Evolution of vertebrate transient receptor potential vanilloid 3 channels: opposite temperature sensitivity between mammals and western clawed frogs

Abstract

Transient Receptor Potential (TRP) channels serve as temperature receptors in a wide variety of animals and must have played crucial roles in thermal adaptation. The TRP vanilloid (TRPV) subfamily contains several temperature receptors with different temperature sensitivities. The TRPV3 channel is known to be highly expressed in skin, where it is activated by warm temperatures and serves as a sensor to detect ambient temperatures near the body temperature of homeothermic animals such as mammals. Here we performed comprehensive comparative analyses of the TRPV subfamily in order to understand the evolutionary process; we identified novel TRPV genes and also characterized the evolutionary flexibility of TRPV3 during vertebrate evolution. We cloned the TRPV3 channel from the western clawed frog Xenopus tropicalis to understand the functional evolution of the TRPV3 channel. The amino acid sequences of the N- and C-terminal regions of the TRPV3 channel were highly diversified from those of other terrestrial vertebrate TRPV3 channels, although central portions were well conserved. In a heterologous expression system, several mammalian TRPV3 agonists did not activate the TRPV3 channel of the western clawed frog. Moreover, the frog TRPV3 channel did not respond to heat stimuli, instead it was activated by cold temperatures. Temperature thresholds for activation were about 16 °C, slightly below the lower temperature limit for the western clawed frog. Given that the TRPV3 channel is expressed in skin, its likely role is to detect noxious cold temperatures. Thus, the western clawed frog and mammals acquired opposite temperature sensitivity of the TRPV3 channel in order to detect environmental temperatures suitable for their respective species, indicating that temperature receptors can dynamically change properties to adapt to different thermal environments during evolution.

Figure 1. Phylogenetic relationship of the TRPV gene subfamily of vertebrates.

Statistical confidence (bootstrap value) is indicated beside the respective branch . The TRPV5/6 genes were used as outgroups. WC frog, AC frog, SG pufferfish, and TS stickleback indicate western clawed frog, African clawed frog, spotted green pufferfish, and three-spined stickleback, respectively.

Figure 2. Conserved gene arrangements in the genomic regions encompassing vertebrate TRPV1-TRPV3 genes.

The gene orders around TRPV1/2 of medaka and torafugu (A) and TRPV1-TRPV3 of vertebrates (B) are shown. The genes are shown as boxes with their directions indicated. Filled, hatched, gray, and striped boxes represent TRPV1, TRPV2, TRPV3, and TRPV7 (only found in platypus genome), respectively. The open boxes indicate non-TRP genes. The orthologous genes among different species are connected by lines. The physical distances of the genomic regions are indicated. Chr., chromosome.

Figure 3. Comparison of TRPV3 among representative terrestrial vertebrate species.

The schematic structure of the TRPV3 channel (A) is shown and the N- (B) and C- (C) terminal regions of amino acid alignments are indicated. The open circles, gray boxes, striped boxes, and black box represent the putative ankyrin repeat, transmembrane, pore loop, and TRP domains, respectively. The amino acids identical to, similar to, and different from the consensus residues are indicated in red, blue, and black letters, respectively. Exon boundaries for human, mouse, platypus, and chicken are indicated by open triangles and those for western clawed frog by filled triangles. (B) The first ankyrin repeat domain is underlined. Gene structures of TRPV3s in several vertebrate species (D). Exons are indicated by open boxes according to their scale. Introns are indicated by solid lines with their lengths (bp). The open reading frame is indicated by a shaded area with its initiation (Met) and termination (Ter) codons. Gene structures of chicken and platypus were predicted in the genome sequence databases (Ensembl) but not supported by cDNA data thus exons for the 5′- and 3′-UTR regions are not known. Gene structures of human and rat TRPV3s are based on full length cDNA nucleotide sequences and that of the western clawed frog TRPV3 is base on the cDNA nucleotide sequence determined in the present study.

Figure 4. Cold temperature activation of the TRPV3 channel of the western clawed frog.

(A and B) Representative current traces (Upper) from oocytes injected with TRPV3 cRNA of the western clawed frog and corresponding temperatures (Lower). The bar indicates the application of 2-ABP (0.2 mM). (C) A representative trace of an oocyte injected with water alone. (D) An Arrhenius plot of the current in panel B. Average temperature threshold for activation was 16.35±0.51°C (n = 14).

Figure 5. The activation and inhibition properties of the TRPV3 channel of the western clawed frog.

The data in panels A-E were obtained from oocytes injected with TRPV3 cRNA of the western clawed frog. (A and B) Oocytes responded to 2-APB in a dose-dependent manner. A representative current trace evoked by 2-APB (A) and its dose-response curve (B). Currents were normalized to the values at 1 mM. The EC₅₀ was 0.54±0.24 mM, and the Hill coefficient was 1.1±0.3. Error bar indicate SEMs. (C) Inhibition of cold-induced currents in the oocytes. Ruthenium red (20 µM) was applied prior to and during the first cold stimulation. The second and third cold stimuli were applied four minutes after washing out the ruthenium red. (D) A 2-APB (0.5 mM)-induced current in the oocyte was inhibited by ruthenium red (10 µM) even in the presence of 2-APB. (E) Current-voltage relationships of cold- and 2-APB-evoked responses in the oocyte. (F and G) Representative current traces in response to initial applications of camphor (8 mM) (F) or eucalyptol (10 mM) (G) with secondary applications of 2-APB (0.5 mM) in oocytes injected with TRPV3 cRNA of the western clawed frog (Left) or mouse (Right).

Figure 6. Expression of TRPV3 in the skin of the western clawed frog.

Transcription profiles of TRPV3 in the western clawed frogs were examined by semi-quantitative RT-PCR. The gene for elongation factor 1α (EF-1α) was used as an internal control. For negative control experiments, RNA samples from toes of both fore- and hind-limbs as well as kidney were used. The upper and lower bands of the size markers were 200-bp and 100-bp, respectively for TRPV3, and 300-bp and 200-bp, respectively for EF-1α.

Figure 7. The evolutionary changes of TRPV3 channels in the vertebrate lineages.

The major evolutionary events are indicated on the respective branches. The amino acid sequences of the N- and C-terminal regions of the TRPV3 channels were conserved among amniote species, while the N- and C-terminal regions of TRPV3 in the western clawed frog were highly diversified from those regions of TRPV3 in other terrestrial vertebrate species. The ancestral states of the terminal regions are ambiguous since teleost fishes have lost the TRPV3 gene. Western clawed frog and mammals acquired opposite temperature sensitivities of TRPV3 channels; however, the timing of the shift is not clearly determined since the temperature sensitivities of TRPV3 channels of birds and reptiles have not been reported.