Fig 1.
Activation of PKA by FSK sensitizes TRPA1.
A Calcium-imaging experiments on hTRPA1-transfected HEK293t cells. TRPA1 was activated with six applications of carvacrol (filled bars; 50 μM; 20 s). Top: control experiment without application of FSK. Bottom: activation of PKA by application of FSK (open bar, 10 μM, 120 s) causes strong sensitization. B To allow the full development of sensitization, the sensitization ratio was calculated as the amplitude of the sixth over the fourth response. Sensitization ratio 1.04 ± 0.02 in control (white) and 1.29 ± 0.09 with FSK (black; p = 0.002, one-way ANOVA with Bonferroni post-hoc analysis). Number of cells under each condition is indicated. ** p < 0.01
Fig 2.
Sensitization of TRPA1 by FSK is PKA-dependent.
Whole-cell patch-clamp recordings on hTRPA1-transfected HEK293t cells in calcium-free solution. A Carvacrol (100 μM) applied for 150 s before switching to repeated 15 s applications (30 s gap) produces a stable response pattern and avoids distortion by agonist-induced sensitization [19]. Calibration bar is the same for all traces. Dotted line shows average of pre-FSK responses 7 and 8. Top: control recording without application of FSK. Middle: Activation of PKA by FSK (10 μM; 2 x 30 s) sensitizes the following responses. Bottom: Application of the PKA inhibitor H89 (10 μM; 4 x 30 s) as indicated blocks sensitization by FSK. B Maximum inward currents at carvacrol applications 7–12 normalized to the average of pre-FSK responses 7 and 8 (dotted line in A). FSK (filled diamonds) causes a marked increase in current amplitude of responses 9–12 compared to control (open circles) and this effect is blocked by H89 (grey squares). C Quantification of B. Control: increase in mean of responses 10–12 was 1.15 ± 0.04; FSK: increase 1.59 ± 0.06 fold (p < 0.001 compared to control); FSK + H89: 1.03 ± 0.05 fold (p < 0.001 compared to FSK, p = 0.99 compared to control); H89: 1.16 ± 0.06 fold (p = 0.002 compared to FSK, p = 0.99 compared to control or compared to FSK + H89; all one-way ANOVA with Bonferroni post-hoc analysis). Total number of cells measured under each condition is indicated. *** p < 0.001.
Fig 3.
Location of the mutated residues on hTRPA1.
Schematic representation of a TRPA1 monomer, indicating the location of the 11 putative PKA phosphorylation sites that were mutated to alanine. Red circles indicate mutations that did not reduce sensitization. Residues that were found to mediate sensitization by PKA are shown in yellow. Grey ovals indicate ankyrin repeat domains, numbered with roman numerals. Single cysteine and lysine residues (C621, C641, C665, K710) that have been found to be involved in channel activation by covalent agonists [38] are represented by blue circles. All positions are approximations based on the hTRPA1 sequence (NP_015628.2).
Fig 4.
FSK-induced sensitization of TRPA1 S/T mutants in patch-clamp recordings.
A Example traces showing sensitization of WT and mutant hTRPA1 by FSK (10 μM; 2 x 30 s). Full trace shown for WT hTRPA1; for mutant TRPA1, only carvacrol responses 7–12 are displayed. Response numbers are indicated. Dotted line shows average of responses 7 and 8. Scale bars in each trace are 200 pA and 30 s. Traces for WT and WT control have been replicated from Fig 2A. B Quantification of A. Values for WT control and WT+FSK are the same as in Fig 2C. In the WT control group (white), responses 10–12 were 1.15 ± 0.04 fold of responses 7 and 8. The sensitization by FSK (black) in the hTRPA1 WT is marked by the broken line (WT+FSK; 1.59 ± 0.06; p < 0.001). The following levels were measured in the hTRPA1 mutants (grey): 1.92 ± 0.22 for S227A (p = 0.048), 1.80 ± 0.05 for T274A, 1.62 ± 0.10 for S1012A, 1.59 ± 0.08 for T1105A, 1.53 ± 0.04 for S804A, 1.45 ± 0.06 for S87A, 1.45 ± 0.14 for S1101A, (all p ≥ 0.6), 1.31 ± 0.06 for S428A (p = 0.005), 1.28 ± 0.09 for S317A (p = 0.017), 1.25 ± 0.05 for S972A (p = 0.023), 1.23 ± 0.05 for S86A (p = 0.009). All one-way ANOVA with Dunnett’s test, compared to WT+FSK. The number of cells recorded in each group is indicated. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 5.
Sensitization of TRPA1 mutants in calcium imaging experiments.
A Representative recordings from HEK293t cells expressing the four TRPA1 mutants which showed significant reduction in PKA sensitization in patch-clamp experiments (see Fig 4). The protocol was identical to that shown in Fig 1A. Only carvacrol responses 4–6 are displayed. B Quantification of A. Values for WT control and WT+FSK have been replicated from Fig 1B. In control experiments (white), sensitization ratio was 1.04 ± 0.02. With application of FSK (black) sensitization ratio was increased to 1.29 ± 0.09 (p = 0.002). Sensitization ratios in the four mutants (grey) were 1.08 ± 0.04 for S972A (p = 0.047), 1.07 ± 0.05 for S86A (p = 0.017), 0.95 ± 0.05 for S428A (p = 0.0002), and 0.92 ± 0.03 for S317A (p < 0.0001). Number of cells measured in each group is indicated. One-way ANOVA with Dunnett’s test, compared to WT+FSK. The number of cells recorded in each group is indicated. # compares to WT control; * compares to WT+FSK. * p < 0.05, *** p < 0.001.
Fig 6.
Alignment of TRPA1 protein sequence surrounding the four identified PKA phosphorylation sites.
Sequence alignment carried out using Clustal Omega from EMBL-EBI (http://www.ebi.ac.uk/Tools/msa/clustalo/). Sequences for TRPA1 from Drosophila melanogaster (GenBank: AY302598.1; Viswanath et al., 2003), Danio rerio (zebrafish; GenBank: AAV37177.1), Mus musculus (mouse; Ref: NP_808449.1), Rattus norvegicus (rat; Ref: NP_997491.1), Macaca mulatta (rhesus macaque; Ref: XP_001083172.1) and human (Ref: NP_015628.2) obtained from NCBI. Shaded background indicates amino acids identical to the human isoform. Alignment is presented for S317 (top), S428 (second), S86 (third) and S972 (bottom). The serine residues of interest are marked in bold font. This alignment reveals that S317 and S428 are highly conserved across mammalian and non-mammalian species, whereas S86 and S972 are conserved across the mammalian species but are not present in Drosophila or zebrafish.