N A T UR E C O M M UN I CA T I O NS | D OI : 1 0. 1 0 3 8 / s 4 1 46 7 - 01 8 - 0 43 2 4 - 3
A R T I C L E
excluded. The average fluorescent ratio of all mCherry-positive cells was measured
at every 2 s interval. The quantitative results of calcium imaging for wild-type and
mutant Trpa1 proteins were graphed with Prism 7.0 (GraphPad, CA, USA). For
confirmation of the expression and functionality of various Trp channels, control
compounds were used to stimulate Ca2+ responses as indicated40. For inhibition of
Trpa1 with antagonist, transfected HEK293T cells were pre-incubated for 10 min
in 50 µM solutions of HC-030031 (Sigma-Aldrich) dissolved in calcium imaging
buffer with 0.2% DMSO, which was vortexed immediately before adding 2MT.
Streptavidin pulldown of alkynyl-TMT-modified HA-Trpa1. Approximately 1 ×
105 HEK293T cells were seeded into a 12-well plate and incubated in DMEM
medium containing 10% FBS and 1% Penicillin/Streptomycin at 37 °C for 18 h. For
transient transfection, 1 µg of pHA-Trpa1-P2A-mCherry construct was mixed with
3 µl Fugene 6 in 60 µl OptiMEM per 12 well. After 24 h of transfection, the cells
were washed with 500 µl calcium solution (125 mM NaCl, 2 mM MgCl2, 4.5 mM
KCl, 10 mM glucose, 20 mM HEPES, 2 mM CaCl2, 0.1% DMSO, pH 7.2), and
incubated with 300 µM alkynyl-TMT in calcium solution at 30 °C for 6 min. After
washing again with calcium solution, the cells were lysed with lysis buffer (50 mM
HEPES, 2 mM MgCl2, 10 mM KCl, 1% SDS, 150 units/ml of Benzonase (Sigma-
Aldrich, USA) pH 8.0, supplemented with complete™, Mini, EDTA-free protease
inhibitor cocktail (Sigma-Aldrich, USA)) for 20 min at room temperature.
The lysate was centrifuged at 12,000 × g for 3 min to pellet cell debris and the
protein concentration was measured by BCA assay (Thermo Fisher Scientific,
MA, USA).
For click chemistry reaction, 2 µl of 50 mM CuSO4 solution was mixed with 2 µl
of 2.5 mM tris(3-hydroxypropyltriazolylmethyl)amine (THPTA, Sigma-Aldrich,
USA), followed by addition of 1 µl of 1.25 mM azide-PEG3-biotin conjugate
(Sigma-Aldrich, USA) and 1 µl of 50 mM tris(2-carboxyethyl)phosphine (TCEP,
Sigma-Aldrich, USA). The 4 µl mixture of CuSO4/THPTA complex was then
mixed with 44 µl cell lysates (equal amount of total proteins) by vortexing and
incubated at room temperature for 2 h with constant agitation. Afterwards, equal
amount of protein samples (~20–40 µg) were diluted with RIPA buffer (10 mM
Tris HCl, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% SDS, 0.5% sodium
deoxycholate, 1% Triton X-100, pH 8.0) containing protease inhibitor cocktail and
incubated with 15 µl of streptavidin agarose (Cat# 69203, EMD Millipore,
Germany) for 2 h at room temperature with constant rotating. After washing the
streptavidin beads three times with 150 µl RIPA buffer, the biotin-conjugated
proteins were eluted with 1× SDS loading buffer with 50 mM DTT at 95 °C for 10
min. The eluted proteins were resolved by SDS-polyacrylamide gel electrophoresis,
transferred to nitrocellulose membrane, and immunoblotted with rabbit anti-HA
antibody (1:50,000, Cat# 3724s, Cell Signaling Technology, USA) or mouse anti-β-
actin antibody (1:50,000, Cat# sc-47778, Santa Cruz Biotechnology, USA).
Synthesis of TMT, alkynyl-TMT, and SBT. All distillations were carried out with
a Büchi Glass Oven B-585 Kugelrohr. 1H and 13C NMR spectra data were obtained
with JEOL JNM-ECS 400 instruments. Chemical shifts (δ) are quoted in parts per
million using tetramethylsilane (δ = 0 ppm) as the reference for 1H NMR spec-
troscopy, and CDCl3 (δ = 77.0 ppm) for 13C NMR spectroscopy. Reagents and
solvents were purchased from the following commercial suppliers: Tokyo Chemical
Ind., Sigma-Aldrich, Inc., Kanto Chemical Co., Inc., Wako Pure Chemical Ind.,
Ltd., and Nacalai Tesque. Thin-layer chromatography was performed on Merck
Kieselgel 60 F254 (0.25 mm) plates. Synthetic schemes of TMT (3a)[61] and
alkynyl-TMT (3b) are shown in Supplementary Fig. 8a and the methods are
described here:
Synthesis of 2,4,5-trimethyl-2,5-dihydrothiazole (TMT, 3a): To a stirring mixture
of 3-mercapto-2-butanone (1, 2.1 ml, 21.1 mmol) and acetoaldehyde (2a, 1.18 ml,
21.1 mmol) was added NH3 gas at 0 ˚C over a period of 2 h (until the reaction
which releases water and heat is finished). After being diluted with diethyl ether,
the inorganic phase was separated. The organic phase was washed with brine, dried
over Na2SO4, and concentrated at pressures above 400 MPa. The residual oil was
distillated with Kugelrohr distillation apparatus (1 mmHg, 50–70 ˚C) to afford 3a
as a 43:57 mixture of conceivable isomers (1.2 g, 9.3 mmol, 44%, colorless oil).
Minor isomer of 3a. 1H NMR (400 MHz, CDCl3) δ 5.61–5.52 (m, CH3-CH(C)-
S, 1H), 4.37–4.20 (m, N-CH(CH3)-S, 1H), 2.06 (m, CH3-C(CH) = N, 3H), 1.56
(dd, J = 6.6, 1.9 Hz, CH3, 3H), 1.46 (dd, J = 7.1, 1.8 Hz, CH3, 3H). 13C NMR (101
MHz, CDCl3) δ 172.22, 75.53, 56.55, 25.24, 21.72, 17.71.
Major isomer of 3a. 1H NMR (400 MHz, CDCl3) δ 5.52–5.44 (m, CH3-CH(C)-
S, 1H), 4.37–4.20 (m, N-CH(CH3)-S, 1H), 2.06 (m, CH3-C(CH) = N, 3H), 1.58
(dd, J = 6.6, 1.9 Hz, CH3, 3H), 1.51 (dd, J = 7.1, 1.8 Hz, CH3, 3H). 13C NMR (101
MHz, CDCl3) δ 172.01, 75.82, 56.78, 26.24, 22.62, 17.68.
c-Fos IHC and double Trpa1/c-Fos staining of TG. Mice were single housed and
habituated in the test environment 24 h before the experiment. A small filter paper
containing 15 μl of water or 2MT (1.57 × 10−4 mole) was placed into the home
cage for 30 min. After the filter paper was removed, test mouse was allowed to
recover in the home cage for another 30 min. Mice were then rapidly anesthetized
with pentobarbital (50 mg/kg, i.p.) and sequentially perfused by PBS and 4% PFA
in PBS. Trigeminal ganglions were dissected and fixed for 24 h in the same fixative
solution and stored at 4 °C, and then cryoprotected with 30% sucrose (wt/vol) in
PBS for 48 h. The tissues were put in the Tissue-Tek O.C.T compound (Sakura
Finetek), and 30-μm-thick coronal sections were cut on a cryostat (CM3050S,
Leica). For c-Fos IHC, the floating TG sections were washed three times with PBS
for 5 min, incubated with 0.3% Triton X-100 in 0.01 M PBS for 15 min, and washed
again with PBS for three times. The slides were treated with 1% H2O2/PBS for 30
min and washed three times with PBS. After incubating in 0.3% of BSA/0.25%
Triton-X-100/ PBS for 1 h, the sections were incubated with rabbit anti-c-Fos
antibody (1:5000, EMD Millipore, ABE457) at 4 °C overnight. After washing six
times with PBST, the sections were incubated with biotinylated rabbit anti-goat IgG
(1:500, Vector BA5000) for 2 h at room temperature and followed by PBS washing
for three times. The sections were incubated with HRP-Streptavidin (ABC reagent,
VECTOR) for 1 h at room temperature. After washing with PBS three times, the
slides were stained in DAB Peroxidase (HRP) Substrate Kit (VECTOR) in the dark
until the color developed. The reaction was stopped by PBS washing and the
sections were mounted onto glass slides for microscopy. All images were acquired
using the Zeiss LSM700 confocal microscope with a 10× objective lens (NA = 0.45)
under the AxioVision4.8 software. The nuclear c-Fos + neurons were counted in all
sections from the same TG. Representative images shown in the figures were
chosen from a similar region of the trigeminal ganglion based on morphology.
For double Trpa1/c-Fos staining of TG, Trpa1 mRNA ISH was performed
before c-Fos IHC. All reagents and solutions were prepared using
diethylpyrocarbonate (DEPC)-treated distilled deionized water (ddH2O) and
RNase-free reagents. The DNA templates for making sense or antisense Trpa1
probes were generated using PCR primers: 5′-GCCTAATACGACTCACTATA
GGCGGCTTGAGGAGGATTCTG-3′ and 5′-TCTGTGAAGCAGGGTCTCCT-3′;
or 5′-GCGGCTTGAGGAGGATTCTG-3′ and 5′-GCCTAAGACTCACTATAGG
GTCTGTGAAGCAGGGTCTCCT-3′. The RNA probes labeled by digoxygenin-
UTP were generated by in vitro transcription by T7-RNA labeling kit (Roche). The
floating TG sections were washed with 1× PBS for 10 min and treated with PFA
(4% in PBS, wt/vol, Nacalai) for 15 min, then the slides were reduced by treatment
of 0.3% of sodium borohydride and washed with PBS 10 min. Tissue slides were
treated with 0.3% Triton X-100 (vol/vol) in PBS for 15 min followed by twice of
PBS washing. The slides were then treated with 0.75% glycine for 15 min followed
by PBS washing and acetylated by adding 1/400 vol of acetic anhydride (vol/vol) in
PBS for 15 min, followed by PBS washing two times. Afterward, the tissue sections
were incubated in the hybridization solution (50% deionized formamide, 2%
blocking reagent, 5× SSC, 0.1% N-lauroylsarcosine, 0.1% SDS) without probes for
1 h at 55 °C, followed by incubation in the hybridization solution with the Trpa1
Synthesis of 2-(but-3-yn-1-yl)-4,5-dimethyl-2,5-dihydrothiazole (Alkynyl-TMT,
3b): To a solution of 4-pentyn-1-ol (1 ml, 10.8 mmol) in CH2Cl2 (15 ml),
trimethylamine (10 ml), DMSO (6 ml), and SO3 pyridine complex (5.2 g, 32.4
mmol) were added at 0 ˚C, and the mixture was stirred at same temperature for 3 h.
After being neutralized by 1.0 M aq. hydrochloric acid, the mixture was extracted
with chloroform, washed with brine, dried over Na2SO4, and concentrated under
reduced pressure. The residual oil was distillated with Kugelrohr distillation
apparatus (760 mmHg, 100–150 ˚C) to afford 4-pentyn-1-ol (2b, 400 mg, 4.9
mmol, 45%, colorless oil).
To a stirring mixture of 3-mercapto-2-butanone (1, 474 µl, 4.9 mmol) and 2b
(400 mg, 4.9 mmol) was added NH3 gas at 0 ˚C over a period of 2 h (until the
reaction which releases water and heat is finished). After being dissolved the white
precipitation with ethyl acetate, the inorganic phase was separated. The organic
phase was washed with brine, dried over Na2SO4, and concentrated at reduced
pressure. The residual oil was distillated with Kugelrohr distillation apparatus (1
mmHg, 50–90 ˚C) to afford the titled compound as a 3:7 mixture of conceivable
isomers (180 mg, 1.1 mmol, 22%).
Minor isomer of 3b. 1H NMR (400 MHz, CDCl3) δ 5.67–5.62 (m, CH3-CH(C)-
S, 1H), 4.28–4.21 (m, N-CH(Alkyl)-S, 1H), 2.38–2.33 (m, –CH2–, 2H), 2.18–2.09
(m, –CH2–, 1H), 2.06–2.05 (m, CH3-C(CH)=N, 3H), 1.97–1.95 (m, C≡C–H, 1H),
1.96–1.86 (m, –CH2–, 1H), 1.46 (d, J = 7.1 Hz, CH3, 3H). 13C NMR (101 MHz,
CDCl3) δ 172.92, 83.48, 79.44, 68.93, 55.85, 37.43, 21.64, 17.68, 15.76.
Major isomer of 3b. 1H NMR (400 MHz, CDCl3) δ 5.59–5.54 (m, CH3-CH(C)-S,
1H), 4.28–4.21 (m, N-CH(Alkyl)-S, 1H), 2.38–2.33 (m, –CH2–, 2H), 2.18–2.09 (m,
–CH2–, 1H), 2.06–2.05 (m, CH3-C(CH)=N, 3H), 1.97–1.95 (m, C≡C–H, 1H),
1.96–1.86 (m, –CH2–, 1H), 1.51 (d, J = 7.0 Hz, CH3, 3H). 13C NMR (101 MHz,
CDCl3) δ 172.74, 83.48, 79.79, 68.98, 56.05, 38.12, 22.41, 17.68, 15.86.
SBT was synthesized as previously reported62 and the method is described
here. To a suspension of 2-aminoethanethiol hydrochloride (4, 3.4 g, 30.0 mmol) in
dry toluene (125 ml) under argon, a solution of triisobuthylaluminium (1.0 M in
toluene, 75.0 ml, 75.0 mmol) was added dropwise at room temperature. After
refluxing for 30 min, methyl DL-2-methylbutyrate (5, 3.3 g, 28.5 mmol) was added
dropwise. After further refluxing for 2 h, the mixture was diluted with 125 ml of
toluene, cooled to room temperature, and quenched with MeOH (50 ml). The
mixture was stirred at room temperature for 30 min, and then a sat. aq. Rochelle’s
salt (100 ml), sat. aq. NaHCO3 (100 ml), and brine (100 ml) were successively
added and stirred for 1 h. After being diluted with diethyl ether, the inorganic
phase was separated. The organic phase was washed with brine, dried over Na2SO4,
and concentrated in vacuo. The residual oil was distillated with Kugelrohr
distillation apparatus (1 mmHg, 60–120 ˚C) to afford 6 (1.82 g, 12.74 mmol, 42%,
colorless oil).
1H NMR (400 MHz, CDCl3) δ 4.21 (ddt, J = 8.3, 0.8 Hz, 2H), 3.24 (t, J = 8.3 Hz,
2H), 2.68–2.58 (m, 1H), 1.74–1.45 (m, 2H), 1.19 (d, J = 6.8 Hz, 3H), 0.92 (t, J = 7.3
Hz, 3H).
13C NMR (101 MHz, CDCl3) δ 176.92, 64.38, 40.93, 33.11, 28.64, 18.84, 11.78.
N A T U R E C OM M U N I C A T I ON S
( 2 0 1 8 ) 9 :2 0 41
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