Structural basis for promiscuous action of monoterpenes on TRP channels

Abstract

Monoterpenes are major constituents of plant-derived essential oils and have long been widely used for therapeutic and cosmetic applications. The monoterpenes menthol and camphor are agonists or antagonists for several TRP channels such as TRPM8, TRPV1, TRPV3 and TRPA1. However, which regions within TRPV1 and TRPV3 confer sensitivity to monoterpenes or other synthesized chemicals such as 2-APB are unclear. In this study we identified conserved arginine and glycine residues in the linker between S4 and S5 that are related to the action of these chemicals and validated these findings in molecular dynamics simulations. The involvement of these amino acids differed between TRPV3 and TRPV1 for chemical-induced and heat-evoked activation. These findings provide the basis for characterization of physiological function and biophysical properties of ion channels.

Fig. 1. Effect of TRPV3-G573S mutation on the agonistic action of menthol, camphor, and 2-APB.

a Location of Y448 (S1), R567, and G573S (S4–S5 linker) in mouse TRPV3 (mTRPV3). b Representative current traces for HEK293T cells expressing WT or mTRPV3-G573S in response to menthol (3 mM) followed by heat stimulation. c Comparison of menthol-activated current densities in HEK293T cells expressing WT or mTRPV3-G573S at ±60 mV (WT: 86.4 ± 12.7 pA/pF at +60 and 26.2 ± 4.3 pA/pF at −60 mV, n = 16; G573S: 14.0 ± 1.9 pA/pF at +60 mV and 6.9 ± 1.5 pA/pF at −60mV, n = 5). d Structure of camphor. e Representative current traces for WT or mTRPV3-G573S in response to camphor (10 mM) followed by heat stimulation. f Comparison of camphor-activated current densities in HEK293T cells expressing WT or mTRPV3-G573S at ±60 mV (WT: 466.0 ± 43.5 pA/pF at +60 mV and 410.8 ± 29.6 pA/pF at −60mV, n = 26; G573S: 18.6 ± 6.7 pA/pF at +60 mV and 9.6 ± 3.9 pA/pF, n = 5). g Structure of 2-APB. h Representative current traces for WT or mTRPV3-G573S in response to 2-APB (300 μM) followed by heat stimulation. i Comparison of 2-APB-activated current densities in HEK293T cells expressing WT or mTRPV3-G573S at ±60 mV (WT: 361.5 ± 48.8 pA/pF at +60 mV and 187.7 ± 25.3 pA/pF at −60mV, n = 12; G573S: 10.7 ± 1.5 pA/pF at +60 mV and 5.1 ± 1.0 pA/pF, n = 5). Insets in b, e, h indicate current–voltage curves obtained at the different time points (1, 2, 3) shown in each trace. Holding potentials were −60 mV with ramp-pulses (−100 to +100 mV, 300 ms) applied every 3 s. Data represent means ± SEM. Statistical analysis was performed by two-sample t-test, ***p < 0.001.

Fig. 2. Agonistic effect of menthol, but not camphor, involves R567 in mouse TRPV3.

a Representative current traces for HEK293T cells expressing wild type (WT) or R567K mutant stimulated with menthol (3 mM) followed by camphor (10 mM). b Comparison of the normalized current densities (I_menthol/I_camphor) at ±60 mV (WT: 0.16 ± 0.03 at +60 mV and 0.08 ± 0.02 at −60 mV, n = 13; R567K: 0.07 ± 0.02 at +60 mV and 0.02 ± 0.01 at −60mV, n = 11). c Comparison of camphor-activated current densities for HEK293T cells expressing WT or mTRPV3-R567K at ±60 mV (WT: 736.5 ± 198.8 pA/pF at +60 mV and 633.5 ± 153.6 pA/pF at −60 mV, n = 6; R567K: 634.9 ± 68.2 pA/pF at +60 mV and 613.6 ± 35.7 pA/pF at −60 mV, n = 9). Holding potentials were −60 mV with ramp-pulses (−100 to +100 mV, 300 ms) every 3 s. Data represent means ± SEM. Statistical analysis was performed by two-sample t-test, *p < 0.05.

Fig. 3. Effect of rat TRPV1 mutations G563S and R557K on the agonistic action of menthol.

a Location of residues Y441 (S1), R557 (bottom of S4), and G563 (S4–S5 linker) in rat TRPV1 (rTRPV1). b Representative current traces for HEK293T cells expressing WT, G563S, or R557K rTRPV1 in response to menthol (3 mM) followed by capsaicin (CAP, 1 μM) stimulation. c Comparison of normalized current densities (I_menthol/I_CAP) for WT, rTRPV1-G563S, or rTRPV1-R557K expressed by HEK293T cells at +60 mV (WT: 0.08 ± 0.01, n = 11; G563S: 0.03 ± 0.003, n = 10; R557K: 0.01 ± 0.002, n = 9). d Comparison of CAP-activated currents densities for WT, rTRPV1-G563S, or rTRPV1-R557K expressed in HEK293T cells at ±60 mV (WT: 547 ± 26.7 pA/pF at +60 mV and 442 ± 29.2 pA/pF at −60 mV, n = 22; G563S: 521 ± 3.7 pA/pF at +60 mV and 388.1 ± 26.4 pA/pF at −60 mV, n = 14; R557K: 633.5 ± 3.3 pA/pF at +60 mV and 476.4 ± 33.8 pA/pF at −60 mV, n = 11). Holding potentials were −60 mV with ramp-pulses (−100 to +100 mV, 300 ms) applied every 3 s. Data represent means ± SEM. Statistical analysis was performed using two-sample t-test, ***p < 0.001.

Fig. 4. Effect of rat TRPV1 mutations G563S and R557K on agonistic action of camphor and 2-APB.

a Representative current traces for HEK293T cells expressing WT, rTRPV1-G563S, or rTRPV1-R557K stimulated with camphor (10 mM) followed by CAP (1 μM). b Representative current traces for HEK293T cells expressing WT, rTRPV1-G563S, or rTRPV1-R557K stimulated with 2-APB (300 μM) followed by CAP (1 μM). c Comparison of the normalized current densities (I_camphor/I_CAP) for WT, rTRPV1-G563S, or rTRPV1-R557K expressed in HEK293T cells at ±60 mV (I_camphor/I_CAP for WT: 0.63 ± 0.07 at +60 mV and 0.28 ± 0.06 at −60 mV, n = 14; G563S: 0.03 ± 0.01 at +60 mV and 0.02 ± 0.01 at −60 mV, n = 8; R557K: 0.03 ± 0.01 at +60 mV and 0.02 ± 0.01 at −60 mV, n = 5). d Comparison of normalized current densities (I_2-APB/I_CAP) in HEK293T cells expressing WT, rTRPV1-G563S, or rTRPV1-R557K at ±60 mV (I_2-APB/I_CAP for WT: 0.84 ± 0.06 at +60 mV and 0.91 ± 0.08 at −60 mV, n = 13; G563S: 0.03 ± 0.004 at +60 mV and 0.02 ± 0.004 at −60 mV, n = 8; R557K: 0.02 ± 0.001 at +60 mV and 0.02 ± 0.002 at −60mV, n = 5). Holding potentials were −60 mV with ramp-pulses (−100 to +100 mV, 300 ms) applied every 3 s. Data represent means ± SEM. Statistical analysis was performed using two-sample t-test, ***p < 0.001.

Fig. 5. Positive charge of R567 is essential for camphor-evoked activation of mouse TRPV3.

a Representative current traces for HEK293T cells expressing WT, mTRPV3-R567A, or mTRPV3-R567F in response to 2-APB (300 μM) treatment followed by camphor (10 mM) stimulation. b Comparison of normalized current densities (I_camphor/I_2-APB) in HEK293T cells expressing WT, mTRPV3-R567K, mTRPV3-R567H, mTRPV3-R567A, or mTRPV3-R567F at ±60 mV (WT: 1.93 ± 0.25 at +60 mV and 2.79 ± 0.48 at −60 mV, n = 16; R567K: 1.82 ± 0.22 at +60 mV and 1.95 ± 0.34 at −60 mV, n = 11; R567H: 1.77 ± 0.17 at +60 mV and 1.92 ± 0.28 at −60 mV, n = 12; R567A: 0.21 ± 0.02 at +60 mV and 0.25 ± 0.03 at −60 mV, n = 9; R567F: 0.13 ± 0.01 at −60 mV and 0.18 ± 0.03 at −60 mV, n = 12). Holding potentials were −60 mV with ramp-pulses (−100 to +100 mV, 300 ms) applied every 3 s. Data represent means ± SEM. Statistical analysis was performed by one-way ANOVA, *p < 0.05 (R567A vs. R567K, R567H at −60 mV), **p < 0.01 (R567F vs. R567K, R567H at −60 mV) and ***p < 0.001 (R567A vs. WT, R567K, R567H at +60 mV; R567F vs. WT, R567K, R567H at +60 mV; R567A vs. WT at −60 mV; R567F vs. WT at −60 mV).

Fig. 6. Heat-evoked currents for WT and mutated mouse TRPV3 and rat TRPV1.

a Representative current (upper) and temperature (lower) traces for HEK293T cells expressing WT, mTRPV3-G573S, or mTRPV3-R567K exposed to heat up to 53 °C. b Comparison of heat-evoked current densities (left) (WT: 183.5 ± 26.1 pA/pF; G573S: 183.6 ± 49.5 pA/pF; R567K: 211.9 ± 44.1 pA/pF), temperature thresholds (middle) (WT: 50.3 ± 0.6 °C, n = 14; G573S: 50.2 ± 1.5 °C, n = 5; R567K: 51.2 ± 0.4 °C), n = 5, and time to current peak (WT: 58.2 ± 5.2 s, n = 14; G573S: 44.2 ± 9.4 s, n = 5; R567K: 28 ± 2.8 s, n = 5) in HEK293T cells expressing WT, mTRPV3-G573S, or mTRPV3-R567K at −60 mV. c Representative current (upper) and temperature (lower) traces for HEK293T cells expressing WT, rTRPV1-G563S, or rTRPV1-R557K exposed to heat up to 45 °C. d Comparison of heat-evoked current densities for HEK293T cells expressing WT, rTRPV1-G563S, or rTRPV1-R557K at −60 mV. Holding potentials were −60 mV (WT: 40.8 ± 10.1 pA/pF, n = 5; G563S: 6.5 ± 0.6 pA/pF, n = 6; R557K: 4.5 ± 0.6 pA/pF, n = 6). Data represent means ± SEM. Statistical analysis was performed by one-way ANOVA, *p < 0.05, and **p < 0.01.

Fig. 7. Molecular dynamics simulation confirming a stable bond between monoterpenes and R567.

Representative snapshots of a the whole WT TRPV3 system, b menthol in WT TRPV3, c camphor in WT TRPV3, d menthol in mTRPV3-G573/R567F, e camphor in mTRPV3-G573/R567F, f menthol and mTRPV3-G573S/R567, g camphor and mTRPV3-G573S/R567. Black frame in a represents the area we focus in b–g. Pink highlights indicate hydrogen bonds between monoterpenes and R567. Time series of distance between h menthol and R567/F567 and i camphor and R567/F567. Time series of distance between j menthol and G573/S573 and k camphor and G573/S573.