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Graphical Abstract
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Figure 1
Chemical structure of chanoclavine and ergonovine and the inhibitory effects on Xenopus oocytes expressing human 5-hydroxytryptamine (5-HT)3A receptors. (A). Structure of chanoclavine (EC) and ergonovine (EG). (B). After flowing 5-HT mixed with bath solution for one minute per 2 mL, 5-HT (100 µM) induced a reversible inward current (I5-HT). The traces indicate the inward current in the presence of 5-HT coapplied with ergonovine and chanoclavine at 100 µM. The representative traces from four different frogs elicited at the â80-mV holding potential. (C). The histogram indicates that the percentage of inhibition by chanoclavine was 42.5 ± 9.7 calculated from the mean of the peak inward current. Each value represents the mean ± S.E.M (n = 8â10 from four different frogs)
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Figure 2
The concentration-dependent response of Xenopus oocytes expressing human 5-HT3A receptors to ergot alkaloids. (A). The traces represent increases in the current inhibition with increases in the ergonovine concentration. Treatment with 5-HT (100 µM) alone induced an inward current followed by various response traces with 5-HT (100 µM) and ergonovine (10, 30, 100, and 300 µM). (B). The plot shows the ergonovine inhibition current fitted according to Hillâs equation, where the maximum inhibition (Imax) was 15 ± 5.7%. (C). The traces induced by chanoclavine with 5-HT (100 µM) appear dependent upon the concentration (10, 30, 100, and 300 µM). (D). Average percentage curves of chanoclavine fitted according to Hillâs equation based on the peak of the inward current. The Imax was 83.6 ± 12.6%, and the inhibition was 4 ± 3.8%, 7.9 ± 4.1%, 42.5 ± 5.7%, 61.6 ± 6.1%, and 75 ± 4.5% at 10, 30, 100, 200, and 300 µM, respectively. The oocytes expressing human 5-HT3A mRNA were held at â80 mV, and each value represents the mean ± SEM (n = 8â10 from four different frogs).
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Figure 3
Concentration-dependent inhibition responses of mutants to chanoclavine. (AâD). Y91A, F130A, N138A, and F130A+N138A mutant receptors are rectified at a â80 mV holding potential. The 5-HT3A mutant receptors expressed in Xenopus oocytes elicit reversible currents using an electrode voltage clamp. All oocytes were exposed to 10, 30, 100, and 300 µM chanoclavine coapplied with 100 µM 5-HT after treatment with 5-HT alone. (E). The inhibition percentage curve according to the EC concentration of the 5-HT3A mutant receptors. Each value represents the mean ± SEM. Additional half-maximal inhibitory response concentrations (IC50), Imax, and Hillâs coefficient values of the other mutants are listed in Table 1 (n = 6â8 from four different frogs).
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Figure 4
Voltage dependency and competition inhibition of chanoclavine in I5-HT in oocytes expressing 5-HT3A receptors. (AâD). Currentâvoltage relationship of I5-HT inhibition by chanoclavine in 5-HT3A receptors. Wild-type 5-HT3A receptor traces showed a currentâvoltage relationship with EC and EG at the same concentration as 5-HT (100 µM). All control trace voltage ramps change with only the bath solution flowing without treatment. Traces were obtained using voltage changes from â80 mV to +60 mV over a duration of one sweep per two seconds. BâD Indicate F130A, N138A, and F130A+N138A, respectively. (E). Concentration-response effect of 5-HT in the absence or presence of chanoclavine on oocytes expressing human 5-HT3A receptors. Control (â) represents various 5-HT concentration currents from 1 to 100 µM in the absence of chanoclavine, and 5-HT at 30 µM (â¡) and 100 µM (â³) chanoclavine represents the change in the current compared to the control. Normalized currents were divided by the Imax of the control current and the Imax of 5-HT alone, with 30 µM and 100 µM EC at 99.1 ± 14.2, 94.6 ± 24.4, and 60 ± 2.2, respectively. Oocytes were exposed to 5-HT alone or with EC over a duration of 2 mL per minute and held at a â80 mV membrane holding potential. Each value represents the mean ± SEM (n = 8â10 from four different frogs).
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Figure 5
Molecular docking modeling of chanoclavine to the 5-HT3A receptor. (AâD). Side and top views of the chanoclavine docking model to the 5-HT3A receptor. (E). Binding pocket in the 5-HT3A receptor region of the extracellular domain membrane of the pocket side. Chanoclavine docked to the extracellular region that forms a loop complex in the 5-HT3A receptor. (F). 2D schematic of the predicted binding mode of chanoclavine in the ligand-binding pocket. (G,H). Binding interactions of the ligand and residues in the wild and mutant types. The replaced mutants changed the interaction activity to varying degrees.
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Figure 1. Chemical structure of chanoclavine and ergonovine and the inhibitory effects on Xenopus oocytes expressing human 5-hydroxytryptamine (5-HT)3A receptors. (A). Structure of chanoclavine (EC) and ergonovine (EG). (B). After flowing 5-HT mixed with bath solution for one minute per 2 mL, 5-HT (100 µM) induced a reversible inward current (I5-HT). The traces indicate the inward current in the presence of 5-HT coapplied with ergonovine and chanoclavine at 100 µM. The representative traces from four different frogs elicited at the â80-mV holding potential. (C). The histogram indicates that the percentage of inhibition by chanoclavine was 42.5 ± 9.7 calculated from the mean of the peak inward current. Each value represents the mean ± S.E.M (n = 8â10 from four different frogs).
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Figure 2. The concentration-dependent response of Xenopus oocytes expressing human 5-HT3A receptors to ergot alkaloids. (A). The traces represent increases in the current inhibition with increases in the ergonovine concentration. Treatment with 5-HT (100 µM) alone induced an inward current followed by various response traces with 5-HT (100 µM) and ergonovine (10, 30, 100, and 300 µM). (B). The plot shows the ergonovine inhibition current fitted according to Hillâs equation, where the maximum inhibition (Imax) was 15 ± 5.7%. (C). The traces induced by chanoclavine with 5-HT (100 µM) appear dependent upon the concentration (10, 30, 100, and 300 µM). (D). Average percentage curves of chanoclavine fitted according to Hillâs equation based on the peak of the inward current. The Imax was 83.6 ± 12.6%, and the inhibition was 4 ± 3.8%, 7.9 ± 4.1%, 42.5 ± 5.7%, 61.6 ± 6.1%, and 75 ± 4.5% at 10, 30, 100, 200, and 300 µM, respectively. The oocytes expressing human 5-HT3A mRNA were held at â80 mV, and each value represents the mean ± SEM (n = 8â10 from four different frogs).
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Figure 3. Concentration-dependent inhibition responses of mutants to chanoclavine. (AâD). Y91A, F130A, N138A, and F130A+N138A mutant receptors are rectified at a â80 mV holding potential. The 5-HT3A mutant receptors expressed in Xenopus oocytes elicit reversible currents using an electrode voltage clamp. All oocytes were exposed to 10, 30, 100, and 300 µM chanoclavine coapplied with 100 µM 5-HT after treatment with 5-HT alone. (E). The inhibition percentage curve according to the EC concentration of the 5-HT3A mutant receptors. Each value represents the mean ± SEM. Additional half-maximal inhibitory response concentrations (IC50), Imax, and Hillâs coefficient values of the other mutants are listed in Table 1 (n = 6â8 from four different frogs).
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Figure 4. Voltage dependency and competition inhibition of chanoclavine in I5-HT in oocytes expressing 5-HT3A receptors. (AâD). Currentâvoltage relationship of I5-HT inhibition by chanoclavine in 5-HT3A receptors. Wild-type 5-HT3A receptor traces showed a currentâvoltage relationship with EC and EG at the same concentration as 5-HT (100 µM). All control trace voltage ramps change with only the bath solution flowing without treatment. Traces were obtained using voltage changes from â80 mV to +60 mV over a duration of one sweep per two seconds. BâD Indicate F130A, N138A, and F130A+N138A, respectively. (E). Concentration-response effect of 5-HT in the absence or presence of chanoclavine on oocytes expressing human 5-HT3A receptors. Control (â) represents various 5-HT concentration currents from 1 to 100 µM in the absence of chanoclavine, and 5-HT at 30 µM (â¡) and 100 µM (â³) chanoclavine represents the change in the current compared to the control. Normalized currents were divided by the Imax of the control current and the Imax of 5-HT alone, with 30 µM and 100 µM EC at 99.1 ± 14.2, 94.6 ± 24.4, and 60 ± 2.2, respectively. Oocytes were exposed to 5-HT alone or with EC over a duration of 2 mL per minute and held at a â80 mV membrane holding potential. Each value represents the mean ± SEM (n = 8â10 from four different frogs).
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Figure 5. Molecular docking modeling of chanoclavine to the 5-HT3A receptor. (AâD). Side and top views of the chanoclavine docking model to the 5-HT3A receptor. (E). Binding pocket in the 5-HT3A receptor region of the extracellular domain membrane of the pocket side. Chanoclavine docked to the extracellular region that forms a loop complex in the 5-HT3A receptor. (F). 2D schematic of the predicted binding mode of chanoclavine in the ligand-binding pocket. (G,H). Binding interactions of the ligand and residues in the wild and mutant types. The replaced mutants changed the interaction activity to varying degrees.
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