The Met268Pro mutation of mouse TRPA1 changes the effect of caffeine from activation to suppression

Abstract

The transient receptor potential A1 channel (TRPA1) is activated by various compounds, including isothiocyanates, menthol, and cinnamaldehyde. The sensitivities of the rodent and human isoforms of TRPA1 to menthol and the cysteine-attacking compound CMP1 differ, and the molecular determinants for these differences have been identified in the 5th transmembrane region (TM5) for menthol and TM6 for CMP1. We recently reported that caffeine activates mouse TRPA1 (mTRPA1) but suppresses human TRPA1 (hTRPA1). Here we aimed to identify the molecular determinant that is responsible for species-specific differences in the response to caffeine by analyzing the functional properties of various chimeras expressed in Xenopus oocytes. We initially found that the region between amino acids 231 and 287, in the distal N-terminal cytoplasmic region of mTRPA1, is critical. In a mutagenesis study of this region, we subsequently observed that introduction of a Met268Pro point mutation into mTRPA1 changed the effect of caffeine from activation to suppression. Because the region including Met-268 is different from other reported ligand-binding sites and from the EF-hand motif, these results suggest that the caffeine response is mediated by a unique mechanism, and confirm the importance of the distal N-terminal region for regulation of TRPA1 channel activity.

Figure 1

Responses to caffeine by mTRPA1 and hTRPA1 expressed in Xenopus oocytes. (A and C) Representative current traces recorded from Xenopus oocytes expressing mTRPA1 (A) or hTRPA1 (C) under a two-electrode voltage clamp. In each trace, a 1.5-s ramp pulse from −100 to +100 mV was applied twice from a holding potential of −20 mV. Each data set consisted of recordings made without caffeine (first run, gray) and then with caffeine (second run, black). Caffeine was present during the time indicated by the bar. (B and D) The ratios of the current amplitudes recorded at +80 mV from oocytes expressing mTRPA1 (B) or hTRPA1 (D) before and after caffeine application plotted against caffeine concentration. Open circles show data from each oocyte, and the solid diamonds and bars indicate means ± SE. The n-values for each point range from 2 to 6. The n-value for 0 mM caffeine is the sum of all the other points, since data before the application of caffeine were recorded as a negative control in all experiments.

Figure 2

Schematic drawings of the mTRPA1-hTRPA1 chimeras. The components of mTRPA1 and hTRPA1 are shown in dark and white, respectively. The positions of the six TM regions are shown in light gray. In the labels on the left, Mo and Hu stand for mouse and human. Mo705Hu, for example, indicates that the N-terminal 705 residues are from mTRPA1 and the rest are from hTRPA1. Mo-Hu-Mo and Hu-Mo-Hu are chimeras in which only the critical region (231–287 in mTRPA1) was swapped.

Figure 3

Current recordings of mTRPA1, hTRPA1, Mo705Hu, Hu703Mo, Mo375Hu, and Hu374Mo. Under a two-electrode voltage clamp, 200-ms ramp pulses from −100 to +100 mV were applied every 5 s from a holding potential of −20 mV. Recordings are from oocytes expressing mTRPA1 WT (A), hTRPA1WT (B), Mo705Hu (C), Hu703Mo (D), Mo375Hu (E), or Hu374Mo (F). The presence of 5 mM caffeine or 100 μM AITC is indicated by the bars. Panels A–D and panels E and F are from different batches of oocytes.

Figure 4

Analysis of the cumulative data from the experiments in Fig. 3. The current amplitude at +100 mV was changed by application of 5 mM caffeine (A, C, D, and F) or by 100 μM AITC (B and E). (A, B, D, and E) Differences in current amplitudes before caffeine application and after the 10th pulse after application are shown. (C and F) Caffeine was applied after the application and subsequent washout of AITC. Differences between current amplitudes before caffeine application and the amplitude of the first pulse after application are shown. Panels A–C and D–F are from different batches of oocytes. Plots depict means ± SE. The data and n-values are shown in Table 1.

Figure 5

Current recordings for Mo327Hu, Mo287Hu, Mo230Hu, Mo254Hu, Mo201Hu, and Mo121Hu. Details of the experiments are the same as in Fig. 3. Panels A–C and D–F are from different batches of oocytes.

Figure 6

Analysis of the cumulative data from the experiments in Fig. 5. Details of the analyses are the same as in Fig. 4. Panels A–C and D–F are from different batches of oocytes. Plots depict means ± SE. The data and n-values are shown in Table 1.

Figure 7

Current recordings and analyses of Mo-Hu-Mo and Hu-Mo-Hu. (A and B) Details of the experiments are the same as in Fig. 3, except that 10 mM caffeine was used. (C–E) Details of the analyses are the same as in Fig. 4, except that 10 mM caffeine was used. Plots depict means ± SE. The data and n-values are shown in Table 1.

Figure 8

Current recordings and analysis of mTRPA1 point mutants. (A) Alignment of partial amino acid sequences of mTRPA1 and hTRPA1. The identified critical region is highlighted in black (amino acids 231–287 of mTRPA1). Asterisks indicate identical amino acid residues. (B) Representative current recordings from oocytes expressing the indicated mTRPA1 point mutant. Under a two-electrode clamp, 1.5-s ramp pulses from −100 to +100 mV were applied twice from a holding potential of −20 mV. The presence of 10 mM caffeine is indicated by the bar. (C) Ratios of the current amplitudes at +80 mV before and after caffeine application. Plots depict means ± SE. The data for Met268Pro were significantly different from WT mTRPA1, but not from hTRPA1. The data and n-values are shown in Table 2.

Figure 9

Caffeine dose-response relationships of mTRPA1 Met-268 point mutants. The ratios of the current amplitudes at +80 mV before and after caffeine application are plotted against caffeine concentration. Data for M268P (A), M268C (B), M268A (C), M268W (D), and M268G (E) are shown. Open circles show data from each oocyte, and the solid diamonds and bars indicate means ± SE. The n-values for each point range from 1 to 4 (n = 1 only for the case in which 0.1 mM was applied to Met268Ala). The n-value of 0 mM caffeine is the sum of all other points, since data before caffeine application were recorded as a negative control in all experiments.

Figure 10

Current recordings and analyses of hTRPA1 point mutants. (A) Details of the experiments are the same as in Fig. 8B. (B) Details of the analyses are the same as in Fig. 8C. Plots depict means ± SE. The data obtained with the mutants did not statistically differ from WT hTRPA1. The data and n-values are shown in Table 2.