Analysis of transient receptor potential ankyrin 1 (TRPA1) in frogs and lizards illuminates both nociceptive heat and chemical sensitivities and coexpression with TRP vanilloid 1 (TRPV1) in ancestral vertebrates

Abstract

Transient receptor potential ankyrin 1 (TRPA1) and TRP vanilloid 1 (V1) perceive noxious temperatures and chemical stimuli and are involved in pain sensation in mammals. Thus, these two channels provide a model for understanding how different genes with similar biological roles may influence the function of one another during the course of evolution. However, the temperature sensitivity of TRPA1 in ancestral vertebrates and its evolutionary path are unknown as its temperature sensitivities vary among different vertebrate species. To elucidate the functional evolution of TRPA1, TRPA1s of the western clawed (WC) frogs and green anole lizards were characterized. WC frog TRPA1 was activated by heat and noxious chemicals that activate mammalian TRPA1. These stimuli also activated native sensory neurons and elicited nocifensive behaviors in WC frogs. Similar to mammals, TRPA1 was functionally co-expressed with TRPV1, another heat- and chemical-sensitive nociceptive receptor, in native sensory neurons of the WC frog. Green anole TRPA1 was also activated by heat and noxious chemical stimulation. These results suggest that TRPA1 was likely a noxious heat and chemical receptor and co-expressed with TRPV1 in the nociceptive sensory neurons of ancestral vertebrates. Conservation of TRPV1 heat sensitivity throughout vertebrate evolution could have changed functional constraints on TRPA1 and influenced the functional evolution of TRPA1 regarding temperature sensitivity, whereas conserving its noxious chemical sensitivity. In addition, our results also demonstrated that two mammalian TRPA1 inhibitors elicited different effect on the TRPA1s of WC frogs and green anoles, which can be utilized to clarify the structural bases for inhibition of TRPA1.

FIGURE 1.

Noxious chemical sensitivities of WC frog TRPA1 expressed in X. laevis oocytes. A, a representative current trace for CA-evoked responses in X. laevis oocytes injected with WC frog TRPA1 cRNA. B, a dose-response curve for activation of WC frog TRPA1 by CA. Currents were normalized to the values at 2 mm. Each data point represents the mean ± S.E. (n = 4–5). C, a representative current trace for carvacrol-evoked responses in X. laevis oocytes injected with WC frog TRPA1 cRNA. D, a dose-response curve for activation of WC frog TRPA1 by carvacrol. Currents were normalized to the values at 2 mm. Each data point represents the mean ± S.E. (n = 4–7). E-G, representative current traces for AITC (E), acrolein (F), and 2-APB (G) stimulation in oocytes injected with WC frog TRPA1 cRNA (AITC, n = 4; acrolein, n = 3; 2-APB, n = 7).

FIGURE 2.

Noxious chemical sensitivities of WC frog TRPA1 expressed in HEK293 cells. A and B, representative traces of [Ca²⁺]_i changes in response to CA in HEK293 cells transfected with the pVenus-NLS vector harboring WC frog TRPA1 (A) and its dose-response curve (B). Each data point represents the mean ± S.E. (n = 10–17). C-E, representative traces of [Ca²⁺]_i changes in response to carvacrol (C), AITC (D), and acrolein (E) in HEK293 cells transfected with the pVenus-NLS vector harboring WC frog TRPA1 (carvacrol, n = 23; AITC, n = 13; acrolein, n = 11).

FIGURE 3.

Noxious chemical sensitivities of green anole TRPA1 expressed in X. laevis oocytes. A-G, all data were obtained by using X. laevis oocytes injected with green anole TRPA1 cRNA. A, a representative current trace for CA-evoked responses. B, a dose-response curve for activation of green anole TRPA1 by CA. Currents were normalized to the values at 4 mm. Each data point represents the mean ± S.E. (n = 5–10). C, a representative current trace for carvacrol-evoked responses. D, a dose-response curve for activation of green anole TRPA1 by carvacrol. Currents were normalized to the values at 2 mm. Each data point represents the mean ± S.E. (n = 4–8). E-G, representative current traces for AITC (E), acrolein (F), 2-APB (G) (AITC, n = 3; acrolein, n = 3; 2-APB, n = 4).

FIGURE 4.

Heat sensitivity of WC frog and green anole TRPA1s. A and B, representative current (upper) and temperature (lower) traces for heat and AITC stimulation in X. laevis oocytes injected with WC frog TRPA1 cRNA. Current-voltage relationships of heat- and AITC-evoked responses in X. laevis oocytes injected with WC frog TRPA1 cRNA (A, inset). B, a representative current trace of X. laevis oocytes injected with WC frog TRPA1 cRNA stimulated sequentially with cold, heat, and CA (n = 4). C, an Arrhenius plot of the current elicited by the first heat stimulation in panel A. The average temperature threshold for heat stimulation was 39.7 ± 0.7 °C (n = 12). D, average changes in [Ca²⁺]_i (solid line) in response to heat, CA, and ionomycin (Ion) and a temperature trace (dashed line) in HEK293 cells transfected with the pcDNA3.1(+) vector containing WC frog TRPA1. Each data point represents the mean ± S.E. (n = 64). E and F, representative current (upper) and temperature (lower) traces for heat (n = 11) or cold (n = 4) stimulation in X. laevis oocytes injected with green anole TRPA1 cRNA. G, an Arrhenius plot of the current elicited by the first heat stimulation in panel E. The average temperature threshold for heat stimulation was 33.9 ± 0.8 °C (n = 11). H, average changes in [Ca²⁺]_i (solid line) in response to heat, CA, and Ion and a temperature trace (dashed line) in HEK293 cells transfected with the pcDNA3.1(+) vector containing green anole TRPA1.

FIGURE 5.

Effects of mammalian TRPA1 inhibitors on WC frog and green anole TRPA1s. A, inhibition of WC frog TRPA1 activity by RR. The CA-induced current in oocytes injected with WC frog cRNA was inhibited by RR even in the presence of CA. The CA-induced current was recovered after washing out RR for about 2 min (n = 3). B, RR inhibited the increase in [Ca²⁺]_i elicited by CA application in HEK293 cells transfected with pVenus-NLS vector containing WC frog TRPA1. First, carvacrol was applied to show the functional expression of WC frog TRPA1 and then CA was applied (upper trace). To examine the effect of RR, the drug was applied prior to and during the application of CA (lower trace). C and E, HC-030031 (C) or AP18 (E) were applied prior to and during CA stimulation in X. laevis oocytes injected with WC frog TRPA1 cRNA (HC-030031, n = 3; AP18, n = 3). D and F, HEK293 cells transfected with pVenus-NLS vector containing WC frog TRPA1 were stimulated with CA (D, upper). HC-030031 (D, lower) or AP18 (F) were applied prior to and during CA application. G, the average increase in [Ca²⁺]_i in HEK293 cells transfected with the pVenus-NLS vector containing WC frog TRPA1. HC and AP represent HC-030031 and AP18, respectively. Each bar represents the mean ± S.E. (CA, n = 17; CA with RR, n = 15; CA with HC, n = 13; CA with AP, n = 18). RR significantly suppressed the average increase in [Ca²⁺]_i elicited by CA stimulation (*, p < 0.01). H and I, HC-030031 (H) or AP18 (I) were applied prior to and during CA stimulation in X. laevis oocytes injected with green anole TRPA1 cRNA (HC-030031, n = 3; AP18, n = 3).

FIGURE 6.

Functional expression of TRPA1 and TRPV1 in WC frog DRG neurons. A and B, pseudocolor images (A) and a representative trace (B) of [Ca²⁺]_i changes in WC frog DRG neurons stimulated with CA, Cap, and 80 mm of K⁺ (80K). Viability of DRG neurons was confirmed by activation with high concentration K⁺ stimulation (80K). B, the number of DRG neurons that responded to CA and/or Cap are summarized (313 DRG neurons from 8 frogs). The number of neurons that did not respond to both chemicals is indicated at the lower right (box). C, the numbers of DRG neurons that responded to AITC (0.3 mm) and/or Cap (0.3 mm) are summarized (70 DRG neurons from 3 frogs). D, a representative trace of [Ca²⁺]_i (upper) and temperature (lower) changes in WC frog DRG neurons stimulated with heat, CA, Cap, and 80K. The number of DRG neurons that responded to CA and/or Cap among the total heat-sensitive DRG neurons (n = 61, from 4 frogs) are summarized. Note that only one heat-sensitive DRG neuron did not respond to CA and Cap. E, representative traces of [Ca²⁺]_i (upper) and temperature (lower) changes of WC frog DRG neurons stimulated with cold, heat, Cap, CA, and 80K (n = 15, from 2 frogs). F, representative traces of [Ca²⁺]_i (upper) and temperature (lower) changes of WC frog DRG neurons stimulated with cold, CA, menthol, and 80K.

FIGURE 7.

Chemical sensitivity of DRG neurons in the WC frog. A, a representative trace of [Ca²⁺]_i changes in DRG neurons from WC frogs stimulated with increasing concentrations of CA. B, a dose-response curve for [Ca²⁺]_i changes by CA in DRG neurons. Each data point represents the mean ± S.E. (n = 10–17, from 3 frogs). C-E, representative traces for [Ca²⁺]_i changes in DRG neurons from WC frogs stimulated with carvacrol (C), AITC (D), or acrolein (E), and sequentially stimulated with CA and 80K (carvacrol, n = 9; AITC, n = 12; acrolein, n = 10; 3 frogs were used for each chemical).

FIGURE 8.

TRPA1 agonists evoked nocifensive behaviors in the WC frog. A, number of jumps induced by CA with different concentrations. Each bar indicates jumping during the 1-min interval before and after application of CA (n = 3–4). B, a concentration-response relationship for the jumps elicited by CA application. The average number of jumps per minute during the 10-min interval after CA application was plotted for each concentration. Each data point represents the mean ± S.E. (n = 3–4). C, number of jumps during the 1-min interval before and after application of carvacrol (upper), acrolein (middle), and AITC (lower). Three frogs were examined for each chemical.

FIGURE 9.

The evolutionary scenario for nociceptive receptors TRPA1 and TRPV1 within the vertebrate lineages. Temperature sensitivities of TRPA1 and TRPV1 in examined vertebrate species are listed. Note that cold sensitivity of mammalian TRPA1 is under debate. The channels that have not been examined are indicated with dashes. Zebrafish have only one gene that is similar to terrestrial vertebrate TRPV1 and TRPV2 (indicated as TRPV1/2). Each deduced evolutionary event occurring in the respective branch is indicated by an arrow. The gene duplication event producing TRPV1 and TRPV2 was not clearly resolved, thus alternative possibilities are indicated by a line with an asterisk.