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Evid Based Complement Alternat Med
2013 Jan 01;2013:323709. doi: 10.1155/2013/323709.
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Molecular mechanisms of large-conductance ca (2+) -activated potassium channel activation by ginseng gintonin.
Choi SH
,
Lee BH
,
Hwang SH
,
Kim HJ
,
Lee SM
,
Kim HC
,
Rhim HW
,
Nah SY
.
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Gintonin is a unique lysophosphatidic acid (LPA) receptor ligand found in Panax ginseng. Gintonin induces transient [Ca(2+)]i through G protein-coupled LPA receptors. Large-conductance Ca(2+)-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 [Ca(2+)]i and are regulated by various Ca(2+)-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 Ca(2+) 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-Ca(2+)-PKC-calmodulin-CaM kinase II pathways and calcium binding to the Ca(2+) bowl and RCK domain. Gintonin could be a novel contributor against blood vessel constriction and over-excitation of neurons.
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].
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