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Ginseng gintonin activates the human cardiac delayed rectifier K+ channel: involvement of Ca2+/calmodulin binding sites.
Choi SH
,
Lee BH
,
Kim HJ
,
Jung SW
,
Kim HS
,
Shin HC
,
Lee JH
,
Kim HC
,
Rhim H
,
Hwang SH
,
Ha TS
,
Kim HJ
,
Cho H
,
Nah SY
.
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Gintonin, a novel, ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand, elicits [Ca(2+)]i transients in neuronal and non-neuronal cells via pertussis toxin-sensitive and pertussis toxin-insensitive G proteins. The slowly activating delayed rectifier K(+) (I(Ks)) channel is a cardiac K(+) channel composed of KCNQ1 and KCNE1 subunits. The C terminus of the KCNQ1 channel protein has two calmodulin-binding sites that are involved in regulating I(Ks) channels. In this study, we investigated the molecular mechanisms of gintonin-mediated activation of human I(Ks) channel activity by expressing human I(Ks) channels in Xenopus oocytes. We found that gintonin enhances IKs channel currents in concentration- and voltage-dependent manners. The EC50 for the I(Ks) channel was 0.05 ± 0.01 μg/ml. Gintonin-mediated activation of the I(Ks) channels was blocked by an LPA1/3 receptor antagonist, an active phospholipase C inhibitor, an IP3 receptor antagonist, and the calcium chelator BAPTA. Gintonin-mediated activation of both the I(Ks) channel was also blocked by the calmodulin (CaM) blocker calmidazolium. Mutations in the KCNQ1 [Ca(2+)]i/CaM-binding IQ motif sites (S373P, W392R, or R539W)blocked the action of gintonin on I(Ks) channel. However, gintonin had no effect on hERG K(+) channel activity. These results show that gintonin-mediated enhancement of I(Ks) channel currents is achieved through binding of the [Ca(2+)]i/CaM complex to the C terminus of KCNQ1 subunit.
Fig. 1. Schematic of the human KCNQ1 subunit topology showing amino acidmutations. (A) A sequence alignment of the KCNQ1 channel protein and the amino acid residues that were mutated CaM-binding sites IQ1 (373, 392) and IQ2 (539).
Fig. 2. Effects of gintonin on IKs channel activity. (A) Gintonin concentration-response curves for IKs channels (mean ± S.E.M; n = 7–10 oocytes each). Inset, representative traces of gintonin-mediated IKs (KCNQ1 + KCNE1) channel activation at various gintonin concentrations. The representative peak outward current amplitude at +30 mV from a holding potential of −80 mV was measured in the absence or presence of gintonin. (B) Effects of gintonin (0.1 and 0.3 μg/ml each) on the current–voltage (I–V) relationship of the IKs channels (mean ± S.E.M; n = 10–12 oocytes each). (C) Voltage-dependence activation curves for the IKs channel. Inset, Left and right current traces are before and after application of 0.1 μg/mlgintoninto IKschannels, respectively. Currents recorded during 3 s depolarizing pulses to membrane potentials of −60 to +50 mV, applied from a holding potential of −80 mV. Tail currents were measured at −70 mV. I–V relationships for normalized IKs tail currents. Data were fitted to a Boltzmann function. (D) Attenuation of gintonin-induced IKs channel activity after treatment with Ki16425. The histogram shows blockage of gintonin-mediated IKs channel activation by the LPA1/3 receptor antagonist, Ki16425. Application of 0.1 and 1 μg/ml gintoninto IKs channels, respectively (mean ± S.E.M; n = 10–12 each) (*P < 0.001, compared to gintonin treatment only). Inset, Currents traces recorded in the absence and presence of 1 μM Ki16425 in oocytes expressing IKs channels; currents were recorded with a 3-s voltage step to +30 mV from a holding potential of −80 mV.
Fig. 3. Signal transduction pathways of gintonin-mediated IKs channel activation. (A, B) Representative recordings of IKs (A) channel currents following application of gintonin (GT) for 30 s in the presence of U73122, an active PLC inhibitor. U73343, an inactive PLC inhibitor, in oocytes expressing IKs channels. Inset, the representative peak outward current amplitude at +30 mV from a holding potential of −80 mV was measured in the presence of gintonin. The active or inactive PLC inhibitor was pretreated for 5 min before gintonin application. (C, D) Time-current relationship after application of gintonin (GT) for 30 s in the presence of 2-APB, an IP3 receptor antagonist, or BAPTA-AM, a membrane permeable Ca2+chelator, in oocytes expressing IKs channels. Inset, the representative peak outward current amplitude at +30 mV from a holding potential of −80 mV was measured in the presence of gintonin. The application of 2-APB or BAPTA preceded the gintonin application by 2 h. Summary histograms show the peak outward IKs channel currents (mean ± S.E.M; n = 13-14 oocytes each) recorded in oocytes expressing the IKs channel in the absence or presence of the indicated agents (*P < 0.001, compared to gintonin alone).
Fig. 4. Involvement of CaM in gintonin-mediated IKs channel activation. (A) Oocytes expressing IKs channels were incubated in the absence or presence of calmidazolium (1.5 μM) for 10 min. Insets, the representative gintonin-mediated mediated peak outward current amplitude at +30 mV from a holding potential of −80 mV was measured in the absence or presence of calmidazolium. Summary histograms show peak outward IKs channel currents recorded in the absence or presence of calmidazolium (mean ± S.E.M; n = 13–14 oocytes each; *P < 0.001, compared to gintonin alone). (B) Oocytes expressing IKs channels mutated at the Ca2+/CaM-binding sites (S373P, W392R, or R539W) were treated with gintonin for 60 s. Mutation of Ca2+/CaM-binding sites resulted in a rightward shift of the gintonin concentration-response curve (mean ± S.E.M; n = 10–12 oocytes each). Insets, the representative peak outward current amplitude at +30 mV from a holding potential of −80 mV was measured in the presence of gintonin. Gintonin-mediated peak outward IKs channel currents recorded in oocytes expressing mutant channels were significantly attenuated (mean ± S.E.M; n = 10–12 oocytes each; *P < 0.001, compared to the wild type).
Fig. 5. Effects of gintonin on IhERG, Itail, and slow Ideactivating-tail. (A) Representative current trace showing hERG K+ channel enhancement by gintonin (10 μg/ml). Currents were in response to 4-s voltage steps to 0 mV from a holding potential of −90 mV, followed by repolarization to −60 mV. (B) I–V relationship for hERG K+ currents measured at the end of the 4-s test pulse before and after application of 10 μg/ml gintonin (n = 5). Currents were normalized to the control current at 0 mV for each oocyte. Data are represented as mean ± S.E.M. (n = 7).
Fig. 6. Gintonin increases IKs of guinea pig ventricular myocytes. (A) Time course of changes in the amplitude of the IKs tail during applications of gintonin (3 μg/ml). (B) I–V relationships obtained before and after application of gintonin. Currents were elicited by voltage steps from −30 mV to +70 mV with a subsequent step to −30 mV for the tail current. Representative recordings are shown in the inset. Data are represented as mean ± S.E. (n = 8). *p < 0.05 versus control condition (paired t test)
Fig. 7. Effects of gintonin on ventricular IKs depend on LPA receptor. (A) LPA1/3 antagonist, Ki16425 (10 μM) significantly blocked the activation of IKs by gintonin (3 μg/ml). (B) Summary of the percent activation of IKs. Values are expressed as means ± S.E. **p < 0.01 versus control condition (n = 3). Representative recordings obtained beforeand after gintonin in Ki16425-pretreated cellsare shown in the inset.
Fig. 8. Diagram comparing gintonin- and ginsenoside Rg3-mediated activation of the IKs channel. Gintonin-mediated IKs activation proceeds via Ca2+/CaM binding to IQ motifs via G protein-coupled LPA1 receptors, whereas ginsenoside Rg3 activates IKs through a direct interaction with specific amino acids located at the pore entryway of channel proteins following depolarization (Choi et al., 2010).
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