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Figure 1. Effects of Ech A on ITRPV3. (a,c) An original trace of the membrane current density (pA/pF) demonstrating the activation of TRPV3 by 100 μM 2-APB and its inhibition by 74a (a) or by Ech A (c). The vertical lines indicate the current density obtained by ramp-like pulses from −100 to 100 mV (holding voltage, −10 mV) applied with an interval of 20 s. The I/V curves are recorded at the points indicated by the numbers in parentheses (b,d). (e) A summary of the inward current densities (pA/pF at −100 mV) activated by 2-APB and with Ech A or 74a (n = 6). (f) A semi-logarithmic plot of the normalized current according to the incremental concentrations of Ech A, fit with a logistic function (IC50, 2.11 ± 1.055 μM, n = 6). The inward current at −100 mV was normalized to the maximum ITRPV3 (the peak inward current subtracted with the residual inward current in the presence of 74a) in each cell.
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Figure 2. Inhibitory effects of Ech A on IOrai1. (a,c) Representative traces of membrane current density (pA/pF) with responses to the repetitive ramp-like voltage clamp pulses from −130 to 50 mV, 0.2 V/s (vertical lines), with a CsCl pipette solution containing 20 μM of InsP3 and 20 mM of BAPTA. After making a whole-cell configuration, the spontaneous activation of IOrai1 was observed, and the concentration-dependent inhibition of IOrai1 by Ech A (c) or by 5 μM of GdCl3 (a,c) was confirmed. (b,d) The I/V curves recorded at the points are indicated by the numbers in parentheses. (e) A summary of the inward current densities (pA/pF) measured at −130 mV in the control and with the incremental concentrations of Ech A (n = 6). (f) A semi-logarithmic plot of the normalized current according to the concentrations of Ech A and fit with a logistic function (IC50, 2.41 ± 1.554 μM, n = 6). The inward current at −130 mV was normalized to the maximum IOrai1 (the peak inward current subtracted with the residual inward current with GdCl3) in each cell.
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Figure 3. Effects of Ech A on ITRPV1. (a) An original trace of the membrane current density (pA/pF) recorded at the holding voltage of −60 mV, demonstrating the activation by capsaicin (1 μM) and the additional increase by the combined application of Ech A (30 μM). The total inhibition by BCTC (1 μM) was confirmed at the end of experiment. (b) A summary of the inward current density of the control (NT), ITRPV1 activated by capsaicin, further increased by Ech A, and inhibition by BCTC (n = 5). Data represent the mean ± SEM. * p < 0.05; *** p < 0.001; **** p < 0.0001.
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Figure 4. Facilitating effects of Ech A on ITREK1 and ITREK2 with 2-APB. (a,c) Representative traces of the membrane current density (pA/pF) recorded with a KCl pipette solution in TREK-1- (a) and TREK-2- (c) overexpressed HEK 293 cells. I/V curves were obtained by ramp-like depolarizing pulses from−100 to 20 mV (b,d). (e,f) A summary of the outward current densities (pA/pF, left vertical axis) measured at 0 mV in the control, 2-APB (100 μM), Ech A (30 μM), and 2-APB+ Ech A (closed bars). The normalized current (I/Imax at 0 mV, right axis) against the peak amplitude induced by the combined application of 2-APB and Ech A is also shown (open bars). n = 6 for TREK-1 (e) and n = 7 for TREK-2 (f). Data represent the mean ± SEM. * p < 0.05; **** p < 0.0001; ns, statistically not significant.
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Figure 5. Facilitating effects of Ech A on ITREK2 and ITRAAK with AA. (a,c) A representative traces of the membrane current density (pA/pF) in TREK-2- (a) and TRAAK- (c) overexpressed HEK 293 cells. I/V curves were obtained by ramp-like depolarizing pulses from −100 to 20 mV (b,d). (e,f) A summary of the outward current densities (pA/pF, left vertical axis) measured at 0 mV in the control, AA (10 μM), Ech A (30 μM), and AA + Ech A (closed bars). The normalized current (I/Imax at 0 mV, right axis) against the peak amplitude induced by the combined application of 2-APB and Ech A is also shown (open bars). n = 5 for TREK-2 (e) and n = 5 for TRAAK (f). Data represent the mean ± SEM. * p < 0.05; **** p < 0.0001.
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Figure 6. Facilitating effects of Ech A on ITREK2 activated by acidic pHe. (a) A representative trace of the membrane current in TREK-2-overexpressed HEK 293 cells. (a) I/V curves were obtained by ramp-like depolarizing pulses from −100 to 20 mV (b). (c) A summary of the outward current densities (pA/pF, left vertical axis) measured at 0 mV in the control, pHe 5.5, and Ech A (30 μM) combined with pHe 5.5 (closed bars). The normalized current (I/Imax at 0 mV, right axis) against the peak amplitude induced by the combined application of pHe 5.5 and Ech A is also shown (open bars, n = 4). Data represent the mean ± SEM. ** p < 0.01.
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Figure 7. Disappearance of the inhibitory effect of NFx on the ITREK2 facilitated by Ech A. (a,b) A representative current trace and I/V curve of ITREK2 activated by 100 μM 2-APB alone, which was almost completely inhibited by 10 μM of NFx. (c,d) A representative current trace and I/V curve of ITREK2 activated by 2-APB and 30 μM of Ech A, which was not inhibited by 10 μM of NFx. The I/V curves were obtained by ramp-like depolarizing pulses from −100 to 20 mV. (e,f) A summary of the outward current densities (pA/pF, left vertical axis) measured at 0 mV in the control, 2-APB, and NFx (closed bars). A summary of the normalized current (I/Imax at 0 mV, right axis) against the peak amplitude is also shown (open bars). The inhibition by 50 μM RR is also shown in the panel (f). n = 5 for (e) and n = 4 for (f). Data represent the mean ± SEM. ** p < 0.01; **** p < 0.0001; ns, not statistically significant.
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Figure 8. Effects of Ech A on ITASK1 and ITRESK. (a,b) A representative current trace and I/V curve of ITASK1 activated by an alkaline pHe of 8.5 and the partial inhibitory effect of Ech A. (c,d) A representative current trace and I/V curve of ITRESK. The application of up to 30 μM of Ech A had no significant effect. The I/V curves were obtained by ramp-like depolarizing pulses from −120 to 50 mV (vertical lines in (a)) and from −100 to 20 mV (vertical lines in (c)). (e,f) A summary of the outward current densities (pA/pF, left vertical axis) measured at 0 mV (closed bars). A summary of the normalized current (I/Imax at 0 mV, right axis) against the peak amplitude is also shown (open bars). n = 9 for TASK-1 (e) and n = 4 for TRESK (f). Data represent the mean ± SEM. * p < 0.05; *** p < 0.001.
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