Molecular Mechanisms of Large-Conductance Ca2+-Activated Potassium Channel Activation by Ginseng Gintonin

Gintonin is a unique lysophosphatidic acid (LPA) receptor ligand found in Panax ginseng. Gintonin induces transient [Ca2+]i through G protein-coupled LPA receptors. Large-conductance Ca2+-activated K+ (BKCa) channels are expressed in blood vessels and neurons and play important roles in blood vessel relaxation and attenuation of neuronal excitability. BKCa channels are activated by transient [Ca2+]i and are regulated by various Ca2+-dependent kinases. We investigated the molecular mechanisms of BKCa channel activation by gintonin. BKCa channels are heterologously expressed in Xenopus oocytes. Gintonin treatment induced BKCa channel activation in oocytes expressing the BKCa channel α subunit in a concentration-dependent manner (EC50 = 0.71 ± 0.08 µg/mL). Gintonin-mediated BKCa channel activation was blocked by a PKC inhibitor, calphostin, and by the calmodulin inhibitor, calmidazolium. Site-directed mutations in BKCa channels targeting CaM kinase II or PKC phosphorylation sites but not PKA phosphorylation sites attenuated gintonin action. Mutations in the Ca2+ bowl and the regulator of K+ conductance (RCK) site also blocked gintonin action. These results indicate that gintonin-mediated BKCa channel activations are achieved through LPA1 receptor-phospholipase C-IP3-Ca2+-PKC-calmodulin-CaM kinase II pathways and calcium binding to the Ca2+ bowl and RCK domain. Gintonin could be a novel contributor against blood vessel constriction and over-excitation of neurons.


Introduction
Ginseng, the root of Panax ginseng C. A. Meyer, has been used as a representative tonic or an adaptogen to promote longevity and to enhance bodily functions against hypertension and as a neuroprotectant for several hundred years in Far East countries like Korea, China, and Japan. Currently, ginseng is one of the most famous and precious herbal medicines consumed around the world [1]. Recently, we isolated and characterized a novel glycolipoprotein, designated as gintonin, from ginseng. Gintonin is a lysophosphatidicacids-(LPAs-) ginseng major latex-like protein (MLP151) and ginseng ribonuclease-like storage protein complex, in which the lysophosphatidic acids (LPAs) bind to ginseng proteins through hydrophobic interactions, and this is the main principle underlying gintonin action [2][3][4][5][6], whereas most of other LPA receptor ligands are derivatives of LPA or LPA analogs [7]. Gintonin induces transient [Ca 2+ ] i through LPA receptor activation via pertussis toxin-(PTX-) sensitive and -insensitive G proteins in animal cells [2][3][4][5][6]. Thus, gintoninmediated transient [Ca 2+ ] i induction via LPA receptors could be further coupled to the regulation of Ca 2+ -dependent enzymes and Ca 2+ -dependent ion channel activities, which play important roles in biological systems.
Large-conductance Ca 2+ -activated K + (BK Ca ) channels are a family of outward K + -selective ion channels activated in response to membrane depolarization. BK Ca channels 2 Evidence-Based Complementary and Alternative Medicine are activated by intracellular Ca 2+ elevation and/or Ca 2+dependent kinases [8,9]. BK Ca channels play key roles in neuronal and nonneuronal cell functions. For example, in neuronal cells, BK Ca channels regulate the frequency of firing, action potentials following hyperpolarization, and neurotransmitter release. In vascular smooth muscle cells, BK Ca channels are one of the main ion channels that are involved in vasorelaxation [10,11].
BK Ca channels are composed of two subunits: the subunit (also called rSlo), which forms the channel pore [12], and the subunit [13,14], which modifies the voltage and calcium sensitivity of the pore-forming subunit [15,16]. The subunit has a large cytoplasmic C terminus and is responsible for the Ca 2+ -dependent activation of the channel. Furthermore, the cytoplasmic C terminus of the subunit has two domains that are responsible for the Ca 2+ -dependent activation of the channel, namely, the Ca 2+ bowl and the regulator of K + conductance (RCK) domain [17][18][19][20][21]. The cytoplasmic C terminus of the subunit has amino acid residues that can be phosphorylated by a variety of protein kinases such as CaM kinase II, PKA, and PKC [8,9]. Accumulating evidence shows that BK Ca channels play key roles in excitable cells and are regulated by diverse Ca 2+ and Ca 2+dependent kinases [10,11]. Although the signaling pathways of LPA as well as gintonin are well characterized through the biochemical and pharmacological experiments [2][3][4][5][6]22], relatively little is known about the molecular mechanisms how gintonin-mediated [Ca 2+ ] i transient is linked to BK Ca channel regulation.
In the present study, we examined how LPA receptor activation by gintonin may regulate BK Ca channel activity in Xenopus oocytes expressing the subunit of BK Ca alone or in Xenopus oocytes coexpressing BK Ca channels and other BK Ca channel regulators. We found that treatment of gintonin induces BK Ca channel activation. Gintoninmediated BK Ca channel activation is achieved through the LPA1 receptor, the phospholipase C-IP 3 -Ca 2+ pathway, and CaM kinase II phosphorylation of the subunit. We further demonstrated that site-directed mutations of the Ca 2+ bowl, RCK domain, and CaM kinase II phosphorylation site of channels greatly attenuated gintonin action. We compared the regulatory modes between gintonin and ginsenoside Rg 3 in BK Ca channel activation. We further discuss how signal coupling of gintonin to the BK Ca channel through the LPA receptor is associated with the beneficial physiological and pharmacological effects of ginseng on blood vessels and the nervous system.

Materials.
Gintonin was isolated from P. ginseng as described previously [23]. In the present study, we used the crude gintonin fraction, which contains about 9.5% LPAs, the majority being LPA C18:2 [2][3][4][5][6]. Ginsenoside Rg 3 was provided by the AMBO Institute (Seoul, Republic of Korea). The stock solution of ginsenoside Rg 3 was prepared and used as described previously [24]. M1 muscarinic acetylcholine receptor was purchased from Guthrie Research Institute (Sayre, PA, USA). CaM kinase II gene was kindly provided by OriGene (Rockville, MD, USA). All other reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA).

2.2.
In Vitro Synthesis of cRNA. Recombinant plasmids containing cDNA inserts for M1 muscarinic receptor, subunit (rSlo), and constitutively active CaM kinase II were linearized by digestion with appropriate restriction enzymes. The cRNAs from linearized templates were obtained with an in vitro transcription kit (mMessage mMachine; Ambion, Austin, TX, USA) using a SP6, T3, or T7 RNA polymerase. The RNA was dissolved in RNase-free water at 1 g/ L, divided into aliquots, and stored at −70 ∘ C.

Preparation of Xenopus Oocytes and Microinjection.
Xenopus laevis was purchased from Xenopus I (Ann Arbor, MI, USA). Their care and handling were in accordance with the highest standard of institutional guidelines of Konkuk University. For the isolation of oocytes, frogs were anesthetized with an aerated solution of 3-amino benzoic acid ethyl ester followed by the removal of ovarian follicles. The oocytes were subsequently treated with collagenase and then agitated for 2 h in Ca 2+ -free OR2 medium containing 82.5 mM NaCl, 2 mM KCl, 1 mM MgCl 2 , 5 mM HEPES, 2.5 mM sodium pyruvate, 100 units/mL penicillin, and 100 g/mL streptomycin. Stages V-VI oocytes were collected and stored in ND96 medium (in mM: 96 NaCl, 2 KCl, 1 MgCl 2 , 1.8 CaCl 2 , and 5 HEPES, pH 7.5) supplemented with 50 g/mL gentamicin. The oocyte-containing solution was maintained at 18 ∘ C with continuous gentle shaking and was renewed daily. Electrophysiological experiments were performed within 5-6 days of oocyte isolation, with gintonin or 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-L microdispenser (VWR Scientific, San Francisco, CA, USA) fitted with a tapered glass pipette tip (diameter, 15-20 m) [25]. Ca and In Vitro Transcription of BK Ca Channel cDNAs. Single amino acid substitutions of the BK Ca channel (Figure 1(a)) were made using a QuikChange XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA), along with Pfu DNA polymerase and 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 transformed into E. coli strain DH5 , 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 prepared using an mMessage mMachine transcription kit (Ambion, Austin, TX, USA) with T7 RNA polymerase. The absence of degraded RNA was confirmed 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, Austin, TX, USA) with SP6 RNA polymerase or T7 polymerase. The final cRNA products were resuspended at a concentration of 1 g/ L in RNase-free water and stored at −80 ∘ C [25].

Data
Recording. Data recording for BK Ca channel currents was performed, as described by Liu et al. [26], to study the detailed downstreams of gintonin-mediated signaling transduction pathways. Therefore, a custom-made Plexiglas net chamber was used for two-electrode voltage-clamp recordings, as previously reported [25]. The oocytes were impaled with two microelectrodes filled with 3 M KCl (0.

Gintonin Induces BK Ca Channel Activation in a Concentration-Dependent and Voltage-Dependent Manner in Xenopus Oocytes Expressing BK Ca Channels.
In the present study, we first examined the effect of gintonin on BK Ca channel activity in Xenopus oocytes expressing BK Ca channel (rSlo) subunits. Application of gintonin to oocytes injected with subunit cRNAs resulted in the activation of the BK Ca channel as monitored with a clamp step to +60 from −80 mV holding potential ( = 7; current examined at 20-s intervals). The mean current activation by gintonin (10 g/mL) was 2855.1 ± 415.0% (Figure 2(a)), whereas gintonin had no effect on the control oocytes that were not injected with cRNA encoding the BK Ca subunit gene (data not shown). Gintonin-induced BK Ca channel activation occurred in a concentration-dependent manner (Figure 2(a)). The EC 50 was observed to be 0.71 ± 0.08 g/mL. Charybdotoxin and iberiotoxin, highly specific inhibitors of maxi-K channels [27,28], greatly attenuated BK Ca channel activation induced by gintonin (data not shown), indicating that BK Ca channels are functional [29]. BK Ca channel activation by gintonin was observed over the entire voltage range examined from 0 mV. Thus, gintonin-induced BK Ca channel activation occurs in a voltage-dependent manner since marked activations at more positive potentials were observed, as shown in a currentvoltage relationship (Figure 2(b)).

Gintonin-Induced BK Ca Channel Activation Is Rapidly Desensitized following Repeated Application of Gintonin and Is
Blocked by a LPA1/3 Receptor Antagonist. We next examined the changes in gintonin-induced BK Ca channel activation

The Signal Transduction Pathway of Gintonin-Mediated
BK Ca Channel Activation. We next examined the signal transduction pathways involved in gintonin-mediated BK Ca channel activation. We first examined the involvement of phospholipase C (PLC) in gintonin-mediated BK Ca channel activation. To test this possibility, the effects of the active PLC inhibitor U-73122 and its inactive analogue U-73343 were examined on gintonin action [31]. Bath application of U-73122 significantly suppressed gintonin action, whereas the current in the presence of U-73343 was not affected (Figures 3(a) and 3(b)). These results indicate that gintonin-mediated BK Ca channel activation requires PLC activation. To see if the IP 3 receptor was involved in gintonin action, oocytes were stimulated with gintonin in the presence of 2-APB, an IP 3 receptor antagonist. We observed that 2-APB treatment also greatly attenuated the effect of gintonin on BK Ca channel activation (Figure 3(c)). These observations suggest that gintonin first induces an activation of the IP 3 receptor to mobilize intracellular Ca 2+ , and then mobilized [Ca 2+ ] i is coupled to BK Ca channel activation. We have demonstrated in a previous report that gintonin-mediated activation of Ca 2+ -activated Cl − channels is dependent on cytosolic Ca 2+ [23]; thus, we examined whether the effect of gintonin on BK Ca channel activation was also dependent on cytosolic Ca 2+ . To this end, we first treated oocytes with BAPTA-AM, a membrane permeable Ca 2+ chelator, to chelate free cytosolic Ca 2+ and examined gintonin effects. We found that BAPTA-AM completely abolished gintonin action on BK Ca channel activation (Figure 3(d)), indicating that gintonin-mediated BK Ca channel activation is also achieved through mobilization of intracellular Ca 2+ from endoplasmic reticulum. However, while ginsenoside Rg 3 (100 M) also enhanced BK Ca channel currents, as shown previously [24], gintonin exhibited a greater (3-4-fold) activation of the BK Ca channel than ginsenoside Rg 3 . In addition, the enhancing effects of ginsenoside Rg 3 on BK Ca channel currents were not sensitive to the PLC inhibitor, the 2-APB antagonist, and BAPTA ( Figure 3(e)), indicating that the regulatory mode on BK Ca channel activity by gintonin differs from that of ginsenoside Rg 3 .

Involvement of PKC but Not PKA in Gintonin-Mediated
BK Ca Channel Activation. The previous reports have shown that the activation of the PLC pathway also produces lipidsoluble 1,2-diacylglycerol (DAG), an endogenous protein kinase C (PKC) activator. Activation of PKC by treatment with PMA, a DAG analogue, causes receptor phosphorylation and receptor uncoupling from PLC-mediated inositol phospholipid metabolism and results in a loss of Ca 2+ -activated Cl − channel activation by 5-HT or muscarinic acetylcholine receptor agonist stimulations in Xenopus oocytes [4,5,12,32]. Similarly, in the present study we also first examined the effects of the PKC activator PMA on the gintoninmediated BK Ca channel activation. As shown in Figure 4(b), we found that treatment with PMA induced a loss of gintonin-mediated BK Ca channel activation. To confirm PKC involvement in gintonin-mediated BK Ca channel activation, we next examined the effect of PKC inhibitor, calphostin, with gintonin action and found that calphostin also prevented gintonin-mediated BK Ca channel activation (Figure 4(c)), indicating that gintonin-mediated BK Ca channel activation is achieved via PKC activation through LPA receptor. We also tested whether mutation of the PKC phosphorylation site affects gintonin-mediated BK Ca channel activation. As shown in Figure 4(d), mutation of Ser1061 to S1061A significantly attenuated gintonin-mediated BK Ca channel activation [33]. Similarly, we examined whether mutation of the PKA phosphorylation site affects gintonin-mediated BK Ca channel activation. Interestingly, we found that mutation of the PKA phosphorylation site did not affect gintoninmediated BK Ca channel activation (Figures 4(e) and 4(f)). Thus, these results indicate that channel protein phosphorylation by PKC, but not PKA, is involved in gintoninmediated BK Ca channel activation. However, the enhancing effects of ginsenoside Rg 3 on BK Ca channel currents were not affected by PMA, calphostin, and mutant BK Ca channels at the PKC phosphorylation site (Figure 4(g)). These results collectively indicate that the gintonin-mediated but not ginsenoside Rg 3 -mediated BK Ca channel activation involves PKC activation.

Involvement of Calmodulin and Calcium-/Calmodulin-Dependent Kinase II (CaM Kinase II) in Gintonin-Mediated
Activation of BK Ca Channels. Since calmodulin and CaM kinase II have been reported to be involved in the regulation of BK Ca channel activation [8], we determined whether calmodulin and CaM kinase II are involved in gintoninmediated BK Ca channel activation. To this end, we first examined gintonin-mediated BK Ca channel activation following treatment with the calmodulin antagonist, calmidazolium. As shown in Figure 5(b), calmidazolium treatment significantly attenuated gintonin-mediated BK Ca channel activation, indicating that calmodulin is involved in gintonin-mediated BK Ca channel activation. In contrast, the enhancing effects of ginsenoside Rg 3 on BK Ca channel currents were not affected by calmidazolium (Figure 5(c)). Since calmodulin is closely related with CaM Kinase II activation, which is known to regulate BK Ca channel activity [34,35], we next examined if gintonin-mediated BK Ca channel activation is achieved through CaM kinase II. To this end, we constructed two different kinds of mutant BK Ca channels at CaM kinase II phosphorylation sites, T462 and S512, by replacing these residues with alanine (T462A and S512A) [26]. As shown in compared to wild-type channels. Thus, the EC 50 was 0.62 ± 0.04, 9.61 ± 0.15, and 1.38 ± 0.25 g/mL in wild-type and T462A and S521A mutants, respectively. Interestingly, gintonin action on BK Ca channel activation was more strongly inhibited in T462A rather than S512A mutants. These results indicate that gintonin induces CaM kinase II activation and links to BK Ca channel activation through BK Ca channel phosphorylation at T462 and S512.

Involvement of the Ca 2+ -Binding Domain (Ca 2+ Bowl) and RCK Domain in Gintonin-Mediated BK Ca Channel Activation.
BK Ca channels have unique structures called the Ca 2+ bowl and the RCK domain. These two domains play important roles in Ca 2+ -dependent regulation of BK Ca channels [36,37].
To confirm the involvement of the Ca 2+ bowl and RCK domains in gintonin-mediated BK Ca channel activation, we mutated residues at these domains since C-terminus mutations have been shown to affect Ca 2+ -mediated regulation of BK Ca channel activity [17,38]. To this end, we constructed six different mutant BK Ca channels in Ca 2+ -bowl residues, D989, D991, D992, D993, D994, and D995, by replacing these residues with alanine (D989A, D991A, D992A, D993A, D994A, and D995A) [39]. Moreover, we constructed two different kinds of mutant BK Ca channels in RCK domain residues such as D433 and M579 by replacing these residues with alanine and isoleucine (D433A and M579I) [21]. We then examined the effects of gintonin on the activity of these mutant channels. In concentration-response curves, the stimulatory effects of gintonin on BK Ca channel activity were observed to be greatly attenuated in oocytes expressing the mutants compared to wild-type channels in the order of D994A > D989A > D922A > D991 > D993A (Figure 6(a)). The EC 50 values were 0.64±0.08, 1.00±0.01, 2.70±0.06, 4.01± 0.06, 1.38 ± 0.03, 11.31 ± 4.62, and 1.35 ± 0.22 g/mL in wildtype, D989A, D991A, D992A, D993A, D994A, and D995A, respectively. Interestingly, gintonin action on BK Ca channel activation was more strongly inhibited in D994A rather than in other Ca 2+ bowl mutants. We also examined the effects of gintonin on RCK domain mutant channels. As shown in Figure 6(b), gintonin-mediated BK Ca channel activation was greatly attenuated in oocytes expressing the mutant channels D433A and M579I compared to wild-type channels. The representative concentration-response curves are also shown in Figure 6(b). The EC 50 values were 0.51 ± 0.07, 10.71 ± 0.60, and 2.26 ± 0.06 g/mL in wild-type, D433A, and M579I mutants, respectively. Interestingly, gintonin action on BK Ca channel activation was more strongly inhibited in D433A rather than M579I RCK domain mutants. As a positive control, we injected cRNA encoding M1 muscarinic acetylcholine receptor (mAChR) into the oocytes, which are reported to induce transient [Ca 2+ ] i via the G q/11 -PLC-IP 3 pathway [40]. As shown in Figure 6(c), in Ca 2+ bowl mutants such as D991A, D992A, and D994A mutants, treatment of acetylcholine caused a right shift in the concentrationresponse curves. In RCK domain mutants, such as D433S and M579I mutants, treatment of acetylcholine also caused a right shift of the concentration-response curves (Figure 6(d)). These results indicate that the released Ca 2+ induced by gintonin or acetylcholine treatment binds to the Ca 2+ bowl and RCK domain and induces BK Ca channel activation.

Dual Mutations of the Ca 2+ Bowl or RCK Domain with BK Ca Channel Phosphorylation Sites Further Attenuate
Gintonin-Mediated BK Ca Channel Activation. We further examined whether dual mutations of amino acid residues in the Ca 2+ bowl or the RCK domain and in CaM kinase II phosphorylation sites in the BK Ca channel further affect gintonin-mediated BK Ca channel activation. As shown in Figure 7(a), double mutations of CaM kinase II and the Ca 2+ bowl (T462/F994A) further attenuated the gintoninmediated BK Ca channel activation with a concomitant right shift of the concentration-response curves (Figure 7(c)). The EC 50 was 31.23 ± 1.20 g/mL. Additionally, double mutations of CaM kinase II and the RCK domain further attenuated the gintonin-mediated BK Ca channel activation (Figure 7(d)). The EC 50 was 22.5 ± 0.80 g/mL, again confirming that gintonin-mediated BK Ca channel activation includes Ca 2+mediated CaM kinase II activation and Ca 2+ binding to the Ca 2+ bowl and RCK region. However, the enhancing effects of ginsenoside Rg 3 on BK Ca channel currents were not affected by the double mutations of CaM kinase II and the Ca 2+ bowl or by mutations in CaM kinase II and the RCK domain (Figures 7(a) and 7(b)).

Discussion
BK Ca channels exist in excitable cells such as neurons and vascular smooth muscle cells. Their main roles are to induce repolarization following depolarization or to restore the resting membrane potential of neurons and vascular smooth muscles. Thus, the physiological functions of BK Ca channels are to regulate synaptic transmission in the nervous system and to relax the blood vessels. The activation of BK Ca channels is closely linked to transient [Ca 2+ ] i induction by voltage-gated Ca 2+ channel activation after depolarization since BK Ca channels colocalize with Ca 2+ channels [41,42]. The cytoplasmic C terminus of the BK Ca channel subunit contains two main Ca 2+ binding sites, that is, the Ca 2+ bowl and a high Ca 2+ affinity RCK domain [37]. In addition, various kinases also regulate BK Ca channel activities through the phosphorylation of BK Ca channel proteins [43,44].
The present study was performed to elucidate the molecular mechanisms coupling gintonin to BK Ca channel activation by using a Xenopus oocyte gene expression system. Our results revealed four major findings. Firstly, we observed that gintonin treatment induced BK Ca channel activation in a concentration-and voltage-dependent manner via LPA receptor activation but the repeated treatment of gintonin induced a rapid desensitization. Secondly, the presence of a PLC inhibitor, an IP 3 receptor, antagonist, an intracellular Ca 2+ chelator, or a PKC inhibitor greatly attenuated the action, of gintonin. Thirdly, treatment with a calmodulin inhibitor attenuated gintonin action and mutations of PKC and CaM kinase II phosphorylation sites, but not by PKA phosphorylation sites, on the BK Ca channel greatly attenuated gintonin action. Fourthly, mutations of amino acid residues in the Ca 2+ bowl and RCK domains greatly attenuated gintonin-mediated enhancement of BK Ca channel currents. Thus, since BK Ca channels play an important role in presynaptic nerve terminals and blood vessel smooth muscle cells, the findings in the present study show the possibility that gintonin may be a novel BK Ca channel regulator in the nervous and vascular systems via the PLC-IP 3 -Ca 2+ and Ca 2+ -PKC-CaM kinase II signal transduction pathways. Interestingly, although gintonin-and acetylcholine-mediated BK Ca channel activations are attenuated by site-directed mutations of amino acid residues of Ca 2+ bowl and RCK domain, it appears in D994A and D433A mutant channels that the degree of gintonin-mediated BK Ca channel activation was more strongly attenuated than that of acetylcholinemediated BK Ca channel activation. These results imply that although both agents use the same signaling pathway for BK Ca channel activation, D994 residue in Ca 2+ bowl and D433 residue in RCK domain might play more important role in gintonin-rather than acetylcholine-mediated BK Ca channel activation. In a previous study, we demonstrated that ginsenoside Rg 3 enhances BK Ca channel currents following depolarization [24]. By comparing the regulatory mode of gintonin action with ginsenoside Rg 3 action for BK Ca channel activation, we determined that gintonin differs from ginsenoside Rg 3 . Ginsenoside Rg 3 -induced enhancement of BK Ca channel currents was not achieved through receptormediated transient [Ca 2+ ] i (Figures 2 and 3). Thus, ginsenoside Rg 3 -induced BK Ca channel current enhancement did not include membrane receptor signaling transduction pathways. Instead, as a kind of dammarane glycosides (Figure 1(b)), ginsenoside Rg 3 -induced enhancement of BK Ca channel currents was abolished by substitution of a Tyr360 residue, located at the channel pore entrance, and the enhancement of BK Ca channel currents by Rg 3 did not show desensitization after repeated treatment [24]. Thus, ginsenoside Rg 3 regulates BK Ca channel activity through direct interaction with channel proteins at the channel pore entrance. In contrast, as a G protein-coupled LPA receptor ligand, gintonin amplifies BK Ca channel activation via a series of signal transductions through membrane bound G protein-coupled LPA receptor activation (Figure 8). Supporting this notion, gintonin, even at much lower concentrations than ginsenoside Rg 3 , induces greater amplitudes of outward BK Ca channel currents (G q/11 ) Figure 8: A comparative drawing of the action modes between gintonin and ginsenoside Rg 3 in BK Ca channel activation. Gintonin activates BK Ca channels via G protein-coupled LPA1 receptors. Gintonin-mediated BK Ca channel activations are mediated by Ca 2+ binding to the Ca 2+ bowl, RCK, or via activations of Ca 2+ -dependent kinases, whereas ginsenoside Rg 3 activates BK Ca channels through direct interaction with a specific amino acid located at the pore entryway of channel proteins following depolarization but not receptor activation [24].
(by 4-5-fold) than ginsenoside Rg 3 (Figure 3). The EC 50 of gintonin is about 35 nM (under the assumption that the molecular weight of gintonin is 20 kDa), whereas that of ginsenoside Rg 3 was about 15 M for BK Ca channel activation [24]. In addition, interruptions of the receptor signaling pathway by inhibitors or mutations abolished or attenuated gintonin-mediated but not ginsenoside Rg 3 -mediated BK Ca channel activation. These results indicate that although ginseng contains two agents with two different action modes for the regulation of BK Ca channel activity, gintonin is more efficient for BK Ca channel activation than ginsenoside Rg 3 (Figure 8). BK Ca channels are widely distributed in nervous and vascular systems [10,45,46]. In vitro gintonin-mediated BK Ca channel activation might be associated with the in vivo pharmacological effects of ginseng. In previous studies, ginseng has exhibited neuroprotective effects against a variety of excitatory neurotransmitters, toxins, or ischemic stroke [1]. In addition, ginseng is also reported to induce relaxation of blood vessels constricted by adrenergic receptor stimulations [47,48]. Thus, gintonin might be utilized for the reduction of overexcitability of the nervous system or to downregulate hyperactivity of blood vessels. Thus, the present studies show the possibility using a Xenopus oocyte gene expression model system that gintonin might participate in the regulation of synaptic transmission in nerve terminals and vascular muscle tone. However, more investigations are needed to extend from Xenopus oocytes to neuron or muscle cells.
In summary, we found that gintonin induces BK Ca channel activation via membrane G protein-coupled LPA receptor signaling pathways. Using site-directed mutagenesis, we further confirmed the molecular mechanisms between the Ca 2+ bowl, RCK domain, and CaM kinase II, which are involved in gintonin-mediated BK Ca channel regulation. These novel findings provide insight into the molecular basis of the pharmacological effects of ginseng in the nervous and vascular systems.