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FIGURE 1. Behavioral responses to heat and thermal sensitivity of sensory neurons differ between X. laevis and X. tropicalis
(A) Each bar represents the number of jumping behaviors in 1-minute intervals before and during the heat stimulations (n = 4 each for both X. laevis and X. tropicalis). The number of jumping behaviors in the first 1 minute-interval during heat stimulation are summarized (lower; n = 4, 5). *t-test, p<0.05. Note that the data for X. tropicalis were adopted from Ohkita et al. (2012, Fig. 7F) (4). (B) Species differences in the heat response of Xenopus DRG neurons. Representative traces of [Ca2+]i changes in response to heat stimulation in DRG neurons from X. laevis (left) and X. tropicalis (right). The thermal activation threshold is defined as the point at which [Ca2+]i exceeds 10 nM from the baseline. (C) Comparison of thermal activation thresholds of DRG neurons between the two Xenopus species (n = 13 for X. laevis and n = 15 for X. tropicalis from three frogs per species). Each data point represents the mean ± S.E.
*t-test, p<0.05.
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FIGURE 2. Phylogenetic relationship of vertebrate TRPV1 and TRPA1 including Xenopus species (A and B) Evolutionary distances were estimated by comparing amino acid sequences and phylogenetic trees were reconstructed with the Neighbor-Joining method (24). Statistical confidence (bootstrap value) is shown beside each node of the phylogenetic tree. Scale bar indicates 0.05% amino acid substitutions per site.
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FIGURE 3. Species differences in heat response of TRPV1 between the two Xenopus species
(A, B, and D) Representative traces for current and temperature in Xenopus oocytes expressing X. laevis (Xla) TRPV1a (A), Xla-TRPV1b (B), and X. tropicalis (Xtr) TRPV1 (D) in response to repeated heat stimulation. (C) Average current amplitudes elicited by Xenopus oocytes expressing Xla-TRPV1a (n = 11) or Xla-TRPV1b (n = 12). For each channel, the same amount of cRNA was injected into the Xenopus oocytes and current measurements were performed 3 days post-injection with three independent preparations. Current amplitudes for Xla-TRPV1a were significantly larger than those for Xla-TRPV1b (two-way repeated measures ANOVA, p < 0.01). (E) Current amplitudes elicited by repeated heat stimulations were normalized to the maximum currents (the first and third heat stimulation for Xla-TRPV1a and Xtr-TRPV1, respectively) obtained from Xenopus oocytes expressing Xla-TRPV1a (n =
15) or Xtr-TRPV1 (n = 14) with three independent preparations. (F) Thermal activation thresholds for Xla-TRPV1a and Xtr-TRPV1. Dots indicate individual values obtained from different Xenopus oocytes (eight independent preparations) expressing Xla-TRPV1a (n = 31) or Xtr-TRPV1 (n = 18). Average values are shown with bars.
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FIGURE 4. Comparison of Xla-TRPV1a and Xtr-TRPV1 heat responses by expression in HeLa cells Average increase in [Ca2+]i in response to heat stimulation in HeLa cells expressing Xla-TRPV1a (n = 10-16) or Xtr-TRPV1 (n = 9-15). A single heat stimulation was applied for each respective thermal stimulation. Each data point represents the mean ± S.E. *t-test, P<0.05
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FIGURE 5. Ankyrin repeat domains (ARD) 1 to 3 in Xenopus TRPV1 are responsible for the species differences in heat response
(A) Schematic structures of the chimeras between the Xla-TRPV1a and Xtr-TRPV1 channels. Orange and blue bars represent the fragments of Xtr-TRPV1 (T) and Xla-TRPV1a (L), respectively. The names of the chimeras represent the combination of the fragments derived from the respective species. For example, in the X. TRPV1 LTL chimera, the central region of Xtr-TRPV1 was introduced onto the Xla-TRPV1a background. (B and C) Averaged normalized currents obtained from repeated heat stimulation in Xenopus oocytes expressing Xla-TRPV1a, Xtr-TRPV1 and various X. TRPV1 chimeras (n = 16 or 18 for Xtr-TRPV1, n = 12, or 14 for X.TRPV1 LTL, n = 15 for Xla-TRPV1a, n = 11 or 12 for X.TRPV1 LLT, n = 9 or 11 for X.TRPV1 LTT, n = 16 or 17 for X.TRPV1 TLTT, n = 9 or 10 for X.TRPV1 TLTTT). For each of the channels, average current amplitudes were calculated for the respective heat stimulation and the current amplitude whose average value was largest was used for normalization.
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FIGURE 6. A single amino acid substitution is involved in the species differences in TRPV1 heat responses in Xenopus
(A) Amino acid sequence alignment of Xenopus TRPV1 within the region that was identified by the chimeric analysis shown in Fig. 5. Conserved residues are shown in red, and the residues substituted to similar or different amino acids are shown in blue or black, respectively. The positions where mutations were introduced are marked with arrows. The positions involved in the species differences in TRPV1 heat responses are shown in red. The numbering of the positions is in accordance with Xtr-TRPV1. Positions of the ARD were based on rat TRPV1 (38). (B) Normalized currents elicited by Xenopus oocytes expressing Xtr-TRPV1 mutants (n = 10 for Xtr-TRPV1, n = 13 for Xtr-TRPV1 N181K, n = 9 for Xtr-TRPV1 S185H, n = 12 for Xtr-TRPV1 P188L, n = 12 for Xtr-TRPV1 S185H/P188L, n = 12 or 13 for Xtr-TRPV1 N181K/S185H/P188L, n = 9 for Xla-TRPV1a). The currents were normalized to the maximum average current among five heat stimulations for each channel (the first for Xla-TRPV1a; the second for Xtr-TRPV1 P188L and Xtr-TRPV1 N181K/S185H/P188L; the third for Xtr-TRPV1 N181K
and Xtr-TRPV1 S185H/P188L; the fourth for Xtr-TRPV1 and Xtr-TRPV1 S185H) (C) Representative traces for current and temperature in response to repeated heat stimulation in Xenopus oocytes expressing Xtr-TRPV1 S185H (left) and Xtr-TRPV1 P188L (right)
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FIGURE 7. Heat-induced activity differs among Xenopus TRPV1 mutants
Average current amplitudes elicited by repeated heat stimulation in Xenopus oocytes expressing Xenopus TRPV1 mutants (A: n = 7 for Xtr-TRPV1, n = 9 for Xtr-TRPV1 N181K, n = 6 for Xtr-TRPV1 S185H, n = 9 for Xtr-TRPV1 P188L, n = 8 for Xtr-TRPV1 S185H/P188L, n = 9 or 10 for Xtr-TRPV1 N181K/S185H/P188L, n = 5 for Xla-TRPV1a, B: n = 5 for Xla-TRPV1a, n = 10 for Xla-TRPV1a K179N, n = 7 for Xla-TRPV1a H183S, n = 7 for Xla-TRPV1a L186P, n = 8 for Xla-TRPV1a H183S/L186P, n = 8 for Xla-TRPV1a K179N/H183S/L186P, n = 7 for Xtr-TRPV1). For each of the channels, the same amount of cRNA was injected into the Xenopus oocytes and current measurements were performed 3 days post-injection.
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FIGURE 8. Two conservative amino acid substitutions are also involved in species differences in the TRPV1 heat response in Xenopus
(A) Normalized currents elicited by repeated heat stimulation in Xenopus oocytes expressing Xtr-TRPV1 mutants (n = 16 or 19 for Xtr-TRPV1, n = 12 or 13 for Xtr-TRPV1 D141E, n = 14 for Xtr-TRPV1 K156R, n = 12 for Xtr-TRPV1 E180D, n = 12 or 14 for Xtr-TRPV1 E232Q, n = 14 or 15 for Xtr-TRPV1 K156R/E232Q). The currents were normalized to the maximum average current among five heat stimulations for each channel (the second for Xtr-TRPV1 K156R and Xtr-TRPV1 K156R/E232Q; the third for Xtr-TRPV1, Xtr-TRPV1 D141E, Xtr-TRPV1 E180D, and Xtr-TRPV1 E232Q) (B) Average current amplitudes elicited by repeated heat stimulation in Xenopus oocytes expressing Xtr-TRPV1 mutants (n = 11 or 14 for Xtr-TRPV1, n = 7 or 8 for Xtr-TRPV1 D141E, n = 9 for Xtr-TRPV1 K156R, n = 7 for Xtr-TRPV1 E180D, n = 8 for Xtr-TRPV1 E232Q, n = 9 or 10 for Xtr-TRPV1 K156R/E232Q). For each of the channels, the same amount of cRNA was injected into the Xenopus oocytes and current measurements were performed 3 days post-injection. (C) Normalized currents elicited by repeated heat stimulation in Xenopus oocytes expressing Xtr-TRPV1 mutants (n = 5 or 7 for Xtr-TRPV1, n = 4 for Xtr-TRPV1 D141E/P188L, n = 4 for Xtr-TRPV1 K156R/P188L, n = 4 for Xtr-TRPV1 E180D/P188L, n = 4 or 5 for Xtr-TRPV1 E232Q/P188L). The currents were normalized to the second stimulation except for Xtr-TRPV1 in which currents were normalized to the third stimulation.
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FIGURE 9. A single amino acid substitution drastically shifted TRPV1 sensitivity to capsaicin between the two Xenopus species
(A) Alignment of amino acid sequences in transmembrane domain 3 of TRPV1. The arrow marks the amino acid known to be involved in TRPV1 sensitivity to capsaicin. The numbering is based on that of
Xla-TRPV1a. (B-D) Representative traces of dose-dependent changes in [Ca2+]i in HeLa cells expressing Xla-TRPV1a (B), Xtr-TRPV1 (C), and Xla-TRPV1a C521Y (D) mutants in response to capsaicin stimulation with various concentrations (E, n = 12-21 for Xla-TRPV1a, n = 9-13 for Xtr-TRPV1, n = 10-12 for Xla-TRPV1 C521Y).
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FIGURE 10. Species differences in sensory neuron and behavioral responses to capsaicin between the two Xenopus species.
(A and B) Representative traces of dose-dependent changes in [Ca2+]i in responses to capsaicin stimulation in DRG neurons from X. laevis (upper) or X. tropicalis (lower) and their dose dependency (B, n = 7-16 for X. laevis DRG and n = 11-14 for X. tropicalis DRG). K+: 80 mM of KCl. (C, upper and middle) Each bar represents the number of jumping behaviors in 1-minute intervals before and during capsaicin stimulation. (C, lower) The number of jumping behaviors in the first three 1-minute intervals during capsaicin stimulation are summarized (n = 4 for X. laevis and n = 4 for X. tropicalis). Note that the data for X. tropicalis was adopted from Ohkita et al. (2012, Fig. 7E) (4).
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FIGURE 11. The heat response of TRPA1 differs between the two Xenopus species (A-C) Representative traces for current and temperature in Xenopus oocytes expressing Xla-TRPA1a (A), Xla-TRPA1b (B) or Xtr-TRPA1 (C) in response to heat and cinnamaldehyde stimulation. (D-F) Representative Arrhenius plots for Xla-TRPA1a (D), Xla-TRPA1b (E), and Xtr-TRPA1 (F). (G) Thermal activation thresholds for Xla-TRPA1a and Xtr-TRPA1. Dots indicate individual values obtained from different Xenopus oocytes (four independent preparations) expressing Xla-TRPA1a (n = 34) or Xtr-TRPA1 (n = 32; unpaired t-test with p < 0.005). (H) Dose-dependency of Xla-TRPA1a or Xtr-TRPA1 in response to cinnamaldehyde (each data point consists of 4-11 observations). (I) Normalized heat-induced currents for Xla-TRPA1a and Xtr-TRPA1. Heat stimulation was applied following 0.3 mM cinnamaldehyde stimulation as shown in panels A and C. The current amplitude elicited by heat was normalized to that of cinnamaldehyde (Xla-TRPA1a: n =29; Xtr-TRPA1: n = 38; four independent preparations; unpaired t-test with p < 0.001).
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