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Biochem Biophys Res Commun
2017 Dec 09;4941-2:194-201. doi: 10.1016/j.bbrc.2017.10.057.
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Chemical and thermal sensitivity of medaka TRPA1 analyzed in heterologous expression system.
Oda M
,
Saito K
,
Hatta S
,
Kubo Y
,
Saitoh O
.
???displayArticle.abstract??? TRPA1 of insects and several tetrapod vertebrates except for those of rodents have been reported to be activated by noxious chemicals and also by high temperature with a relatively clear threshold. We previously analyzed the characteristics of two TRPA1 paralogs of zebrafish (zTRPA1a, b) and demonstrated that zTRPA1a is specialized for chemical sensing while zTRPA1b responds to thermal stimulations, that zTRPA1b responds to both cold and heat stimuli, and that heat stimulation gradually activates zTRPA1b without a clear threshold. In the medaka genome, a single TRPA1 (olTRPA1) gene is present. To examine if functional properties of olTRPA1 are similar to TRPA1 of land animals or either of zTRPA1a or zTRPA1b, we isolated a TRPA1 cDNA from medaka and performed functional analyses. OlTRPA1 showed a sensitivity to four noxious chemicals (allyl isothiocyanate, caffeine, carvacrol, methyl anthranilate). We observed that cold stimulation does not activate olTRPA1, but heat stimulation gradually activates olTRPA1 with an unclear threshold. Results suggested that a single TRPA1 functions as a chemical and thermal sensor in medaka, and that a gradual heat-activation without clear threshold might be a common feature for TRPA1 of fish living in water.
Fig. 1. Chemical response of oocytes expressing medaka TRPA1. (A) Effects of chemical stimulation on ionic currents in Xenopus oocytes expressing medaka TRPA1 (olTRPA1) were examined. Allyl isothiocyanate (AITC), caffeine, carvacrol, methyl anthranilate (MA) were used as noxious chemicals. Experimental conditions were as described in the materials and methods section. (B) Effects of chemicals on ionic currents in oocytes expressing olTRPA1 or mouse TRPA1 (mTRPA1) were examined. Average current at 8 s after chemical stimulation was plotted as a function of chemical concentration. Each data point represents the mean ± S. E.
Fig. 2. Effect of TRPA1 blocker on medaka TRPA1. (A) Oocytes expressing mouse or medaka TRPA1 were stimulated twice with caffeine and further treated with caffeine and TRPA1 blocker, HC-030031. Experimental conditions were same as Fig. 1. (B) Average currents at 8 s after stimulation were compared. Each data point represents the mean ± S. E. Statistical significance for difference was determined by Student's unpaired t-test and was indicated by * (p < 0.05).
Fig. 3. Behavior response of medaka fish to MA. (A) Three adult fish of medaka were placed into MA-containing water and the locomotor activity of each fish was monitored by video camera. (B) During the first 10 s, irritant-induced movements/second were measured and the mean values were obtained. Statistical significance for differences between multiple groups was determined by the Tukey-Kramer method, and was indicated by * (p < 0.05) for the focused groups.
Fig. 4. Thermal sensitivities of medaka TRPA1. (A) Effects of cold and heat stimulation on ionic currents in Xenopus oocytes expressing olTRPA1 were examined. Experimental conditions were as described in the materials and methods section. (B) Peak average current for cold stimulation and at 40 °C were compared. Each data point represents the mean ± S. E. (C) Elicited outward currents shown in (A) were plotted as a function of temperature. (D) Arrhenius plots of the current elicited by cold and heat stimulation. Statistical significance for differences between multiple groups was determined by Student’s unpaired t-test, and was indicated by ** (p < 0.01) for the focused groups.
