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. 2016 Jun 28:6:28763.
doi: 10.1038/srep28763.

Human TRPA1 is a heat sensor displaying intrinsic U-shaped thermosensitivity

Lavanya Moparthi  1 , Tatjana I Kichko  2 , Mirjam Eberhardt  3 , Edward D Högestätt  4 , Per Kjellbom  1 , Urban Johanson  1 , Peter W Reeh  2 , Andreas Leffler  3 , Milos R Filipovic  5   6 , Peter M Zygmunt  4
Affiliations

Human TRPA1 is a heat sensor displaying intrinsic U-shaped thermosensitivity

Lavanya Moparthi et al. Sci Rep. .

Abstract

Thermosensitive Transient Receptor Potential (TRP) channels are believed to respond to either cold or heat. In the case of TRP subtype A1 (TRPA1), there seems to be a species-dependent divergence in temperature sensation as non-mammalian TRPA1 is heat-sensitive whereas mammalian TRPA1 is sensitive to cold. It has been speculated but never experimentally proven that TRPA1 and other temperature-sensitive ion channels have the inherent capability of responding to both cold and heat. Here we show that redox modification and ligands affect human TRPA1 (hTRPA1) cold and heat sensing properties in lipid bilayer and whole-cell patch-clamp recordings as well as heat-evoked TRPA1-dependent calcitonin gene-related peptide (CGRP) release from mouse trachea. Studies of purified hTRPA1 intrinsic tryptophan fluorescence, in the absence of lipid bilayer, consolidate hTRPA1 as an intrinsic bidirectional thermosensor that is modified by the redox state and ligands. Thus, the heat sensing property of TRPA1 is conserved in mammalians, in which TRPA1 may contribute to sensing warmth and uncomfortable heat in addition to noxious cold.

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Figures

Figure 1
Figure 1. The purified hTRPA1 is a warmth receptor.
Temperatures above 22 °C evoked steady state outward and inward hTRPA1 single channel currents at a test potential of +60 and −60 mV as shown by representative traces and the corresponding amplitude histograms. Purified hTRPA1 was inserted into planar lipid bilayers and channel currents were recorded with the patch-clamp technique in a symmetrical K+ solution (c indicates the closed channel state).
Figure 2
Figure 2. The purified hTRPA1 displays intrinsic U-shaped thermosensitivity.
(a) Traces are part of a continuous recording (13 min) of hTRPA1 single channel currents at various temperatures at a test potential of +60 mV. (b) Left graph shows single-channel mean open probability values at different temperatures. Data points marked by asterisk were published previously. Right graph shows Arrhenius plot for the heat responses (25, 30 and 35 °C). Data were obtained at a steady state test potential of +60 mV (see Fig. 1) and analysed only for a first exposure to the indicated temperatures. Data are represented as mean ± s.e.m. (numbers of experiments within parentheses). (c) Black trace shows hTRPA1 channel openings when separated from orange trace (baseline) at both negative and positive test potentials (2-s voltage ramps from −100 to +100 mV). (d) The selective TRPA1 antagonist HC030031 and the non-selective TRP channel pore blocker ruthenium red inhibited cold and heat hTRPA1 responses at a test potential of +60 mV (n = 3). Purified hTRPA1 was inserted into planar lipid bilayers and channel currents were recorded with the patch-clamp technique in a symmetrical K+ solution (c indicates the closed channel state).
Figure 3
Figure 3. Redox modification influences the responsiveness of purified hTRPA1 to cold and heat.
(a) The Cy3-dye disulphide labeling fluorescence assay (top gel) revealed that the purified hTRPA1 used for functional studies was partially oxidized (control) as reducing (DTT, 1 mM) and oxidizing (H2O2, 100 μM) agents abolished and increased the disulphide bond formation, respectively. Coomassie blue staining (bottom gel) shows the amount of protein used for analysis of respective treatment. (b) Cold and heat responses of hTRPA1 were abolished by the reducing agents DTT and TCEP. (c) H2O2 (100 μM) activated hTRPA1 at 22 °C (see also Table 1) and the activity was abolished with TCEP. Notably, H2O2 reduced heat and increased cold responses. (b,c) Purified hTRPA1 was inserted into planar lipid bilayers and channel currents were recorded with the patch-clamp technique in a symmetrical K+ solution at a test potential of +60 mV (c indicates the closed channel state). (d) Summary of the effect of DTT (5 mM), TCEP (1 mM) and H2O2 (100 μM) on hTRPA1 single channel mean open probability (Po) examined at 30 °C and 15 °C at a test potential of +60 mV. Shown is also the effect of TCEP (1 mM) on hTRPA1 single channel activity evoked by H2O2 (100 μM) at 22 °C at a test potential of +60 mV. Data are represented as mean ± s.e.m. of paired comparisons before and after treatment (numbers of experiments are shown within bars). *P < 0.05, **P < 0.01 and ***P < 0.001 indicate statistically significant differences using the Student’s paired t test. (e) As shown by analysis of hTRPA1 intrinsic tryptophan fluorescence, cold and heat produced lipid bilayer-independent conformational changes of hTRPA1 (control) that were potentiated and inhibited by H2O2 (100 μM) and DTT (500 μM), respectively (n = 3). The hump (*) was only observed at temperatures above 22 °C. The fluorescence intensity, emitted at 335 nm, for each indicated temperature was related to that of 22 °C and expressed as Relative Fluorescence Intensity. Data are represented as mean ± s.e.m. Spectra of control is shown below.
Figure 4
Figure 4. Redox modification influences the activity of purified hTRPA1 without its N-terminal ankyrin repeat domain (Δ1-688 hTRPA1).
Representative traces show single channel activity for Δ1-688 hTRPA1 when inserted into planar lipid bilayers and exposed to cold (15 °C) or the oxidant H2O2 (100 μM) at 22 °C. The cold-evoked channel activity was inhibited by the reducing agents TCEP (1 mM, n = 4) and DTT (5 mM, n = 3), and as shown with TCEP the effect was reversible. The single channel mean open probability (Po) and conductance (Gs) values for cold were 0.46 ± 0.05 and 31 ± 6 pS, respectively (n = 6). The Po and Gs values for H2O2 were 0.16 ± 0.05 and 57 ± 8 pS, respectively (n = 6). The channel activity evoked by H2O2 was inhibited by TCEP (1 mM, n = 3). Channel currents were recorded with the patch-clamp technique a test potential of +60 mV in a symmetrical K+ solution (c indicates the closed channel state).
Figure 5
Figure 5. The cold and heat sensitivity of hTRPA1 is influenced by the redox state and ligands.
(a) H2O2 triggered cold-evoked inward currents at −60 mV and in 500 ms voltage ramps (n = 12). (b) Carvacrol also triggered hTRPA1 cold responses, but only in cells pre-exposed to heat (n = 5–8). (c,d) Increasing the temperature from 25 °C to 35 °C evoked heterologously expressed hTRPA1 outward currents in HEK293t cells that were blocked by the selective TRPA1 antagonist HC030031 (91 ± 5%, n = 5), whereas only minor currents were observed in non-transfected cells (n = 10–19). (e) At 40 °C, outward currents were not different from those in non-transfected cells (n = 6–8). (f) H2O2, but not acrolein or carvacrol (see Supplementary Fig. 5), prevented a further rise in outward currents when increasing the temperature from 25 °C to 35 °C. (g) Bar graph summarizing the effect of cold and heat in the absence (control) and presence of the various treatments at either −100 or +100 mV (n = 5–7). Data are represented as mean ± s.e.m. *P < 0.05, **P < 0.01 and ***P < 0.001 indicate statistically significant differences using ANOVA and Tukey’s honest significant difference test.
Figure 6
Figure 6. Mouse TRPA1 heat sensitivity is influenced by the redox state.
(a) Raising the temperature from 22 °C to 36 °C caused a small TRPA1-dependent calcitonin gene-related peptide (CGRP) release from mouse trachea. After pre-incubation of trachea with subliminal concentrations of H2O2 + NaOCl, producing singlet oxygen, there was a substantial increase in CGRP release at 36 °C that was inhibited by the selective TRPA1 antagonist HC030031 (50 μM) and in TRPA1 knock-out mice. (b) At 37 °C, acute exposure to various concentrations of H2O2 and NaOCl combined caused a concentration-dependent release of CGRP that was dependent on TRPA1 but not TRPV1. Under these conditions, H2O2 and NaOCl (each at 100 μM) alone evoked minor CGRP release compared to their combination. Interestingly, the highest concentration of H2O2 and NaOCl combined (each 1 mM) was less effective causing CGRP release possibly due to desensitization of TRPA1. Data are represented as mean ± s.e.m. (numbers of experiments within bars). *P < 0.05, **P < 0.01 and ***P < 0.001 indicate statistically significant differences using ANOVA Tukey’s honest significant difference Tukey’s honest significant difference test.
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