Analysis of Drosophila TRPA1 reveals an ancient origin for human chemical nociception

Abstract

Chemical nociception, the detection of tissue-damaging chemicals, is important for animal survival and causes human pain and inflammation, but its evolutionary origins are largely unknown. Reactive electrophiles are a class of noxious compounds humans find pungent and irritating, such as allyl isothiocyanate (in wasabi) and acrolein (in cigarette smoke). Diverse animals, from insects to humans, find reactive electrophiles aversive, but whether this reflects conservation of an ancient sensory modality has been unclear. Here we identify the molecular basis of reactive electrophile detection in flies. We demonstrate that Drosophila TRPA1 (Transient receptor potential A1), the Drosophila melanogaster orthologue of the human irritant sensor, acts in gustatory chemosensors to inhibit reactive electrophile ingestion. We show that fly and mosquito TRPA1 orthologues are molecular sensors of electrophiles, using a mechanism conserved with vertebrate TRPA1s. Phylogenetic analyses indicate that invertebrate and vertebrate TRPA1s share a common ancestor that possessed critical characteristics required for electrophile detection. These findings support emergence of TRPA1-based electrophile detection in a common bilaterian ancestor, with widespread conservation throughout vertebrate and invertebrate evolution. Such conservation contrasts with the evolutionary divergence of canonical olfactory and gustatory receptors and may relate to electrophile toxicity. We propose that human pain perception relies on an ancient chemical sensor conserved across approximately 500 million years of animal evolution.

Figure 1. dTrpA1 mediates gustatory responses to reactive electrophiles

a, Chemical structures. b, Proboscis extension response (PER) frequency at five sequential tastant offerings, ingestion permitted. (*p<0.05, **p<0.01, unpaired t-test.) c, PER, tastant contacts only legs. Five sequential offerings combined (n≥10 flies). d, PER, ingestion permitted: light blue, first offering; dark blue, second to fifth offerings combined. Statistically distinct groups marked by different letters (Tukey HSD, α=0.01). Data are mean +/− SEM. All studies use 12% (350mM) sucrose, alone or with 100 mM caffeine, 2 mM AITC, 10 mM NMM, or 6 mM CA. n=3 groups of ≥7 flies, unless noted.

Figure 2. dTrpA1 functions in chemosensors

a–c, dTRPA1 expression. Arrows denote cell bodies, arrowheads distal neurites. Cuticle autofluorescence visible. (b), LSO sensilla numbered. apn: accessory pharyngeal nerve. d. dTrpA1 mutants lack dTRPA1 staining. e. eso, esophagus. f–i, Gr66a-Gal4 and dTRPA1 co-expression in LSO. (f) Nuclear and (g,h) membrane GFPs expressed using Gr66a-Gal4. j–l, PER to 350 mM sucrose containing 10mM NMM. (j, k) Ingestion permitted. l, tarsal contact only. j, dTRPA1 knockdown. k, dTRPA1 rescue. l, dTRPA1 gain-of-function. *: α=0.05, **: α=0.01, differ from Gal4 and UAS controls, Tukey HSD. j,k, n=3 groups of 7–8 flies, l, n≥10 flies.

Figure 3. Insect TRPA1s are reactive electrophile sensors

a–e. Representative responses of dTRPA1 (a–d) and agTRPA1 (e) expressed in oocytes. Left panels, currents at −60 and +60 mV. Perfusion buffer containing indicated chemical (a, c, d, 100 μM; b, e, 40 μM) was applied for 60–80 sec. 100 μM ruthenium red (RR) applied as noted. Right panels show I–V relationships at points marked on left panels. f, g, Ectopic dTRPA1 expression confers electrophile sensitivity upon motor neurons. f, Motor neuron-driven excitatory junction potentials (EJPs) from third instar larval muscles. g. Mean EJP frequencies. In controls, no EJPs were observed.

Figure 4. TRPA phylogeny

a, Conservation of residues implicated in electrophile detection. choannoflag, choannoflagellate. b, TRPA1-wt (wild type) and dTRPA1-2C channels in Xenopus oocytes. 60 sec pulses of AITC (0.1, 0.5, and 1.0 mM) were applied with 25 sec intervals. c. +60 mV currents normalized to channel's response to 1.0 mM AITC. *p<0.05, **p<0.001, unpaired t-test. d, Bayesian consensus phylogeny for TRPAs. Internal branches labeled by posterior probability (<0.5 branches collapsed). Red dot denotes ancestor of TRPA1 clade. e, Cladogram showing TRPA complements, including numbers of channels. Red dot denotes bilaterian ancestor. f, PAML residue identity estimates for ancestral TRPA1.