Bimodal action of menthol on the transient receptor potential channel TRPA1

Abstract

TRPA1 is a calcium-permeable nonselective cation transient receptor potential (TRP) channel that functions as an excitatory ionotropic receptor in nociceptive neurons. TRPA1 is robustly activated by pungent substances in mustard oil, cinnamon, and garlic and mediates the inflammatory actions of environmental irritants and proalgesic agents. Here, we demonstrate a bimodal sensitivity of TRPA1 to menthol, a widely used cooling agent and known activator of the related cold receptor TRPM8. In whole-cell and single-channel recordings of heterologously expressed TRPA1, submicromolar to low-micromolar concentrations of menthol cause channel activation, whereas higher concentrations lead to a reversible channel block. In addition, we provide evidence for TRPA1-mediated menthol responses in mustard oil-sensitive trigeminal ganglion neurons. Our data indicate that TRPA1 is a highly sensitive menthol receptor that very likely contributes to the diverse psychophysical sensations after topical application of menthol to the skin or mucous membranes of the oral and nasal cavities.

Figure 1.

Bimodal modulation of TRPA1 by menthol. A, C, E, Representative time course of the whole-cell currents at −80 (bottom trace) and +80 mV (top trace) during voltage ramps, elicited by 1 mm (A), 30 μm (C), and 1 μm (E) menthol. A 500 ms voltage ramp protocol from −150 to +150 mV from a holding of 0 mV was applied every 2 s. B, D, F, Current–voltage (I–V) relationships obtained at the times indicated in A, C, and E, respectively.

Figure 2.

Effects of menthol on the gating properties of TRPA1. A, Representative whole-cell TRPA1 currents in response to the indicated voltage protocol under control conditions and during application of 10 and 100 μm and 1 mm menthol. B, I–V relationships obtained at the end of the 300 ms voltage steps shown in A. C, Dose–response relationships of menthol on TRPA1 at the indicated voltages. Dotted lines indicate constitutive currents before menthol application. D, Tail currents at −150 mV in control and during application of the different concentrations of menthol (n = 4–7). Continuous lines represent a global fit of the data using Equation 4. Error bars indicate SEM. I_ss, Steady-state current.

Figure 3.

Effect of menthol isomers, menthyl chloride, and thymol on TRPA1. A, Chemical structures of (−)-menthol, (+)-menthol, (−)-neomenthol, (+)-neomenthol, (−)-menthyl chloride, and thymol. B–D, Dose–response relationship of (−)- and (+)-menthol (B), (−)- and (+)-neomenthol and (−)-menthyl chloride (C), and thymol (D) on TRPA1 (n ≥ 4). Error bars indicate SEM.

Figure 4.

Modulation of TRPA1 single-channel properties by menthol. A, Representative cell-attached patch-clamp recordings using a step protocol (top) and different concentrations of menthol. Before washout, 1000 μm menthol was used. B, Corresponding ensemble average currents from 30 to 80 single traces recorded. C, Corresponding all-point amplitude histograms at +80 and −80 mV.

Figure 5.

Kinetics of TRPA1 single channels in dependence of the menthol concentration. A, Dose dependence of the effect of menthol on the fraction of null traces in cell-attached patch-clamp recording, using the step protocol indicated in Figure 4. n.d., No data. B, Dose dependence of the effect of menthol on the TRPA1 burst probability at +80 and −80 mV (n = 5). C, D, Representative example of probability density function (pdf) of open channel dwell time (t_open) at +80 mV in the absence (C) and presence (D) of 100 μm menthol. Black lines represent a biexponential (C) and monoexponential (D) fit to the data. Error bars indicate SEM. *p < 0.05; n = 3–5.

Figure 6.

Menthol responses in mouse trigeminal neurons. A, Ratiometric images of fura-2 AM-loaded neurons in control conditions and during application of 30 μm menthol, 100 μm MO, or 45 mm KCl. B, Individual [Ca²⁺]_i traces from the neurons indicated in A. C, Percentages of MO-sensitive and MO-insensitive neurons that responded to different menthol concentrations. D, Exemplary traces of MO-sensitive cell responses to stimulation with 30 μm and 1 mm menthol normalized to the MO amplitude. Arrow indicates the “off response.” E, Mean maximal amplitudes of menthol responses in MO-sensitive cells normalized to the MO amplitude. Error bars indicate SEM. *p < 0.05; **p < 0.01.

Figure 7.

Menthol responses in mouse trigeminal neurons. A, Individual [Ca²⁺]_i traces from single neurons derived from wild-type animals and TRPA1 knock-out mice. B, Percentages of neurons from WT and TRPA1 knock-out animals that responded to different stimuli, including 100 μm MO, 100 μm menthol, and 1 μm capsaicin (Caps.). C, Comparison of the time course of the response to 100 μm menthol in MO-sensitive and MO-insensitive cells from wild-type and TRPA1^−/− mice. D, The time for the calcium signal to decline to 60% of the maximal menthol response.

Figure 8.

Cross-modulatory effect of menthol and MO on TRPA1. A, Cross-desensitizing effect of 100 μm menthol on MO-activated current. B, I–V relationships obtained at the times indicated in A. C, Inhibition of MO-stimulated trigeminal neurons by 1 mm menthol in calcium imaging measurements (thick line, mean values; thin lines, SEM; n = 4). D, Representative time course of serial application of menthol and MO. Note the opposite effects of 100 μm menthol before and after MO application. E, I–V relationships obtained at the times indicated in D. F, Prior application of 100 μm MO prevents responses of trigeminal neurons to 30 μm menthol (thick line, mean values; thin lines, SEM; n = 11).