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Biochem Biophys Res Commun
2020 Jan 22;5214:914-920. doi: 10.1016/j.bbrc.2019.10.203.
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Unique high sensitivity to heat of axolotl TRPV1 revealed by the heterologous expression system.
Hori S
,
Saitoh O
.
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The thermosensation mechanism plays critical roles in various animals living in different thermal environment. We focused on an axolotl, which is a tailed amphibian originally from Lake Xochimilco area in the Vally of Mexico, and examined its behavior response to heat stimulation. Mild heat at 33 °C induced noxious locomotive activity to axolotls, but the noxious response of another tailed amphibian, Iberian ribbed newt, was not observed at 33 °C. To explore the mechanism for the temperature sensitivity of axolotls, we isolated a cDNA of TRPV1. Using the degenerate primer PCR method, we identified the DNA fragment encoding axolotl TRPV1 (axTRPV1), and then cloned a full-length cDNA. We studied the chemical and thermal sensitivities of axTRPV1 by two-electrode voltage clamp method using Xenopus oocyte expression system. Capsaicin, acid, and 2-aminoethoxydiphenylborane apparently activated axTRPV1 channels in a dose-dependent manner. The analysis of thermal sensitivity showed that axTRPV1 was significantly activated by heat but not by cold. The average temperature threshold for heat-activation was 30.95 ± 0.12 °C. This thermal activation threshold of axTRPV1 is unique and significantly low, when compared with the known thresholds of TRPV1s from various animals. Further, this threshold of axTRPV1 is well consistent with the observation of heat-induced behavior of axolotls at 33 °C, demonstrating that axolotl shows noxious response to mild heat mediated through axTRPV1.
Fig. 1. Behavior response to heat of axolotls.
An axolotl or an Iberian ribbed newt (RN) was placed into a metallic tray with pre-warmed (25 °C, 33 °C, or 39 °C) the tap water. Heat-induced movements per 2 s (axolotl: n = 4) or 1 s (NR: n = 3) were measured and the mean movement (cm/min) was calculated. Statistical significance for differences between two groups was determined by Student’s unpaired t-test and was indicated by * (p < 0.05) and ** (p < 0.01).
Fig. 2. The expression of axolotl TRPV1.
(A) Total RNA was isolated from various tissues and subjected to reverse transcription with random primers. The reverse-transcribed cDNA was used as a template for PCR (+). Total RNA treated under the same conditions without reverse-transcriptase was used as a negative control (−). The primers used for β-actin and axolotl TRPV1 were described in the section of Materials and Methods
(B) Total RNA was isolated from developing embryos (st.32 (late tailbud), st.40 (pre-hatching)) and hatching larvae (Hatch) and the expression of β-actin and axTRPV1 was examined as indicated above.
Fig. 3. Chemical response of oocytes expressing axolotl TRPV1
(A) Effects of capsaicin on ionic currents in Xenopus oocytes expressing axTRPV1 were examined. 1 μM–100 μM capsaicin were used. Average current at 30 s after capsaicin stimulation was plotted as a function of chemical concentration. Each data point represents the mean ± S. E (n = 5).
(B) Effects of acid on ionic currents of axTRPV1 were similarly examined and analyzed (n = 5).
(C) Effects of 2-aminoethoxydiphenyl borate (2-APB) on ionic currents of axTRPV1 were similarly examined and analyzed (n = 5)
Fig. 4. Thermal sensitivities of axolotl TRPV1
(A) Effects of cold and heat stimulation on ionic currents in Xenopus oocytes expressing axTRPV1 were examined. Experimental conditions were as described in the materials and methods section. (B) Peak average currents for heat stimulation at 30 °C were compared. Each data point represents the mean ± S. E (n = 8). Statistical significance for difference was determined by Student’s unpaired t-test and was indicated by *** (p < 0.001). (C) The elicited currents at −80 mV shown in (A) were plotted as a function of temperature. (D) Arrhenius plots of the current of axTRPV1 elicited by heat stimulation. (E) Electrophysiological response of oocytes expressing axTRPV1 to the bath solution at 20 °C, 25 °C, 30 °C, and 35 °C. Voltage ramps from −100 mV to +100 mV were applied every 2 s.
Supplemental Fig. 1. Phylogenetic tree of vertebrate TRPV family.
Amino acid sequences of TRPA1 from human (NP_015628.2), TRPV1s from human (NP_542435.2), mouse (NP_001001445.1), rat (NP_114188.1), chicken (NP_989903.1), rattlesnake (ADD82931.1), western clawed frog (wcfrog, NP_001243521.1), medaka (XP_011482044.1), and zebrafish (NP_001119871.1), TRPV2s from human (NP_057197.2), mouse (EDL10354.1), rat (NP_058903.2), chicken (XP004946742.1), and wcfrog (XP_002938302.2), TRPV3s from human (NP_001245134.1) and rat (NP_001020928.2), TRPV4s from human (NP_001170904.1) and rat (NP_076460.1), TRPV5s from human (NP_062815.3) and rat (NP_446237.2), and TRPV6s from human (NP_061116.5) and rat (NP_446138.1) were used for a maximum likelihood analysis.
Supplemental Fig. 2. Effects of blockers on capsaicin-induced currents of axTRPV1.
(A) In Xenopus oocytes, axTRPV1 was expressed and current traces were monitored. After the application of 20 μM capsaicin, blocker (SB366791 or capsazepine) was further applied to oocytes. Capsaicin and blocker were applied at the time indicated by the bars. (B) Using currents at −80 mV, the inhibition of rat or axolotl TRPV1 by blocker was estimated by [(capsaicin-induced currents after 60 s) – (currents after the 60 s treatment with blocker)] / (capsaicin-induced currents after 60 s). Each data point represents the mean ± S. E (n = 3). Differences between multiple groups were examined by the Tukey-Kramer method, with significance being indicated by a, b, and c (p < 0.05). When two letters are different, they are significantly different.
