Activation of Drosophila melanogaster TRPA1 Isoforms by Citronellal and Menthol

Abstract

Background: The transient receptor potential ankyrin 1 (TRPA1) cation channels function as broadly-tuned sensors of noxious chemicals in many species. Recent studies identified four functional TRPA1 isoforms in Drosophila melanogaster (dTRPA1(A) to (D)), but their responses to non-electrophilic chemicals are yet to be fully characterized.

Methods: We determined the behavioral responses of adult flies to the mammalian TRPA1 non-electrophilic activators citronellal and menthol, and characterized the effects of these compounds on all four dTRPA1 channel isoforms using intracellular Ca²⁺ imaging and whole-cell patch-clamp recordings.

Results: Wild type flies avoided citronellal and menthol in an olfactory test and this behavior was reduced in dTrpA1 mutant flies. Both compounds activate all dTRPA1 isoforms in the heterologous expression system HEK293T, with the following sensitivity series: dTRPA1(C) = dTRPA1(D) > dTRPA1(A) ≫ dTRPA1(B) for citronellal and dTRPA1(A) > dTRPA1(D) > dTRPA1(C) > dTRPA1(B) for menthol.

Conclusions: dTrpA1 was required for the normal avoidance of Drosophila melanogaster towards citronellal and menthol. All dTRPA1 isoforms are activated by both compounds, but the dTRPA1(B) is consistently the least sensitive. We discuss how these findings may guide further studies on the physiological roles and the structural bases of chemical sensitivity of TRPA1 channels.

Keywords: AITC; Drosophila melanogaster; HC-030031; TRPA1; avoidance; citronellal; isoform; menthol; olfaction; repellent.

Figure 1

Adult Drosophila melanogaster flies avoid citronellal and menthol in a dTrpA1-dependent manner. (a) Direct airborne repellent test. Thirty minutes after loading the vehicle and the test solutions at the locations indicated by the blue and green crosses respectively, the flies found in vehicle and test areas (marked by the blue and green shades) were counted and the avoidance index (A.I.) was calculated using the formula shown on the right. #Flies in Exp and #Flies in Veh stand for the number of flies found in the regions corresponding to the experimental compound and vehicle, respectively. Positive values of A.I. indicate preference and negative values indicate avoidance. (b) Examples of the positions of WT (left) and dTrpA1¹ (right) flies exposed to vehicle (blue; 20 µL DMSO) and citronellal solutions (green; 20 µL of 1 mM citronellal in DMSO). The bar graph on the right shows the average A.I. to citronellal for WT flies (n = 5 plates, one-sample t-test vs. 0, p = 0.026 indicated with *) and dTrpA1¹ flies (n = 6 plates, one-sample t-test vs. 0, p = 0.4). WT vs. dTrpA1¹, two sample t-test, p = 0.26. (c) Examples of WT (left) and dTrpA1¹ (right) fly positions after exposure to vehicle (blue; 20 µL ethanol) and the menthol solutions (green; 20 µL of 1 mM menthol in ethanol). The bar graph on the right displays the average A.I. to menthol for WT (n = 4 plates, one-sample t-test vs. 0, p = 0.004 indicated by ***) and dTrpA1¹ flies (n = 4 plates, one-sample t-test vs. 0, p = 0.26). WT vs. dTrpA1¹, two sample t-test, p = 0.0004.

Figure 2

Basal currents recorded in HEK293T cells expressing dTRPA1 isoforms. (a) Examples of basal (non-stimulated) currents recorded in non-transfected cells and in cells expressing either of the four dTRPA1 channel isoforms. (b) Average basal current density determined at +75 mV (filled bars) and −75 mV (open bars) in non-transfected and in cells expressing dTRPA1(A), dTRPA1(B), dTRPA1(C) or dTRPA1(D) (n = 4, 66, 12, 16 and 12, respectively). (c) Average rectification index determined for the same cells as in (b). The * symbols indicate p < 0.05 compared to non-transfected cells, with Kruskal-Wallis test. The # symbols indicate p < 0.05 compared to 1, with Wilcoxon signed rank test.

Figure 3

Citronellal increases intracellular [Ca²⁺] in cells transfected with dTRPA1 isoforms. (a–d) Example traces of changes of intracellular Ca²⁺ concentration induced by citronellal (0.001–1 mM) and AITC (100 µM) in HEK293T cells expressing either dTRPA1(A), dTRPA1(B), dTRPA1(C) or dTRPA1(D). (e,f) Concentration dependence of the average amplitude of intracellular Ca²⁺ increase induced by citronellal; panel (e) raw data and panel (f) data obtained by normalizing to the amplitude of the response to AITC in each cell. n = 102–298, 21–75, 30 and 39 for isoforms A to D, respectively.

Figure 4

Citronellal increases whole-cell currents in the four dTRPA1 isoforms. (a–d) Examples of the effects of citronellal (1 mM), HC-030031 (HC; 100 µM) and AITC (100 µM) on the currents recorded at +75 mV (circles) and −75 mV (squares) in whole-cell patch-clamp experiments in HEK293T cells expressing either dTRPA1(A), dTRPA1(B), dTRPA1(C) or dTRPA1(D). A solution where all cations were replaced by N-methyl-D-glucamine (NMDG⁺) was used to assess the presence of leak currents [44]. The middle insets represent current traces recorded at the time points indicated by the corresponding colors in the left panels. The dot plots on the right represent the change in current density amplitude with respect to baseline values (recorded at +75 and −75 mV). The horizontal lines represent medians and the boxes represent the 25–75 percentiles. Application of citronellal (1 mM, red symbols) induced whole-cell current increases in cells expressing: dTRPA1(A) (p = 0.003, n = 5), dTRPA1(B) (p = 0.01, n = 7), dTRPA1(C) (p = 0.0016, n = 6) or dTRPA1(D) (p = 0.01, n = 8, one-sample t-test vs. 0). The TRPA1 blocker HC-030031 (HC; 100 µM) inhibited citronellal-induced (1 mM) currents (blue symbols) in cells expressing dTRPA1(A) (p = 0.003, n = 4–5), dTRPA1(D) (p = 0.024, n = 7–8; two-sample t-test), dTRPA1(C) (p = 0.00006, n = 6) or dTRPA1(B) (p = 0.018, n = 7; one tailed paired sample t-test).

Figure 5

Menthol increases intracellular Ca²⁺ in cells transfected with dTRPA1 isoforms. (a–d) Example traces of changes of intracellular Ca²⁺ concentration induced by menthol (0.3–1 mM) and AITC (100 µM) in HEK293T cells expressing: dTRPA1(A), dTRPA1(B), dTRPA1(C) or dTRPA1(D). (e,f) Concentration dependence of the average amplitude of intracellular Ca²⁺ increase induced by menthol; panel (e) raw data and panel (f) data obtained by normalizing to the amplitude of the response to AITC in each cell. n = 127, 210, 43 and 65 for isoforms A to D, respectively.

Figure 6

Menthol increases whole-cell currents in the four dTRPA1 isoforms. (a–d) Example of the effects of menthol (1 mM), HC-030031 (HC; 100 µM) and AITC (100 µM) on the amplitude of whole-cell currents measured at +75 mV (circles) and −75 mV (squares) in HEK293T cells expressing either dTRPA1(A), dTRPA1(B), dTRPA1(C) or dTRPA1(D). An extracellular solution where all cations were replaced by N-methyl-d-glucamine (NMDG⁺) was used to assess the size of leak currents. The middle insets show current traces recorded at the time points indicated by the corresponding colors in the left panels. The dot plots on the right show the change in current density amplitude with respect to baseline values (+75 mV and −75 mV). The horizontal lines represent the medians and the boxes represent the 25–75 percentiles. Application of menthol (1 mM, red symbols) increased whole-cell current in cells expressing: dTRPA1(A) (p = 0.001, n = 8), dTRPA1(B) (p = 0.0003, n = 5), dTRPA1(C) (p = 0.0006, n = 10) or dTRPA1(D) (p = 0.003, n = 7, one-sample t-test vs. 0). The TRPA1 blocker HC-030031 (HC; 100 µM) partly inhibited menthol-induced currents (blue symbols) in cells expressing dTRPA1(A) (p = 0.04, n = 6–8), dTRPA1(B) (p = 0.024, n = 4–5; two-sample t-test), dTRPA1(C) (p = 0.0004, n = 10) or dTRPA1(D) (p = 0.004, n = 7; one tailed paired sample t-test). * p < 0.05.

Figure 7

Schematic representation of dTRPA1 sequences and proteins. Diagram of the genomic locus of dTrpa1 (left) resulting in different isoforms of dTRPA1 proteins (right). In the middle: the nomenclature that we maintain and the corresponding GenBank reference. ARD = ankyrin repeat domains, TM = trans-membrane segments.