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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 = 7â10 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 â80 mV was measured in the absence or presence of gintonin. (B) Effects of gintonin (0.1 and 0.3 μg/ml each) on the currentâvoltage (IâV) relationship of the IKs channels (mean ± S.E.M; n = 10â12 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 â60 to +50 mV, applied from a holding potential of â80 mV. Tail currents were measured at â70 mV. IâV 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 = 10â12 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 â80 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 â80 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 = 13â14 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 = 10â12 oocytes each). Insets, the representative peak outward current amplitude at +30 mV from a holding potential of â80 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 = 10â12 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 â90 mV, followed by repolarization to â60 mV. (B) IâV 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) IâV relationships obtained before and after application of gintonin. Currents were elicited by voltage steps from â30 mV to +70 mV with a subsequent step to â30 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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