Beltrán LR et al. (2013), The pungent substances piperine, capsaicin, 6-g...

XB-ART-47745

Front Pharmacol 2013 Jan 01;4:141. doi: 10.3389/fphar.2013.00141.

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The pungent substances piperine, capsaicin, 6-gingerol and polygodial inhibit the human two-pore domain potassium channels TASK-1, TASK-3 and TRESK.

Beltrán LR , Dawid C , Beltrán M , Gisselmann G , Degenhardt K , Mathie K , Hofmann T , Hatt H .

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For a long time, the focus of trigeminal chemoperception has rested almost exclusively on TRP channels. However, two-pore domain (K2P) potassium channels have recently been identified as targets for substances associated with typical trigeminal sensations, such as numbing and tingling. In addition, they have been shown to be modulated by several TRP agonists. We investigated whether the pungent substances piperine, capsaicin, 6-gingerol and polygodial have an effect on human K2P channels. For this purpose, we evaluated the effects of these pungent substances on both wild-type and mutant K2P channels by means of two-electrode voltage-clamp experiments using Xenopus laevis oocytes. All four pungent substances were found to inhibit the basal activity of TASK-1 (K2P 3.1), TASK-3 (K2P 9.1), and TRESK (K2P 18.1) channels. This inhibitory effect was dose-dependent and, with the exception of polygodial on TASK-1, fully reversible. However, only piperine exhibited an IC50 similar to its reported EC50 on TRP channels. Finally, we observed for TASK-3 that mutating H98 to E markedly decreased the inhibition induced by piperine, capsaicin, and 6-gingerol, but not by polygodial. Our data contribute to the relatively sparse knowledge concerning the pharmacology of K2P channels and also raise the question of whether K2P channels could be involved in the pungency perception of piperine.

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Species referenced: Xenopus laevis
Genes referenced: dtl kcnk10 kcnk18 kcnk3 kcnk9 tff3.7

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	FIGURE 1. Chemical structure of (A) piperine, (B) capsaicin, (C) 6-gingerol and (D) polygodial.
	FIGURE 2. (A) Left (from left to right) absolute currents presented by: uninjected Xenopus oocytes, oocytes injected with cRNA coding for hTREK-1, hTASK-1, hTRAAK, hTASK-3, hTREK-2 and hTRESK, before and after the application of 300 Î¼M piperine. The currents were registered at the final 50 ms of the +50 mV constant (see methods for a description of the ramp protocol used). Note that the amount of current carried by uninjected oocytes is negligible in comparison to oocytes expressing a K2P channel. Right. Normalized currents showing the effect of 300 Î¼M piperine on Xenopus oocytes injected with cRNA coding for hTREK-1, hTASK-1, hTREK-1, hTASK-3, hTREK-2 and hTRESK. For each group, the current was normalized to the current registered prior to the application of 300 Î¼M piperine (dotted line). All data are expressed as the mean +/- SEM. N of 3â6 for injected oocytes, N of 10 for uninjected oocytes. P < 0.05, P < 0.01, **P < 0.001. (B) Representative voltage clamp recordings of Xenopus laevis oocytes expressing hTASK-1 (top), hTASK-3 (middle) and hTRESK (bottom), before, during and after the application of 100 Î¼M piperine. In each case, piperine induced a fully reversible inhibition; for TASK-1, however, due to the prolonged recovery time, 40 ramps were not included. Dotted line represents zero current. (C) Dose-response curves for piperine on hTASK-1 (red circles), hTASK-3 (orange circles) and hTRESK (green circles). The IC50 values are listed in the text and in Table 1. N of 3-6 oocytes. For each measurement, the current was normalized to the current registered prior to the application of piperine. (D) hTASK-1 currents recorded before (gray bars) and after (green bars) the application of 300 Î¼M piperine. The currents were elicited by voltage pulses from -140 to +80 mV in 20-mV steps, 500 ms duration from a holding potential of -80 mV. Values were obtained during the last 50 ms of the voltage pulses. N of 6 oocytes. (E) Left: representative voltage clamp recording of a Xenopus oocyte expressing hTASK-1 repetitively exposed to 30 Î¼M piperine. Right: current clamp recording of a Xenopus oocyte expressing hTASK-1 exposed to 100 Î¼M piperine, which led to a membrane depolarization of approximately 15 mV.
	FIGURE 3. (A) Left (from left to right) absolute currents presented by: uninjected Xenopus oocytes, oocytes injected with cRNA coding for hTREK-1, hTASK-1, hTRAAK, hTASK-3, hTREK-2 and hTRESK, before and after the application of 100 Î¼M capsaicin. Right: normalized currents showing the effect of 100 Î¼M capsaicin on Xenopus oocytes injected with cRNA coding for hTREK-1, hTASK-1, hTRAAK, hTASK-3, hTREK-2 and hTRESK. For each group, the current was normalized to the current registered prior to the application of 100 Î¼M capsaicin (dotted line). All data are expressed as the mean +/- SEM. N of 3â6 oocytes. P < 0.05, *P < 0.01. (B) Representative voltage clamp recordings of Xenopus laevis oocytes expressing TASK-1 (top), TASK-3 (middle) and TRESK (bottom), before, during and after the application of 100 Î¼M capsaicin, showing in every case a fully reversible inhibition. Dotted line represents zero current. (C) Dose-response curves for capsaicin on TASK-1 (red circles), TASK-3 (orange circles) and TRESK (green circles). The IC50 values are listed in the text and in Table 1. N of 3â6 oocytes. For each measurement, the current was normalized to the current registered prior to the application of capsaicin. (D) hTASK-3 currents recorded before and after the application of 100 Î¼M capsaicin. The currents were elicited by voltage pulses from -140 to +80 mV in 20 mV steps, 500 ms duration from a holding potential of -80 mV. Values were obtained during the last 50 ms of the voltage pulses. N of 6 oocytes. (E) Representative voltage clamp recording of a Xenopus oocyte expressing TASK-3 repetitively exposed to 30 Î¼M capsaicin. (F) Current clamp recording of a Xenopus oocyte expressing TASK-3 exposed to 100 Î¼M capsaicin, which led to a membrane depolarization of approximately 30 mV.
	FIGURE 4. (A) Left (from left to right) absolute currents presented by: uninjected Xenopus oocytes, oocytes injected with cRNA coding for hTREK-1, hTASK-1, hTRAAK, hTASK-3, hTREK-2 and hTRESK, before and after the application of 300 Î¼M 6-gingerol. Right normalized currents showing the effect of 300 Î¼M 6-gingerol on Xenopus oocytes injected with cRNA coding for hTREK-1, hTASK-1, hTRAAK, hTASK-3, hTREK-2 and hTRESK. For each group, the current was normalized to the current registered prior to the application of 300 Î¼M 6-gingerol (dotted line). All data are expressed as the mean +/- SEM. N of 9â18 oocytes. P < 0.05, P < 0.01, **P < 0.001. (B) Representative voltage clamp recordings of Xenopus laevis oocytes expressing TASK-1 (top), TASK-3 (middle) and TRESK (bottom), before, during and after the application of 300 Î¼M 6-gingerol, showing fully reversible inhibition in all cases. Dotted line represents zero current. (C) Dose-response curves for 6-gingerol on TASK-1 (red circles), TASK-3 (orange circles) and TRESK (green circles). N of 3â19 oocytes. For each measurement, the current was normalized to the current registered prior to the application of 6-gingerol.
	FIGURE 5. (A) Left (from left to right) absolute currents presented by: uninjected Xenopus oocytes, oocytes injected with cRNA coding for hTREK-1, hTASK-1, hTRAAK, hTASK-3, hTREK-2 and hTRESK, before and after the application of 1 mM polygodial. Right. Normalized currents showing the effect of 1 mM polygodial on Xenopus oocytes injected with cRNA coding for hTREK-1, hTASK-1, hTRAAK, hTASK-3, hTREK-2 and hTRESK. For each group, the current was normalized to the current registered prior to the application of 1 mM polygodial (dotted line). All data are expressed as the mean +/- SEM. N of 3 to 6 oocytes. *P < 0.05. (B) Representative voltage clamp recordings of Xenopus laevis oocytes expressing TASK-1 (top), TASK-3 (middle) and TRESK (bottom), before, during and after the application of 1 mM polygodial, showing a partially reversible inhibition for TASK-1 and TASK-3. Dotted line represents zero current. (C) Dose-response curves for polygodial on TASK-1 (red circles) and TASK-3 (orange circles). The IC50 values are listed in the text and in Table 1. N of 3â5 oocytes. For each measurement, the current was normalized to the current registered prior to the application of polygodial.
	FIGURE 6. (A) Human TASK-3 membrane topology diagram. Four transmembrane segments (M1 to M4) and two pore regions (P1 and P2) are shown. Mutated amino acids are indicated either with a star or as the replaced sequence. (B) Comparative effects of pH 5.0, 100 Î¼M capsaicin, 300 Î¼M piperine, 1 mM 6-gingerol and 1 mM polygodial on wild-type human TASK-3 (gray bars) and the H98E mutant (green bars). N of 4â6 oocytes. P < 0.05, P < 0.01, **P < 0.001. (C) Comparative effects of the four pungent substances under evaluation in wild-type hTASK-3 (gray bars) vs. TREK-2 chimera (light blue bars). N of 4-5 oocytes. (D) Comparative effects of pH 5.0, 100 Î¼M capsaicin, 300 Î¼M piperine, 1 mM 6-gingerol and 1 mM polygodial on wild-type human TASK-3 (gray bars) and the E30C mutant (purple bars). N of 4â6 oocytes. (E) Representative voltage clamp recordings of a Xenopus laevis oocyte expressing TASK-3 WT exposed to a pH of 6.5 and then to 300 Î¼M piperine.
	FIGURE 7. Dose response relationships for the inhibition induced by pungent substances on TASK-3 WT (gray bars) vs. H98E (green bars) for (A) piperine, N of 5â12 oocytes; (B) capsaicin, N of 5â10 oocytes; (C) 6-gingerol, N of 3â19 oocytes and (D) polygodial N of 3â9 oocytes. P < 0.05, P < 0.01, **P < 0.001.

References [+] :

André, Pharmacological characterisation of the plant sesquiterpenes polygodial and drimanial as vanilloid receptor agonists. 2006, Pubmed

	FIGURE 1. Chemical structure of (A) piperine, (B) capsaicin, (C) 6-gingerol and (D) polygodial.
	FIGURE 2. (A) Left (from left to right) absolute currents presented by: uninjected Xenopus oocytes, oocytes injected with cRNA coding for hTREK-1, hTASK-1, hTRAAK, hTASK-3, hTREK-2 and hTRESK, before and after the application of 300 Î¼M piperine. The currents were registered at the final 50 ms of the +50 mV constant (see methods for a description of the ramp protocol used). Note that the amount of current carried by uninjected oocytes is negligible in comparison to oocytes expressing a K2P channel. Right. Normalized currents showing the effect of 300 Î¼M piperine on Xenopus oocytes injected with cRNA coding for hTREK-1, hTASK-1, hTREK-1, hTASK-3, hTREK-2 and hTRESK. For each group, the current was normalized to the current registered prior to the application of 300 Î¼M piperine (dotted line). All data are expressed as the mean +/- SEM. N of 3â6 for injected oocytes, N of 10 for uninjected oocytes. P < 0.05, P < 0.01, **P < 0.001. (B) Representative voltage clamp recordings of Xenopus laevis oocytes expressing hTASK-1 (top), hTASK-3 (middle) and hTRESK (bottom), before, during and after the application of 100 Î¼M piperine. In each case, piperine induced a fully reversible inhibition; for TASK-1, however, due to the prolonged recovery time, 40 ramps were not included. Dotted line represents zero current. (C) Dose-response curves for piperine on hTASK-1 (red circles), hTASK-3 (orange circles) and hTRESK (green circles). The IC50 values are listed in the text and in Table 1. N of 3-6 oocytes. For each measurement, the current was normalized to the current registered prior to the application of piperine. (D) hTASK-1 currents recorded before (gray bars) and after (green bars) the application of 300 Î¼M piperine. The currents were elicited by voltage pulses from -140 to +80 mV in 20-mV steps, 500 ms duration from a holding potential of -80 mV. Values were obtained during the last 50 ms of the voltage pulses. N of 6 oocytes. (E) Left: representative voltage clamp recording of a Xenopus oocyte expressing hTASK-1 repetitively exposed to 30 Î¼M piperine. Right: current clamp recording of a Xenopus oocyte expressing hTASK-1 exposed to 100 Î¼M piperine, which led to a membrane depolarization of approximately 15 mV.
	FIGURE 3. (A) Left (from left to right) absolute currents presented by: uninjected Xenopus oocytes, oocytes injected with cRNA coding for hTREK-1, hTASK-1, hTRAAK, hTASK-3, hTREK-2 and hTRESK, before and after the application of 100 Î¼M capsaicin. Right: normalized currents showing the effect of 100 Î¼M capsaicin on Xenopus oocytes injected with cRNA coding for hTREK-1, hTASK-1, hTRAAK, hTASK-3, hTREK-2 and hTRESK. For each group, the current was normalized to the current registered prior to the application of 100 Î¼M capsaicin (dotted line). All data are expressed as the mean +/- SEM. N of 3â6 oocytes. P < 0.05, *P < 0.01. (B) Representative voltage clamp recordings of Xenopus laevis oocytes expressing TASK-1 (top), TASK-3 (middle) and TRESK (bottom), before, during and after the application of 100 Î¼M capsaicin, showing in every case a fully reversible inhibition. Dotted line represents zero current. (C) Dose-response curves for capsaicin on TASK-1 (red circles), TASK-3 (orange circles) and TRESK (green circles). The IC50 values are listed in the text and in Table 1. N of 3â6 oocytes. For each measurement, the current was normalized to the current registered prior to the application of capsaicin. (D) hTASK-3 currents recorded before and after the application of 100 Î¼M capsaicin. The currents were elicited by voltage pulses from -140 to +80 mV in 20 mV steps, 500 ms duration from a holding potential of -80 mV. Values were obtained during the last 50 ms of the voltage pulses. N of 6 oocytes. (E) Representative voltage clamp recording of a Xenopus oocyte expressing TASK-3 repetitively exposed to 30 Î¼M capsaicin. (F) Current clamp recording of a Xenopus oocyte expressing TASK-3 exposed to 100 Î¼M capsaicin, which led to a membrane depolarization of approximately 30 mV.
	FIGURE 4. (A) Left (from left to right) absolute currents presented by: uninjected Xenopus oocytes, oocytes injected with cRNA coding for hTREK-1, hTASK-1, hTRAAK, hTASK-3, hTREK-2 and hTRESK, before and after the application of 300 Î¼M 6-gingerol. Right normalized currents showing the effect of 300 Î¼M 6-gingerol on Xenopus oocytes injected with cRNA coding for hTREK-1, hTASK-1, hTRAAK, hTASK-3, hTREK-2 and hTRESK. For each group, the current was normalized to the current registered prior to the application of 300 Î¼M 6-gingerol (dotted line). All data are expressed as the mean +/- SEM. N of 9â18 oocytes. P < 0.05, P < 0.01, **P < 0.001. (B) Representative voltage clamp recordings of Xenopus laevis oocytes expressing TASK-1 (top), TASK-3 (middle) and TRESK (bottom), before, during and after the application of 300 Î¼M 6-gingerol, showing fully reversible inhibition in all cases. Dotted line represents zero current. (C) Dose-response curves for 6-gingerol on TASK-1 (red circles), TASK-3 (orange circles) and TRESK (green circles). N of 3â19 oocytes. For each measurement, the current was normalized to the current registered prior to the application of 6-gingerol.
	FIGURE 5. (A) Left (from left to right) absolute currents presented by: uninjected Xenopus oocytes, oocytes injected with cRNA coding for hTREK-1, hTASK-1, hTRAAK, hTASK-3, hTREK-2 and hTRESK, before and after the application of 1 mM polygodial. Right. Normalized currents showing the effect of 1 mM polygodial on Xenopus oocytes injected with cRNA coding for hTREK-1, hTASK-1, hTRAAK, hTASK-3, hTREK-2 and hTRESK. For each group, the current was normalized to the current registered prior to the application of 1 mM polygodial (dotted line). All data are expressed as the mean +/- SEM. N of 3 to 6 oocytes. *P < 0.05. (B) Representative voltage clamp recordings of Xenopus laevis oocytes expressing TASK-1 (top), TASK-3 (middle) and TRESK (bottom), before, during and after the application of 1 mM polygodial, showing a partially reversible inhibition for TASK-1 and TASK-3. Dotted line represents zero current. (C) Dose-response curves for polygodial on TASK-1 (red circles) and TASK-3 (orange circles). The IC50 values are listed in the text and in Table 1. N of 3â5 oocytes. For each measurement, the current was normalized to the current registered prior to the application of polygodial.
	FIGURE 6. (A) Human TASK-3 membrane topology diagram. Four transmembrane segments (M1 to M4) and two pore regions (P1 and P2) are shown. Mutated amino acids are indicated either with a star or as the replaced sequence. (B) Comparative effects of pH 5.0, 100 Î¼M capsaicin, 300 Î¼M piperine, 1 mM 6-gingerol and 1 mM polygodial on wild-type human TASK-3 (gray bars) and the H98E mutant (green bars). N of 4â6 oocytes. P < 0.05, P < 0.01, **P < 0.001. (C) Comparative effects of the four pungent substances under evaluation in wild-type hTASK-3 (gray bars) vs. TREK-2 chimera (light blue bars). N of 4-5 oocytes. (D) Comparative effects of pH 5.0, 100 Î¼M capsaicin, 300 Î¼M piperine, 1 mM 6-gingerol and 1 mM polygodial on wild-type human TASK-3 (gray bars) and the E30C mutant (purple bars). N of 4â6 oocytes. (E) Representative voltage clamp recordings of a Xenopus laevis oocyte expressing TASK-3 WT exposed to a pH of 6.5 and then to 300 Î¼M piperine.
	FIGURE 7. Dose response relationships for the inhibition induced by pungent substances on TASK-3 WT (gray bars) vs. H98E (green bars) for (A) piperine, N of 5â12 oocytes; (B) capsaicin, N of 5â10 oocytes; (C) 6-gingerol, N of 3â19 oocytes and (D) polygodial N of 3â9 oocytes. P < 0.05, P < 0.01, **P < 0.001.