Choi SH , Lee BH , Hwang SH , Kim HJ , Lee SM , Kim HC , Rhim HW , Nah SY .

	Figure 1. The primary amino acid sequence of the mutated BKCa channel α subunit and the chemical structure of ginsenoside Rg3. (a) The brief sequence alignment of the BKCa channel and the mutated amino acid residues in the CaM kinase II phosphorylation sites (462, 512), PKA (939), PKC (1061), or Ca2+ binding sites of the Ca2+ bowl (989, 991, 992, 993, 994, 995) and RCK domains (433, 579). (b) The chemical structure of ginsenoside Rg3. Glc: glucose.
	Figure 2. Effects of gintonin on BKCa channel activity. (a) Gintonin concentration-response curve (mean ± S.E.M; n = 13–15 oocytes each). Inset, the representative traces of gintonin-mediated BKCa channel activation at various concentrations. (b) Effects of gintonin (10 μg/mL) on the current-voltage (I-V) relationship of the wild-type BKCa channel (mean ± S.E.M; n = 13–15 oocytes each). (c) Time-current relationship plotted against time before and after repeated applications of gintonin (10 μg/mL) for 30 s in oocytes expressing the BKCa channel. Inset, the representative peak outward current amplitude at +40 mV from a holding potential of −80 mV was measured in the absence or presence of gintonin. (d) Summary histograms show that repeated application of gintonin induces the desensitization of gintonin-mediated BKCa channel activation (*P < 0.001, compared to 1st gintonin treatment). (e) The representative traces on blockage of gintonin-mediated BKCa channel activation by the LPA1/3 receptor antagonist, Ki16425. (f) Summary histograms show that gintonin-mediated activation of the BKCa channel is blocked by the LPA1/3 receptor antagonist, Ki16425 (mean ± S.E.M; n = 12–14 each) (*P < 0.001, compared to gintonin alone).
	Figure 3. The signal transduction pathways in gintonin-mediated BKCa channel activation. ((a) and (b)) Time-current relationship following application of gintonin (10 μg/mL) or ginsenoside Rg3 (100 μM) for 30 s in the presence of U73122, an active PLC inhibitor, or U73343, an inactive PLC inhibitor, in oocytes expressing BKCa channels. Inset, the representative peak outward current amplitude at +40 mV from a holding potential of −80 mV was measured in the presence of gintonin or ginsenoside Rg3. The active or inactive PLC inhibitor was pretreated for 5 min before gintonin or ginsenoside Rg3 application. ((c) and (d)) Time-current relationship after application of gintonin (10 μg/mL) or ginsenoside Rg3 (100 μM) for 30 s in the presence of 2-APB, an IP3 receptor antagonist, or BAPTA, an intracellular Ca2+ chelator, in oocytes expressing BKCa channels. Inset, the representative peak outward current amplitude at +40 mV from a holding potential of −80 mV was measured in the presence of gintonin or ginsenoside Rg3. The application of 2-APB or BAPTA preceded the gintonin application by 2 h. (e) Summary histograms show the peak outward BKCa channel currents (mean ± S.E.M; n = 13-14 oocytes each) recorded in oocytes expressing the BKCa channel in the absence or presence of the indicated agents. (*P < 0.001, compared to gintonin alone).
	Figure 4. Involvement of PKC but not PKA in gintonin-mediated BKCa channel activation. (a) Gintonin or ginsenoside Rg3 induces activation of BKCa channels in oocytes. ((b)–(d)) Time-current relationships show the effects of gintonin or ginsenoside Rg3 in the pretreatment of PMA (1 μM) for 10 min, calphostin (Cal 1.5 μM) for 10 min, or the mutation of the PKC phosphorylation site (S1061A). Peak outward currents were recorded during bath application of gintonin (10 μg/mL). Insets, the representative gintonin-mediated or ginsenoside Rg3-mediated peak outward current amplitude at +40 mV from a holding potential of −80 mV was measured in the presence of PMA, calphostin, or mutant BKCa channels. (e) Time-current relationships following the application of gintonin (10 μg/mL) or ginsenoside Rg3 (100 μM) for 30 s in oocytes expressing S939A mutant BKCa channels. (f) Concentration dependency of wild-type and S939A mutant BKCa channels on gintonin-mediated BKCa channel activation. (g) Summary histograms show that peak outward BKCa channel currents (mean ± S.E.M; n = 11-12 oocytes each) recorded in oocytes expressing the BKCa channel in the absence or presence of the indicated agents or mutation (*P < 0.001, compared to gintonin alone).
	Figure 5. Involvement of calmodulin and CaM kinase II in gintonin-mediated BKCa channel activation. ((a) and (b)) Oocytes expressing the BKCa channel were incubated in the absence (a) or presence (b) of calmidazolium (1.5 μM) for 10 min. Insets, the representative gintonin-mediated or ginsenoside Rg3-mediated peak outward current amplitude at +40 mV from a holding potential of −80 mV was measured in the absence or presence of calmidazolium. (c) Summary histograms show peak outward BKCa channel currents recorded in oocytes expressing the BKCa channel in the absence or presence of the calmidazolium (CMZ) (mean ± S.E.M; n = 13-14 oocytes each; *P < 0.001, compared to gintonin alone). (d) The oocytes expressing mutant BKCa channel at the CaM kinase II phosphorylation site (T462A or S521A) were treated with gintonin by bathing application for 60 s. Mutation of CaM kinase II phosphorylation sites resulted in a rightward shift of the gintonin concentration-response curve (mean ± S.E.M; n = 10–12 oocytes each). (e) Summary histograms show that the gintonin-mediated peak outward BKCa channel currents recorded in oocytes expressing mutant BKCa channel at the CaM kinase II phosphorylation site (T462A or S521A) were significantly attenuated (mean ± S.E.M; n = 10–12 oocytes each; *P < 0.001, compared to wild-type).
	Figure 6. Involvement of the Ca2+ bowl and RCK domain in gintonin-mediated BKCa channel activation. (a) The oocytes expressing wild-type or various mutant BKCa channels at the Ca2+ bowl were treated by bath application of gintonin (10 μg/mL) for 60 s, and peak outward currents were recorded. Mutations of the Ca2+ bowl caused a rightward shift of the gintonin concentration-response curve. (b) The oocytes expressing wild-type or RCK domain mutant BKCa channels, D433A or M579I, were treated with gintonin by bathing application for 60 s. Mutation of the RCK domain also caused a rightward shift of the gintonin concentration-response curve. (c) The oocytes coexpressing M1 muscarinic receptor or various mutant BKCa channels with M1 muscarinic receptor were treated by bath application of acetylcholine (100 μM), and peak outward currents were recorded. Mutations of the calcium bowl caused a rightward shift of the acetylcholine concentration-response curve. (d) Oocytes coexpressing wild-type with M1 muscarinic receptor or mutant BKCa channels at the RCK domain such as D433A or M579I with M1 muscarinic receptor were treated with gintonin by bathing application for 60 s. Mutations in the RCK domain caused a rightward shift of the gintonin concentration-response curve (mean ± S.E.M; n = 13-14 oocytes each).
	Figure 7. Double mutations of CaM kinase II and Ca2+ bowl or RCK domain further attenuate gintonin-mediated BKCa channel activation. (a) Time-current relationship after application of gintonin (10 μg/mL) or ginsenoside Rg3 (100 μM) for 60 s in oocytes coexpressing BKCa channels and CaM kinase II + Ca2+ bowl mutants. Insets, the representative peak outward current amplitude at +40 mV from a holding potential of −80 mV was measured in the presence of gintonin or ginsenoside Rg3. (b) Time-current relationship after application of gintonin (10 μg/mL) or ginsenoside Rg3 (100 μM) for 60 s in oocytes coexpressing BKCa channels and CaM kinase II + RCK domain mutants. Inset, the representative peak outward current amplitude at +40 mV from a holding potential of −80 mV was measured in the presence of gintonin or ginsenoside Rg3. (c) Coexpression of BKCa channels with CaM kinase II + Ca2+ bowl mutants caused a further rightward shift of the gintonin concentration-response curve. (d) Coexpression of BKCa channels with CaM kinase II + RCK domain mutants caused a further rightward shift of the gintonin concentration-response curve (mean ± S.E.M; n = 13 oocytes each).
	Figure 8. A comparative drawing of the action modes between gintonin and ginsenoside Rg3 in BKCa channel activation. Gintonin activates BKCa channels via G protein-coupled LPA1 receptors. Gintonin-mediated BKCa channel activations are mediated by Ca2+ binding to the Ca2+ bowl, RCK, or via activations of Ca2+-dependent kinases, whereas ginsenoside Rg3 activates BKCa channels through direct interaction with a specific amino acid located at the pore entryway of channel proteins following depolarization but not receptor activation [24].

Bao, Elimination of the BK(Ca) channel's high-affinity Ca(2+) sensitivity. 2002, Pubmed, Xenbase

Bao, Elimination of the BK(Ca) channel's high-affinity Ca(2+) sensitivity. 2002, Pubmed , Xenbase
Bao, Mapping the BKCa channel's "Ca2+ bowl": side-chains essential for Ca2+ sensing. 2004, Pubmed , Xenbase
Berkefeld, Repolarizing responses of BKCa-Cav complexes are distinctly shaped by their Cav subunits. 2008, Pubmed , Xenbase
Candia, Mode of action of iberiotoxin, a potent blocker of the large conductance Ca(2+)-activated K+ channel. 1992, Pubmed
Chang, Differential expression of the alpha and beta subunits of the large-conductance calcium-activated potassium channel: implication for channel diversity. 1997, Pubmed
Choi, Ginsenoside Rg3 enhances large conductance Ca2+-activated potassium channel currents: a role of Tyr360 residue. 2011, Pubmed , Xenbase
Dworetzky, Phenotypic alteration of a human BK (hSlo) channel by hSlobeta subunit coexpression: changes in blocker sensitivity, activation/relaxation and inactivation kinetics, and protein kinase A modulation. 1996, Pubmed
Gao, Interaction of agitoxin2, charybdotoxin, and iberiotoxin with potassium channels: selectivity between voltage-gated and Maxi-K channels. 2003, Pubmed
Ghatta, Large-conductance, calcium-activated potassium channels: structural and functional implications. 2006, Pubmed
Hwang, Gintonin, newly identified compounds from ginseng, is novel lysophosphatidic acids-protein complexes and activates G protein-coupled lysophosphatidic acid receptors with high affinity. 2012, Pubmed
Im, Pharmacological tools for lysophospholipid GPCRs: development of agonists and antagonists for LPA and S1P receptors. 2010, Pubmed
Ishii, Muscarinic acetylcholine receptors. 2006, Pubmed
Jiang, Crystal structure and mechanism of a calcium-gated potassium channel. 2002, Pubmed
Kaczorowski, High-conductance calcium-activated potassium channels; structure, pharmacology, and function. 1996, Pubmed
Kang, Ginsenosides of the protopanaxatriol group cause endothelium-dependent relaxation in the rat aorta. 1995, Pubmed
Kim, Ginsenoside Rg3 mediates endothelium-dependent relaxation in response to ginsenosides in rat aorta: role of K+ channels. 1999, Pubmed
Kimura, Two novel Xenopus homologs of mammalian LP(A1)/EDG-2 function as lysophosphatidic acid receptors in Xenopus oocytes and mammalian cells. 2001, Pubmed , Xenbase
Latorre, Large conductance Ca2+-activated K+ (BK) channel: activation by Ca2+ and voltage. 2006, Pubmed
Lee, Ginsenoside Rg3 inhibits human Kv1.4 channel currents by interacting with the Lys531 residue. 2008, Pubmed , Xenbase
Liu, Presynaptic Ca2+/calmodulin-dependent protein kinase II modulates neurotransmitter release by activating BK channels at Caenorhabditis elegans neuromuscular junction. 2007, Pubmed , Xenbase
Lupu-Meiri, Dual regulation by protein kinase C of the muscarinic response in Xenopus oocytes. 1989, Pubmed , Xenbase
McManus, Functional role of the beta subunit of high conductance calcium-activated potassium channels. 1995, Pubmed , Xenbase
Moran, Protein kinase C modulates neurotransmitter responses in Xenopus oocytes injected with rat brain RNA. 1989, Pubmed , Xenbase
Mutoh, Insights into the pharmacological relevance of lysophospholipid receptors. 2012, Pubmed
Nah, Ginsenosides: are any of them candidates for drugs acting on the central nervous system? 2007, Pubmed
Nelson, Relaxation of arterial smooth muscle by calcium sparks. 1995, Pubmed
Orellana, Guanosine 5'-O-(thiotriphosphate)-dependent inositol trisphosphate formation in membranes is inhibited by phorbol ester and protein kinase C. 1987, Pubmed
Piskorowski, Calcium activation of BK(Ca) potassium channels lacking the calcium bowl and RCK domains. 2002, Pubmed , Xenbase
Salkoff, High-conductance potassium channels of the SLO family. 2006, Pubmed
Sansom, Regulation of Ca(2+)-activated K(+) channels by multifunctional Ca(2+)/calmodulin-dependent protein kinase. 2000, Pubmed
Schreiber, Transplantable sites confer calcium sensitivity to BK channels. 1999, Pubmed , Xenbase
Schreiber, A novel calcium-sensing domain in the BK channel. 1997, Pubmed , Xenbase
Singer, Short- and long-term desensitization of serotonergic response in Xenopus oocytes injected with brain RNA: roles for inositol 1,4,5-trisphosphate and protein kinase C. 1990, Pubmed , Xenbase
Tanaka, Molecular constituents of maxi KCa channels in human coronary smooth muscle: predominant alpha + beta subunit complexes. 1997, Pubmed , Xenbase
Tanaka, MaxiK channel roles in blood vessel relaxations induced by endothelium-derived relaxing factors and their molecular mechanisms. 2004, Pubmed
Thompson, The aminosteroid U-73122 inhibits muscarinic receptor sequestration and phosphoinositide hydrolysis in SK-N-SH neuroblastoma cells. A role for Gp in receptor compartmentation. 1991, Pubmed
Toro, Maxi-K(Ca), a Unique Member of the Voltage-Gated K Channel Superfamily. 1998, Pubmed
Wang, Simultaneous binding of two protein kinases to a calcium-dependent potassium channel. 1999, Pubmed
Wang, Calcium/calmodulin-dependent protein kinase II phosphorylates and regulates the Drosophila eag potassium channel. 2002, Pubmed , Xenbase
Wang, Regulation of synaptic transmission by presynaptic CaMKII and BK channels. 2008, Pubmed
Weiger, Modulation of calcium-activated potassium channels. 2002, Pubmed
Xia, Multiple regulatory sites in large-conductance calcium-activated potassium channels. 2002, Pubmed
Yazejian, Direct measurements of presynaptic calcium and calcium-activated potassium currents regulating neurotransmitter release at cultured Xenopus nerve-muscle synapses. 1997, Pubmed , Xenbase
Zeng, Divalent cation sensitivity of BK channel activation supports the existence of three distinct binding sites. 2005, Pubmed , Xenbase
Zhu, Mutation of protein kinase C phosphorylation site S1076 on alpha-subunits affects BK(Ca) channel activity in HEK-293 cells. 2009, Pubmed