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. 2022 Nov 18;23(22):14297.
doi: 10.3390/ijms232214297.

Potent Activation of Human but Not Mouse TRPA1 by JT010

Masaki Matsubara  1 , Yukiko Muraki  1 , Noriyuki Hatano  1 , Hiroka Suzuki  1 , Katsuhiko Muraki  1
Affiliations

Potent Activation of Human but Not Mouse TRPA1 by JT010

Masaki Matsubara et al. Int J Mol Sci. .

Abstract

Transient receptor potential (TRP) ankyrin repeat 1 (TRPA1), which is involved in inflammatory pain sensation, is activated by endogenous factors, such as intracellular Zn2+ and hydrogen peroxide, and by irritant chemical compounds. The synthetic compound JT010 potently and selectively activates human TRPA1 (hTRPA1) among the TRPs. Therefore, JT010 is a useful tool for analyzing TRPA1 functions in biological systems. Here, we show that JT010 is a potent activator of hTRPA1, but not mouse TRPA1 (mTRPA1) in human embryonic kidney (HEK) cells expressing hTRPA1 and mTRPA1. Application of 0.3-100 nM of JT010 to HEK cells with hTRPA1 induced large Ca2+ responses. However, in HEK cells with mTRPA1, the response was small. In contrast, both TRPA1s were effectively activated by allyl isothiocyanate (AITC) at 10-100 μM. Similar selective activation of hTRPA1 by JT010 was observed in electrophysiological experiments. Additionally, JT010 activated TRPA1 in human fibroblast-like synoviocytes with inflammation, but not TRPA1 in mouse dorsal root ganglion cells. As cysteine at 621 (C621) of hTRPA1, a critical cysteine for interaction with JT010, is conserved in mTRPA1, we applied JT010 to HEK cells with mutations in mTRPA1, where the different residue of mTRPA1 with tyrosine at 60 (Y60), with histidine at 1023 (H1023), and with asparagine at 1027 (N1027) were substituted with cysteine in hTRPA1. However, these mutants showed low sensitivity to JT010. In contrast, the mutation of hTRPA1 at position 669 from phenylalanine to methionine (F669M), comprising methionine at 670 in mTRPA1 (M670), significantly reduced the response to JT010. Moreover, the double mutant at S669 and M670 of mTRPA1 to S669E and M670F, respectively, induced slight but substantial sensitivity to 30 and 100 nM JT010. Taken together, our findings demonstrate that JT010 potently and selectively activates hTRPA1 but not mTRPA1.

Keywords: JT010; calcium channel; dorsal root ganglion; synoviocytes; transient receptor potential ankyrin repeat 1.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The hTRPA1-specific activation by JT010. (A,B) JT010 at a concentration range from 0.3 to 100 nM induced Ca2+ responses in representative HEK-hTRPA1 cells (A) and the peak JT010-induced Ca2+ response (Δratio) in HEK-hTRPA1 cells (five independent experiments) is summarized as a concentration-response relationship (B). At the end of each experiment, 100 μM AITC was applied to confirm hTRPA1 expression. (C,D) JT010 and AITC at 10 nM and 100 μM, respectively, were applied to HEK-hTRPA1 and HEK-mTRPA1 cells, and the measured Ca2+ response (C) and the peak JT010- and AITC-induced Ca2+ response (five independent experiments each) (D) are summarized. Two-way analysis of variance (ANOVA): * p = 0.0279, F = 5.85 (species); ** p < 0.0001, F = 84.5 (drugs); * p = 0.0312, F = 5.58 (interaction). Vertical bars = SEM.
Figure 2
Figure 2
(A–C) JT010-induced membrane currents at −90 and +90 mV in a representative HEK-hTRPA1 cell which was superfused with SBS without Ca2+ (A) and the peak JT010-induced currents are summarized as a concentration-response relationship (C, five independent experiments). The current and voltage (I–V) relationships under each experimental condition are also shown (B). Repeated measures one-way ANOVA: * p = 0.0116. F = 4.28 (+90 mV). Post hoc Tukey–Kramer test: # p = 0.0285 (+90 mV vs. cont), # p = 0.0229 (+90 mV vs. A96). Repeated measures one-way ANOVA: * p = 0.0291. F = 3.37 (−90 mV). Post hoc Tukey–Kramer test: # p = 0.0495 (−90 mV vs. cont), # p = 0.0468 (−90 mV vs. A96). The pipette solution contained 0.3 μM Ca2+ to maintain TRPA1 activity. Ramp waveform voltage pulses from −110 to +90 mV for 300 ms were applied every 5 s at a holding potential of −10 mV. To confirm the hTRPA1 activity, cells were treated with A96 at 5 μM at the end of the agonist application. (D–F) No effects of JT010 on mTRPA1. The experimental conditions were identical to those demonstrated in (A), except HEK-mTRPA1 cell and additional applications of 30 μM AITC and 5 μM A96 (D). At the end of each experiment, AITC and A96 were applied to confirm the expression of mTRPA1. The peak JT010-and AITC-induced currents are summarized as a concentration-response relationship (F, four independent experiments). Repeated measures one-way ANOVA: ** p < 0.0001. F = 8.54 (+90 mV). Post hoc Tukey–Kramer test: ## p < 0.0001 (+90 mV, vs. 1st A96 and 2nd A96), ## p = 0.00038 (+90 mV, vs. cont), ## p = 0.00039 (+90 mV, vs. 10 nM JT010), ## p = 0.00045 (+90 mV, vs. 30 nM JT010), ## p = 0.00062 (+90 mV vs. 100 nM JT010), ## p = 0.00069 (+90 mV vs. 2nd cont). Repeated measures one-way ANOVA: ** p < 0.0001. F = 12.2 (−90 mV). Post hoc Tukey–Kramer test: ## p < 0.0001 (−90 mV vs. all). The ‘ns’ shows no significance by the Tukey–Kramer test. Vertical bars = SEM.
Figure 3
Figure 3
Effects of JT010 on endogenous TRPA1 in human and mouse cells. (A) JT010 at 10 nM and AITC at 100 μM were applied to human FLSs with or without inflammation. FLSs were treated with 10 U IL-1α or vehicle for 24 h and then exposed to JT010 and AITC, and Ca2+ response was monitored ((A), each representative cell). The peak JT010- and AITC-induced Ca2+ response (Δratio) in FLSs with or without IL-1α (six independent experiments each) are summarized in the lower panel. Two-way ANOVA: ** p < 0.0001, F = 167 (pretreatments); ** p < 0.0001, F = 27.0 (drugs); ** p = 0.00015, F = 21.7 (interaction) (B) JT010 at 10 nM and AITC at 100 μM were applied to mouse DRGs, and Ca2+ response was monitored ((B), a representative cell). The peak JT010- and AITC-induced Ca2+ response (Δratio) in DRGs are summarized (lower panel, five independent experiments). Paired Student’s t-test: ## p = 0.00042. Vertical bars = SEM.
Figure 4
Figure 4
Comparison of JT010-induced TRPA1 response among mutants of N- and C-terminal cysteine residues of hTRPA1 and mTRPA1. (A) Alignment of amino acid sequence between hTRPA1 and mTRPA1. C621 and C665 in hTRPA1 (homologous to mTRPA1 C622 and C666) shown by boxes indicate critical cysteines for electrophilic TRPA1 agonist modification. Bold and underlined letters (C59, C1021, C1025 in human, indicated by an arrow) show cysteines substituted in mutant mTRPA1 (Y60C, H1023C, N1027C), whose effect was examined. Yellow color boxes indicate potential critical amino acids for JT010-sensitivity, whose importance is examined in Figure 5 and Figure 6. (B–D) Ca2+ responses of mutant hTRPA1 with C621S mutation and mutant mTRPA1s with Y60C, H1023C, and N1027C mutations to 10 nM JT010. To confirm the channel expression, 100 µM AITC was applied at the end of the experiment. Each representative Ca2+ response was superimposed (B) and the peak JT010- and AITC-induced Ca2+ response (Δratio) is summarized (C, five independent experiments). Paired Student’s t-test: ** p < 0.0001, ** p = 0.00092, ** p < 0.0001, and ** p = 0.00064 for hC621S, mY60C, mH1023C, and mN1027C, respectively (D) Ca2+ response to JT010 was normalized with that to AITC and is summarized. The responses of wild hTRPA1 and mTRPA1 were also included as a comparison (the same data set as Figure 1). Unpaired Student’s t-test: ## p < 0.0001. The ‘ns’ shows no significance by the Tukey–Kramer test. Vertical bars = SEM.
Figure 5
Figure 5
Critical importance of F669 for JT010-induced hTRPA1 response. (A–C) JT010 (10 to 100 nM)- and AITC (30 µM)-induced currents at −90 and +90 mV were compared between hTRPA1 (A) and 669M-hTRPA1 (B), and are summarized (C, four-five independent experiments). Following agonist treatment, 5 µM A96 was added to block TRPA1 channel current components. In the lower panel of (A,B), I–V relationships under each experimental condition are shown. Cells were superfused with SBS without Ca2+ and dialyzed with Cs-aspartate rich pipette solution including 0.3 µM Ca2+. Ramp waveform voltage pulses from −110 to +90 mV for 300 ms were applied every 5 s. Two-way ANOVA (+90 mV): ** p < 0.0001, F = 33.3 (hTRPA); ** p < 0.0001, F = 17.1 (drugs); ** p = 0.00045, F = 4.54 (interaction). Two-way ANOVA (−90 mV): ** p < 0.0001, F = 28.6 (hTRPA); ** p < 0.0001, F = 11.4 (drugs); ** p = 0.00846, F = 3.06 (interaction). Vertical bars = SEM.
Figure 6
Figure 6
S669 and M670 are the potential amino acids that determine the low sensitivity of mTRPA1 to JT010. Cells were superfused with SBS without Ca2+ and dialyzed with a Cs-aspartate-rich pipette solution including 0.3 µM Ca2+. Ramp waveform voltage pulses from −110 to +90 mV for 300 ms were applied every 5 s. (A,B) JT010 was commutatively applied to HEK cells with an M670F substitution in mTRPA1 (A, 670F-mTRPA1) and double substitutions of S669E and M670F (B, 669E, 670F-mTRPA1) to examine the effects on membrane currents at −90 and +90 mV, and the pooled data of the peak currents evoked are summarized (C, four to six independent experiments including the same data set as in Figure 2F). After applying 100 nM JT010, 5 µM A96 was added to block the TRPA1 channel current components. For comparison, 30 µM AITC was used. In the middle and right panels of (A,B), the I-V relationships under each experimental condition are shown. (D) Each current amplitude shown in (C) was normalized to that of the control without JT010 and exhibited the relative amplitude change under each treatment. Dunnett’s multiple comparisons test was performed for each TRPA1 gene. * p = 0.0116 and ** p = 0.00829 for 30 and 100 nM JT010, respectively in 669E, 670F-mTRPA1 (+90 mV). * p = 0.0327 for 100 nM JT010 in 669E, 670F-mTRPA1 (−90 mV). Vertical bars = SEM.
Figure 7
Figure 7
Structure modeling of the interaction of JT010 with F669 on hTRPA1 (PDB ID: 6PQO) and M670 on mTRPA1. Ribbon diagrams colored grey of the hTRPA1 atomic model for residues 447–1079 are shown on the left. The Modeller optimized model of mTRPA1 for residues 446–1074 and JT010 are colored orange and red, respectively. A close-up view of the JT010 binding sites is shown on the right. The methoxyphenyl group of JT010 potentially interacts with the phenyl of F669 in hTRPA1 in a π-π interaction manner. The coordination is shown by the dotted line with a 4.9 Å distance. Out of 100 models, the structure with the lowest zDOPE score (2.69) was adopted (see also Materials and Methods).
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