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Figure 3 Naked Mole-Rat TrkA Is Impaired in TRPV1 Current Potentiation Show full caption (A) Schematic representation of the transfection and recording conditions used. (B) NGF causes substantial sensitization of proton-gated rat TRPV1 currents when signaling through rat TrkA, but this effect is reduced in the oocytes co-expressing the naked mole-rat TrkA receptor. (C) Sensitization levels across NGF concentrations were calculated as the ratio of a current immediately after and before NGF superfusion. High NGF concentration rescues the sensitization through NGF TrkA. For all the measurements, at least three pH stimuli were applied before the NGF superfusion in order to obtain the stable current responses, while at least two acid-gated currents were recorded post-NGF application. Between 2 and 18 oocytes were recorded for every NGF concentration. Inset: ten oocytes, injected with equal concentration of rat or naked mole-ratTrkA cRNA, were lysed, and pelleted membranes were subjected to western blotting. Prior to blotting, the protein concentration was measured to ensure equal sample loading. TrkA is expressed as a 140-kDa protein. (D and E) 100 ng/mL NGF potentiated acid-gated TRPV1 currents recorded in X. laevis oocytes via chimeric TrkA (D), but the sensitization level was significantly smaller than for rat TrkA and not different from the NMR TrkA receptor quantified in (E). Two-way ANOVA with Sidak’s multiple comparison was used in (C), and one-way ANOVA with Bonferroni’s multiple comparison test was used in (E) (∗p < 0.05; ∗∗∗p < 0.001). Data are presented as mean ± SEM, except in (C) for NMR TrkA at 1 ng/mL NGF (only mean current plotted), where two oocytes were recorded.

We cloned the naked mole-rat TrkA cDNA from mRNA isolated from sensory neurons (nmrTrkA). The nmrTrkA sequence was identical to that predicted from the naked mole-rat genome assembly (). The predicted naked mole-rat TrkA peptide sequence was aligned with orthologous sequences from 26 other mammalian species ( Figure S2 ). There was significant sequence divergence in the extracellular TrkA domains, including the juxtamembrane NGF-binding domain; however, the intracellular sequences within the kinase domain were highly conserved ( Figure S2 B). All tyrosine residues important for receptor activation were conserved in all the species, including the naked mole-rat. We reasoned that at least some of the amino acid variants in the kinase domain of nmrTrkA may be common variants found in African mole-rats (family Bathyergidae). In order to screen for such variants, we obtained TrkA sequences from five further African mole-rat species: the Damaraland mole-rat (Fukomys damarensis), the Mashona mole-rat (Fukomys darlingi), the giant mole-rat (Fukomys mechowii), the Natal mole-rat (Cryptomys hottentotus natalensis), and Emin’s mole-rat (Heliophobius emini) ( Figure S3 A). We used genomic DNA from these species to PCR amplify the exonic regions of the TrkA gene, guided by variants found in nmrTrkA. However, we also assembled TrkA transcripts from published RNA sequencing (RNA-seq) data from African mole-rat species (). In addition, we obtained RNA from the brains of three Mashona mole-rats and performed RNA-seq followed by de novo transcriptome assembly ( Table S1 ). An African mole-rat phylogeny was constructed including the new transcriptome data from the Mashona mole-rat ( Figure S3 C), and this was in close agreement with previous analyses that had not included this species (). Alignment of the available predicted TrkA amino acid sequences from African mole-rats revealed that the nmrTrkA kinase domain has accumulated at least three amino acid variants that are either absent or rare in the animal kingdom, including African mole-rats ( Figure S3 B). There was just one amino acid change that appeared to be unique to naked mole-rat, which was a leucine (rat) to cysteine substitution at position 774 ( Figure S2 B). The accumulation of amino acid variants in the nmrTrkA kinase domain encouraged us to carry out a functional analysis of the ability of this receptor to participate in nociceptor sensitization. To do this, we tested the ability of the naked mole-rat TrkA receptor to sensitize TRPV1 using electrophysiology with X. laevis oocytes as the heterologous expression system. Oocytes were injected with a ratTrpv1cRNA and cRNAs coding for either ratTrkA or nmrTrkA. We observed that 1 μM capsaicin causes substantial and long-lasting desensitization of TRPV1 currents in oocytes and thus decided to record proton-gated TRPV1 currents to quantify NGF sensitization, as others have done (). Using a two-electrode voltage clamp, we showed that an acidic stimulus (pH 5.8) produced robust inward currents in TRPV1-expressing oocytes that were absent in non-injected oocytes (data not shown). In oocytes injected with ratTrkA and ratTrpv1 cRNA, superfusion of NGF (100 ng/mL, 5 min) caused a robust sensitization of acid-gated currents ( Figure 3 B). However, the same NGF concentration produced a significantly smaller sensitization of TRPV1 currents in oocytes injected with nmrTrkA and ratTrpv1 cRNA ( Figures 3 B and 3E). Comparable amounts of rat and naked mole-rat TrkA protein were present in membranes isolated from X. laevis oocytes ( Figure 3 C), indicating that differences in TrkA protein levels was unlikely to account for the reduced TRPV1 sensitization. We next varied NGF concentration (1–1,000 ng/mL) but kept the superfusion time constant (5 min). TrkA is a high-affinity NGF receptor with a dissociation constant Kof less than 10M (). When oocytes were stimulated with 1,000 ng/mL NGF, activation of the naked mole-rat TrkA receptor produced a degree of sensitization similar to that observed with rat TrkA ( Figure 3 C). These results strongly suggest that the naked mole-rat TrkA molecule is less efficient at initiating sensitization with NGF concentrations of ∼100 ng/mL, which was shown to be saturating in adult rat sensory neurons (). It is conceivable that recombinant human NGF used in this study (rhNGF) displays stronger binding affinity to rat TrkA than to the naked-mole-rat TrkA. To test this idea, we cloned chimeric TrkA receptors containing the N-terminal, extracellular part of the receptor from rat TrkA together with the transmembrane domain and entire intracellular kinase domain from the naked mole-rat molecule ( Figures 3 D and 3E). HEK293 cells were transiently transfected with either rat or chimeric TrkA construct to assess NGF-stimulated TrkA activation ( Figure S4 ). An antibody raised against extracellular rat TrkA domain was used to measure the total level of TrkA protein in cell lysates (total TrkA), and two antibodies that recognize phosphorylated tyrosine residues in the TrkA kinase domain were employed to study receptor activation. Anti-phospho-TrkA (Tyr674/675; numbering for human TrkA) was used to measure the phosphorylation levels of the activation loop tyrosines (), and an anti-phospho-TrkA (Tyr490) was used that recognizes the activated putative Shc binding site (). NGF stimulation triggered rapid phosphorylation of Tyr674/675 in rat TrkA, but not in chimeric TrkA ( Figures S4 A and S4B). In contrast to rat TrkA, NGF treatment did not have any effect on activation of Tyr674/675 in the chimeric TrkA receptor. However, the Tyr674/675 residues in both chimeric TrkA and rat TrkA displayed strong basal receptor phosphorylation in the absence of NGF, probably triggered by receptor dimerization events due to overexpression. This observation is in agreement with previous findings that an antibody against the TrkA extracellular domain can itself crosslink two receptors, causing their activation in PC12 cells (). NGF triggered increased phosphorylation of the Tyr490 residue in the rat TrkA molecule after 1 min but did not have any apparent effect on the phosphorylation level of the chimeric TrkA Tyr490 residue ( Figures S4 C and S4D). Next, we tested chimeric TrkA in the context of NGF-mediated TRPV1 sensitization. Proton acid-gated TRPV1 currents in X. laevis oocytes co-expressing chimeric TrkA could only be moderately sensitized with 100 ng/mL NGF; indeed, the mean level of sensitization observed was not significantly larger than that found with the full-length nmrTrkA ( Figure 3 E). In contrast, sensitization of TRPV1 proton currents by NGF-stimulated oocytes co-expressing ratTrkA was at least twice as large as with full-length nmrTrkA or chimeric receptors. These results strongly suggest that a hypo-functional naked mole-rat TrkA kinase domain underlies the lack of TRPV1 sensitization in this species.