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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.
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Figure 2. Effects of gintonin on BKCa channel activity. (a) Gintonin concentration-response curve (meanâ±âS.E.M; n = 13â15 oocytes each). Inset, the representative traces of gintonin-mediated BKCa channel activation at various concentrations. (b) Effects of gintonin (10âμg/mL) on the current-voltage (I-V) relationship of the wild-type BKCa channel (meanâ±âS.E.M; n = 13â15 oocytes each). (c) Time-current relationship plotted against time before and after repeated applications of gintonin (10âμg/mL) for 30âs in oocytes expressing the BKCa channel. Inset, the representative peak outward current amplitude at +40âmV from a holding potential of â80âmV 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 (meanâ±âS.E.M; n = 12â14 each) (*P < 0.001, compared to gintonin alone).
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Figure 3. The signal transduction pathways in gintonin-mediated BKCa channel activation. ((a) and (b)) Time-current relationship following application of gintonin (10âμg/mL) or ginsenoside Rg3 (100âμM) for 30âs 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 +40âmV from a holding potential of â80âmV 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 (10âμg/mL) or ginsenoside Rg3 (100âμM) for 30âs 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 +40âmV from a holding potential of â80âmV 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 (meanâ±âS.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).
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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)â(d)) Time-current relationships show the effects of gintonin or ginsenoside Rg3 in the pretreatment of PMA (1âμM) for 10âmin, calphostin (Cal 1.5âμM) for 10âmin, or the mutation of the PKC phosphorylation site (S1061A). Peak outward currents were recorded during bath application of gintonin (10âμg/mL). Insets, the representative gintonin-mediated or ginsenoside Rg3-mediated peak outward current amplitude at +40âmV from a holding potential of â80âmV was measured in the presence of PMA, calphostin, or mutant BKCa channels. (e) Time-current relationships following the application of gintonin (10âμg/mL) or ginsenoside Rg3 (100âμM) for 30âs 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 (meanâ±âS.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).
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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 (1.5âμM) for 10âmin. Insets, the representative gintonin-mediated or ginsenoside Rg3-mediated peak outward current amplitude at +40âmV from a holding potential of â80âmV 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) (meanâ±âS.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 (meanâ±âS.E.M; n = 10â12 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 (meanâ±âS.E.M; n = 10â12 oocytes each; *P < 0.001, compared to wild-type).
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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 (10âμg/mL) for 60âs, 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 60âs. 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 (100âμM), 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 60âs. Mutations in the RCK domain caused a rightward shift of the gintonin concentration-response curve (meanâ±âS.E.M; n = 13-14 oocytes each).
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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 (10âμg/mL) or ginsenoside Rg3 (100âμM) for 60âs in oocytes coexpressing BKCa channels and CaM kinase IIâ+âCa2+ bowl mutants. Insets, the representative peak outward current amplitude at +40âmV from a holding potential of â80âmV was measured in the presence of gintonin or ginsenoside Rg3. (b) Time-current relationship after application of gintonin (10âμg/mL) or ginsenoside Rg3 (100âμM) for 60âs in oocytes coexpressing BKCa channels and CaM kinase IIâ+âRCK domain mutants. Inset, the representative peak outward current amplitude at +40âmV from a holding potential of â80âmV was measured in the presence of gintonin or ginsenoside Rg3. (c) Coexpression of BKCa channels with CaM kinase IIâ+âCa2+ bowl mutants caused a further rightward shift of the gintonin concentration-response curve. (d) Coexpression of BKCa channels with CaM kinase IIâ+âRCK domain mutants caused a further rightward shift of the gintonin concentration-response curve (meanâ±âS.E.M; n = 13 oocytes each).
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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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