RESUMEN

Drosophila TrpA1 (transient receptor potential ankyrin 1) transcripts are alternatively spliced at two distinct sites each with a choice of mutually exclusive exons. The first site determines exon1 encoding the amino terminus to produce either nucleophile-, electrophile- and noxious temperature-gated TRPA1(A) or electrophile- and innocuous warmth-gated TRPA1(B). The second site selects for exon10, resulting in TrpA1 variants with either exon10a or exon10b encoding a domain between the N-terminal ankyrin repeats and the transmembrane segments. Although unbiased assembly would generate TRPA1 with four different domain combinations, the functional impact of these alternative domains remains to be thoroughly examined. Here, we find that there is a relatively strong linkage in mRNA splicing between the two sites in case of TrpA1(B), but not TrpA1(A), transcripts. Our semi-quantitative assay, consisting of reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing, revealed that exon10b is little coupled with TrpA1(B) transcripts, suggesting that only three isoforms, TRPA1(A)-exon10a [denoted as TRPA1(A)], TRPA1(A)-exon10b [TRPA1(A)10b] and TRPA1(B)-exon10a [TRPA1(B)], are present at detectable levels using our method. Interestingly, heterologously expressed TRPA1(A)10b showed elevated sensitivity to low concentrations of N-methyl maleimide (NMM), a cysteine-modifying electrophile, compared with other isoforms. Equivalent isoforms in malaria-transmitting Anopheles gambiae displayed a similar pattern of isoform-dependent NMM dose dependences, suggesting that the chemosensory regulation by selective domain assembly is conserved in insect TRPA1s. Thus, alternative RNA splicing of exon10 is coordinated in conjunction with the first exons, regulating chemical sensitivity of insect TRPA1s.

RESUMEN

Nociception is the sensory perception of noxious chemical stimuli. Repellent behavior to avoid noxious stimuli is indispensable for survival, and this mechanism has been evolutionarily conserved across a wide range of species, from mammals to insects. The transient receptor potential ankyrin 1 (TRPA1) channel is one of the most conserved noxious chemical sensors. Here, we describe the heterologous stable expression of Tribolium castaneum TRPA1 (TcTRPA1) in human embryonic kidney (HEK293) cells. The intracellular Ca2+ influx was measured when two compounds, citronellal and l-menthol, derived from plant essential oils, were applied in vitro using a fluorescence assay. The analysis revealed that citronellal evoked Ca2+ influx dose-dependently for TcTRPA1, whereas l-menthol did not. In combination with our present and previous results of the avoidance-behavioral assay at the organism level, we suggest that TcTRPA1 discriminates between these two toxic compounds, and diversification in the chemical nociception selectivity has occurred in TRPA1 channel among insect taxa.

RESUMEN

Chemically induced nociception has not yet been studied intensively in genetically tractable models. Hence, our goal was to establish a Drosophila assay that can be used to study the cellular and molecular/genetic bases of chemically induced nociception. Drosophila larvae exposed to increasing concentrations of hydrochloric acid (HCl) produced an increasingly intense aversive rolling response. HCl (0.5%) was subthreshold and provoked no response. All classes of peripheral multidendritic (md) sensory neurons (classes I-IV) are required for full responsiveness to acid, with class IV making the largest contribution. At the cellular level, classes IV, III and I showed increases in calcium following acid exposure. In the central nervous system, Basin-4 second-order neurons are the key regulators of chemically induced nociception, with a slight contribution from other types. Finally, chemical nociception can be sensitized by tissue damage. Subthreshold HCl provoked chemical allodynia in larvae 4 h after physical puncture wounding. Pinch wounding and UV irradiation, which do not compromise the cuticle, did not cause chemical allodynia. In sum, we developed a novel assay to study chemically induced nociception in Drosophila larvae. This assay, combined with the high genetic resolving power of Drosophila, should improve our basic understanding of fundamental mechanisms of chemical nociception. This article is part of the Theo Murphy meeting issue 'Evolution of mechanisms and behaviour important for pain'.

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RESUMEN

Solar irradiation including ultraviolet (UV) light causes tissue damage by generating reactive free radicals that can be electrophilic or nucleophilic due to unpaired electrons. Little is known about how free radicals induced by natural sunlight are rapidly detected and avoided by animals. We discover that Drosophila Transient Receptor Potential Ankyrin 1 (TRPA1), previously known only as an electrophile receptor, sensitively detects photochemically active sunlight through nucleophile sensitivity. Rapid light-dependent feeding deterrence in Drosophila was mediated only by the TRPA1(A) isoform, despite the TRPA1(A) and TRPA1(B) isoforms having similar electrophile sensitivities. Such isoform dependence re-emerges in the detection of structurally varied nucleophilic compounds and nucleophilicity-accompanying hydrogen peroxide (H2O2). Furthermore, these isoform-dependent mechanisms require a common set of TRPA1(A)-specific residues dispensable for electrophile detection. Collectively, TRPA1(A) rapidly responds to natural sunlight intensities through its nucleophile sensitivity as a receptor of photochemically generated radicals, leading to an acute light-induced behavioral shift in Drosophila.

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