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.
Highlights
transient receptor potential ankyrin 1 (TRPA1) is a Ca2+-permeable non-selective cation channel found in vertebrates and invertebrates [1,2,3]
We focused on the non-electrophilic compounds citronellal and menthol because the four dTRPA1 isoforms have been shown to respond to the electrophilic agonist AITC [21,38]
We focused on the non-electrophiles citronellal and menthol, as they have been shown to trigger avoidance responses in Drosophila melanogaster [11,14,34,40]
Summary
TRPA1 is a Ca2+-permeable non-selective cation channel found in vertebrates and invertebrates [1,2,3]. We tested whether these in vivo effects could be mediated by activation of dTRPA1 by characterizing the responses of the different channel isoforms using HEK293T cells as heterologous expression system. These experiments yielded that all four dTRPA1 isoforms can be activated by citronellal and menthol, albeit with markedly different sensitivities. The currents recorded at −75 mV in cells transfected with dTRPA1(C) or dTRPA1(D) were not significantly larger than in non-transfected cells, but outward currents were significantly larger (Figure 2a,b), which resulted in larger rectification indexes (~6 and ~5, respectively; Figure 2c) These data show that isoforms A, C and D display constitutive activity when heterologously expressed in HEK293T cells. For the B isoform, the evidence of constitutive activity is limited to the finding that the currents showed significant outward rectification, in contrast to the currents recorded in non-transfected cells
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