in Figs. 3C and 3D, pretreatment of BAPTA-AM (10 μM, 3 h) to oocytes had no affect Rg₃-mediated BK_Ca channel currents. Rather, Rg₃ enhanced BK_Ca channel currents with concentration-dependent manner, indicating that Rg₃-mediated enhancement of BK_Ca channel currents is not desensitized and independent of intracellular Ca²⁺.

Fig. 3. Rg₃-mediated BK_Ca channel current enhancements were not desensitized and were independent of intracellular Ca²⁺. (A) The representative peak outward current amplitude at +40 mV from a holding potential of -80 mV was measured and plotted against time before and after repeated applications of Rg₃ (100 μM) for 30 s. (B) The normalized currents after repeated Rg₃ treatment. BK_Ca channel currents were not desensitized after repeated treatment of Rg₃. Data represented the mean ± S.E.M. (n = 6). (C) Oocytes were first treated with BAPTA-AM (10 μM, 3 h). The representative peak outward current amplitude at +40 mV from a holding potential of -80 mV was measured and plotted against time before and after repeated applications of the indicated concentration of Rg₃. (D) The normalized currents after treatment of various concentrations of Rg₃ in oocytes pre-treated with BAPTA-AM. Data represented the mean ± S.E.M. (n = 6).

The presence of Rg₃ caused a rightward shift of TEA- mediated concentration curve on BK_Ca channel currents and vice versa

Since we demonstrated in the previous report that the regulatory effects of Rg₃ on human Kv1.4 channel currents ex-pressed in Xenopus oocytes was affected by extracellular application of TEA (Lee et al., 2008), we first examined the effects of TEA on BK_Ca channel currents. As shown in Fig. 4A, in wild-type chan-nels, TEA inhibited BK_Ca channel currents in a con-centration-dependent manner. We next examined Rg₃ effects on TEA-mediated inhibition of BK_Ca channel currents. Interest-ingly, the presence of Rg₃ (30 μM) caused a rightward shift of the TEA concentration curve on BK_Ca channel current inhibitions from 0.8 ± 0.5 to 20.1 ± 2.3 mM (p < 0.001, compared to the absence of Rg₃) (Fig. 4A). Second, we examined the effects of TEA on Rg₃-mediated enhancement of BK_Ca channel cur-rents. The presence of TEA (300 μM) caused a rightward shift of the Rg₃-mediated concentration curve on BK_Ca channel cur-rents from 16.0 ± 1.8 to 224.2 ± 13.8 μM (p < 0.001, compared to the absence of TEA) (Fig. 4B). In experiments using TEA, our results indicated that Rg₃ enhancement of BK_Ca channel currents is achieved through interaction from the outside of the cell.

Fig. 4. Effect of TEA on Rg₃ action and vice versa in wild-type channel and Y360I mutant effects on TEA and Rg₃ action. (A) Concentration-response curves for TEA in the absence or presence of Rg₃ (30 μM) in wild-type BK_Ca channels. The presence of Rg₃ caused a significant rightward shift of TEAconcentration-response curves from 0.9 ± 0.1 to 20.1 ± 2.3 mM (p < 0.001, compared to the absence of Rg₃). In addition, mutation of Y360 to Y360I in the absence of Rg₃ shifted TEA concentration-response curve rightward from 0.9 ± 0.1 to 34.2 ± 2.6 mM (p < 0.001, compared to wild-type). (B) Concentration-response curves for Rg₃ in the presence of TEA (300 μM) in wild-type BK_Ca channels. The presence of TEA caused a significant rightward shift of Rg₃ concentration-response curves from 16.0 ± 1.8 to 224.2 ± 13.9 μM (p < 0.001, compared to the absence of TEA). (C) Effect of Rg₃ on TEA concentration-response curve in Y360I mutant channel. Rg₃ almost had no effect on TEA concentration-response curve in Y360I mutant channels. Data represented the mean ± S.E.M. (n = 8).

The pore entryway of channel is involved in Rg₃-mediated enhancement of BK_Ca channel currents

Since Rg₃ regulates human Kv1.4 and KCNQ1 plus KCNE1 channels through interaction with amino acids at the channel pore entryway (Choi et al., 2010; Lee et al., 2008), we subsequently examined the possibility of whether Rg₃-induced regulations of BK_Ca channel activity were also mediated through interactions with the analogous amino acids from Tyr360 to The363 in the pore entryway (Fig. 1B). The mutants constructed are listed in Table 1. Since mutation Y360A attenuated Rg₃-induced enhancement of BK_Ca channel currents (Table 1), we further substituted Y360 with other amino acid residues, including glutamine, isoleucine, lysine, serine, and valine (generating Y360Q, Y360I, Y360K, Y360S and Y360V, respectively), and examined the effect of Rg₃ on BK_Ca channel currents in oocytes expressing these mutants. Our results revealed that mutation Y360I greatly attenuated Rg₃-induced enhancement of BK_Ca channel currents (Figs. 5A and 5B) and was followed by order of Y360S > Y360E > Y360A. These results indicate that substitutions of Y360 with amino acid residues such as alanine, isoleucine, serine, or glutamate attenuate the Rg₃-induced enhancement of BK_Ca channel currents compared to wild-type. However, substi-tution of other amino acid residues in other positions such as charybdotoxin binding site (F332A) had no discernable effects (Gao et al., 2003, Table 1).

Table 1.

Effect of Rg₃ on wild-type and various mutant BK_Ca channels expressed in Xenopus laevis oocytes. Currents were elicited by single-step voltage pulses from -80 to +40 mV.

	Imax	EC50	nH
Wild-type	370.4 ± 35.1	15.1 ± 3.1	1.7 ± 0.5
F332A	371.4 ± 52.4	41.6 ± 13.7	2.7 ± 1.9
V359A	641.3 ± 67.9	26.8 ± 7.4	1.4 ± 0.5
Y360A	287.2 ± 12.8#	43.8 ± 4.5*	2.5 ± 0.4
Y360I	93.4 ± 0.1*	46.8 ± 0.1*	1.1 ± 0.0
Y360K	409.6 ± 29.9	15.5 ± 2.6	1.4 ± 0.2
Y360Q	440.0 ± 36.4	16.4 ± 3.2	1.3 ± 0.2
Y360S	181.8 ± 27.5*	34.4 ± 10.7#	1.3 ± 0.3
Y360V	598.0 ± 97.5	43.9 ± 17.2*	1.5 ± 0.6
Y360E	214.2 ± 25.9*	13.6 ± 3.4	2.5 ± 1.3
A361T	373.9 ± 43.4	14.7 ± 3.3	1.2 ± 0.3
K362A	341.1 ± 57.6	12.5 ± 3.0	1.1 ± 0.2
T363A	311.1 ± 45.9	15.0 ± 1.0	2.2 ± 0.2

I_max (%), maximal activation by Rg₃. Rg₃ activation was determined as the % difference of currents in the presence and absence of Rg₃. EC₅₀, Values were mean ± S.E.M (n = 8-10/group). n_H, Hill coefficient. ^#p < 0.05, *p < 0.01 compared with the wild-type BK_Ca channel.

Fig. 5. Effects of Rg₃ on mutant BK_Ca channel activities. (A) The representative traces on Rg₃-mediated BK_Ca channel current enhancement in Y360I mutant BK_Ca channels. Rg₃ slightly enhanced Y360I channel currents. (B) Concentration-response curves of Rg₃ on wild-type and various mutant BK_Ca channel currents at Y360 residue. The representative outward current was measured from an oocyte injected with cRNAs that encoded BK_Ca channel wild-type or mutant α subunit. Solid lines have been fitted to the Hill equation. Currents were in response to 400 ms voltage steps to +40 mV from a holding potential of -80 mV. Data represented the mean ± S.E.M. (n = 6-7).

Mutation of Y360 to Y360I caused TEA concentration-response curve to rightward shift and abolished Rg₃ effects on TEA concentration-response curve observed in wild-type channel

Interestingly, the previous report showed that Y308 residue of Drosophila BK_Ca (dSlo) channel is the extracellular TEA binding site and that mutation of Y308 to Y308V almost abolished TEA sensitivity (Shen et al., 1994). Similarly, mutation of Y360, which is the analogous amino acid of Y308 residue of Drosophila BK_Ca (dSlo) channel, to Y360I also caused a shift of the TEA concentration response curve towards the right and we meas-ured IC₅₀ values of 0.9 ± 0.1 and 34.2 ± 2.5 mM (p < 0.001, compared to wild-type) in wild-type and Y360I mutant, respec-tively (Fig. 4A). In addition, we examined Rg₃ effects on TEA-mediated BK_Ca channel regulation using Y360I mutant channel. As shown in Fig. 4C, Rg₃ had almost no effect on TEA-mediated inhibition of mutant BK_Ca channel currents. These results indicated that mutation of Y360 to Y360I caused a loss of Rg₃ effects on TEA-mediated BK_Ca channel regulations (Lagrutta et al., 1998), again showing a possibility that Rg₃ enhanced BK_Ca channel currents by sharing the extracellular TEA binding site of the channel.

Rg₃ activated wild-type but not mutant Y360I BK_Ca channels in outside-out configuration

Since Rg₃ enhanced BK_Ca channel currents, we next examined Rg₃ effects on BK_Ca channel currents using outside-out patch clamp configuration in wild-type and Y360I mutant channels. In current-voltage (I-V) relationships the application of 10 μM Rg₃ slightly enhanced wild-type BK_Ca channel currents but the application of 30 μM Rg₃ caused a large enhancement of wild-type BK_Ca channel currents (Fig. 6A, right panel). However, in mutant Y360I channels, the enhancing effect of Rg₃ (30 μM) was greatly attenuated (Fig. 6B, right panel). The G-V relationship was also constructed for Rg₃ action in wild-type and Y360I mutant channels. In the absence of Rg₃ the half-activation volt-age (V_1/2) was 136.4 ± 0.4 mV. The presence of 10 and 30 μM Rg₃ caused a shift of V_1/2 to 123.3 ± 0.3 and 96.30 ± 0.4 mV in wild-type, respectively (n = 5, p < 0.05, compared to the presence of 30 μM Rg₃). In Y360I mutant channels, V_1/2 was 140.4 ± 0.4 mV in the absence of Rg₃. The presence of 10 and 30 μM Rg₃ did not cause a significant shift of V_1/2 to 131.8 ± 0.5 and 120.0 ± 1.0 mV, respectively (n = 5) (Figs. 6C and 6D). These results again indicate that Rg₃ activates BK_Ca channels by inducing more negative shift of voltage-activation profile and that mutation of Y360 to Y360I attenuates Rg₃ action.

Fig. 6. Effects of Rg₃ on wild-type and Y360I mutant BK_Ca channel currents in outside-out patch clamp configuration. (A and B) Effects of 30 μM Rg₃ on the macroscopic I-V relationship of wild-type and Y360I mutant BK_Ca channel in the presence of 1 μM Ca²⁺. Representative current traces of macroscopic wild-type (A) or Y360I mutant (B) BK_Ca channel currents. Their respective concentrantion-dependent I-V relationships are shown in right panel for wild-type and Y360I mutant channels. Ionic currents were elicited with 200-ms step-pulses of different voltage protocols: -30 to 200 mV in 10-mV in-crements from holding potentials of -50 mV. Each data point in I-V relationships represents the mean current value measured over 198 ± 1.5 ms of test pulses. (C and D) Effects of Rg₃ on the G-V relationships of wild-type (C) or Y360I mutant (D) BK_Ca channel currents. Conductance values were obtained from peak tail currents and normalized to the maximum conductance observed at 10 and 30 μM Rg₃. V_½ values were obtained by fitting independent data sets with Boltzmann function. Each symbol represents the conduc-tance values obtained from two different concentration of Rg₃. Each data point represents the mean value ± S.E.M. (n = 5-6 each).

DISCUSSION

In the nervous system, stimulations above threshold by various stimuli cause activation of voltage-dependent Ca²⁺ channels that induce an elevation of [Ca²⁺]_i and its resulting elevation initiates signaling pathways for the release of a variety of neuro-transmitters from presynaptic sites and Ca²⁺-dependent cellular events (Hille, 2001). In addition to the role as a second messenger, the elevation of [Ca²⁺]_i in presynaptic sites is also coupled to activation of BK_Ca channels, which are usually co-localized with voltage-dependent Ca²⁺ channels in presynaptic sites for repolarization or return to resting membrane potential (Berkefeld et al., 2006). Thus, since BK_Ca channels play a negative feedback control with [Ca²⁺]_i, BK_Ca channel activators or openers could be utilized as neuroprotective agents against excessive Ca²⁺ influx through depolarization or excitatory neurotransmitters (Lawson, 2000). Although BK_Ca channels are involved in synaptic regulation in the nervous system, relatively little has been studied about the molecular mechanisms by which ginsenosides regulate BK_Ca channel activity.

In the present study, we made three major findings. First, we observed that Rg₃ among various ginsenosides enhanced BK_Ca channel currents in concentration- and voltage-dependent man-ners, while Rg₃-enhancement of BK_Ca channel currents was independent of intracellular Ca²⁺. Second, the presence of TEA significantly shifted the concentration response curve of Rg₃ towards the right and vice versa in wild-type channel. And third, mutation of the channel pore entry site (Y360 to Y360I), which is also known as extracellular TEA binding site, greatly attenu-ated Rg₃ enhancement of BK_Ca channel currents and caused a rightward shift of the TEA concentration-response curve similar to Rg₃, indicating that Rg₃ may interact with the external TEA biding site via residue Tyr360.

BK_Ca channels consist of α and β subunits. The four α subunits assemble to form the channel pore and the auxiliary β subunits function to provide the biophysical and pharmacological properties of channels, including Ca²⁺ and voltage sensitivity, and gating kinetics (Ha et al., 2006; Qian et al., 2002; Valverde et al., 1999; Wallner et al., 1999; Xia et al., 1999). Some BK_Ca channel openers such as NS-1619, BMS-204352 and TIBC act independently from the β subunit (Coghlan et al., 2001; Dick et al., 2002; Ha et al., 2006; Imaizumi et al., 2002; Valverde et al., 1999; Vergara et al., 1998), whereas other openers such as dehydrosoyasaponin-I and 17-β-estradiol require β subunit for their action (Giangiacomo et al., 1998; Valverde et al., 1999). In the present study, we found that the Rg₃-enhancement of BK_Ca channel current was independent of β subunit (Fig. 2), suggesting that Rg₃ effects on BK_Ca channel are achieved through interaction with α subunit.

Therefore, we next sought to examine the possible mecha-nisms by which Rg₃ achieved the enhancement of BK_Ca chan-nel currents through interaction with α subunit. As shown in Fig. 1A, Rg₃ is a type of glycoside and consists of a carbohydrate portion, a steroid backbone and an alkene side chain (Lee et al., 2008). In a previous report we demonstrated using homology modeling methods that carbohydrate portions of Rg₃ form hy-drogen bonds and maintain stable interactions with amino acids of channel proteins (Lee et al., 2008). In addition, we also demonstrated that Rg₃ inhibits Kv1.4 channel currents through interaction with K531 residue, which is located at the channel pore entryway and is also one of the extracellular TEA binding and K⁺ activation sites. In contrast, Rg₃ activates KCNQ + KCNE channel through interaction with Lys and Val residues at channel pore entryway (Choi et al., 2010). Based on previous experimental data (Choi et al., 2010; Lee et al., 2008), we first constructed mutant channels at the channel pore entryway of α subunit and found that Rg₃ effects on BK_Ca channel current enhancements were also greatly attenuated in Y360I mutant channels.

We observed some consistent results in Rg₃-mediated K⁺ channel regulation experiments. As shown in Fig. 1B, K⁺ channels exhibit a common feature in that they all have pore-lining P-loop with a consensus amino acid sequence -TXGYGD- which is called the K⁺ channel “signature sequence” (Heginbotham et al., 1992; 1994). These residues, repeated in each of the four α subunits, form the K⁺ selectivity filter. In previous studies, mutations of first or second amino acid after -TXGYGD- abolished Rg₃-mediated inhibition (i.e., Kv1.4 channel) (Lee et al., 2008) or Rg₃-mediated activation (i.e., KCNQ + KCNE) (Choi et al., 2010). These results indicate that Rg₃ interacts with amino acid residues in common interaction regions near the “signature sequence” in subsets of K⁺ channels examined.

However, it could not be excluded that the Rg₃ might regulate BK_Ca channel activity through allosteric mechanisms. In this case, the mutations introduced in Y360 residue might induce the conformational changes and interfere the interaction of Rg₃ with channel proteins, thus attenuating Rg₃-enhancement of BK_Ca channel currents. Taken together, although Y360 residue plays an important role in Rg₃-mediated BK_Ca channel activation, further studies will be required to elucidate the detailed mecha-nisms how Rg₃ regulates BK_Ca channels.

In summary, we found that Rg₃ enhances BK_Ca channel currents without desensitization after repeated treatment and with a Ca²⁺-independent manner. We have used site-directed muta-genesis to further characterize the Rg₃ interaction site with BK_Ca channel. We found that the Y360 residue at the channel pore entryway of brain BK_Ca channel is involved in Rg₃-media-ted BK_Ca channel regulations. These novel findings provide insight into the molecular basis of the pharmacological effects of ginseng in nervous systems.