Resistance in taxa bearing this neurotoxin and a few predators appears to come from convergent replacements in specific Nav residues that interact with TTX. This poison is broadly employed as a potent antipredator defense, blocking voltage‐gated sodium channels (Nav) in muscles and nerves, paralyzing and sometimes killing predators. The repeated evolution of tetrodotoxin (TTX) resistance provides a model for testing hypotheses about the mechanisms of convergent evolution. The online version of this article includes the following figure supplement(s) for figure 5: Figure 5 continued on next page Data were fit with a Hill equation to estimate IC 50 values. (D) Dose-response curves showing the proportion of Na + current elicited during a step depolarization from À100 to À20 mV for wild-type, individual mutants, and triple-mutant Na v 1.6 channels exposed to increasing concentrations of TTX. 10 mM, p<0.0001), while mutated Na v 1.6 was unaffected (repeated measures ANOVA, p=0.879).
Wild-type Na v 1.6 was blocked by TTX (Tukey's multiple comparisons test with Bonferroni correction: control vs. (C) Current-voltage (I-V) relationships showing normalized currents for wild-type (n = 21) and mutant Na v 1.6 (n = 20) channels.
(B) Representative currents from wild-type mouse Na v 1.6 or Na v 1.6 with newt substitutions Y371A, V1407I, and I1699V treated with 1 mM (blue) or 10 mM (orange) TTX. Sequence alignment of Na v 1.6 pore-loop motifs revealed three amino acid differences in newts from Oregon or Idaho populations. (A) Predicted topology of Na v 1.6 with mutations in domains I, III, and IV. Newts possess Na v channel mutations that confer physiological resistance to TTX. Parallel evolution of DIII and DIV P-loop substitutions in Na v 1.6 of toxic newts and TTX resistant garter snakes. GenBank accession numbers of vertebrate Na v channel protein sequences used in multiple sequence alignments and analysis. The online version of this article includes the following source data and figure supplement(s) for figure 4: Source data 1. The approximate locations of newt mutations are shown as orange circles, and the amino acid site of each mutation is numbered based on Na v 1.6 from Mus musculus. Data are missing for DI and DII of Na v 1.1 in newts, which we did not recover in our sequencing efforts. Putative TTX resistance mutations are highlighted in orange mutations that are not highlighted are either synapomorphic in a gene clade or are present in TTX sensitive channels. Sequence alignment of S5-S6 P-loops from newts and other vertebrates showing amino acid substitutions relative to the P-loop consensus sequence for each Na v channel shown here. Protein alignment of Na v channels across representative vertebrates. This study highlights the complex interactions among adaptive physiology, animal-bacterial symbiosis, and ecological context. Additionally, we sequenced the Nav channel gene family in toxic newts and found that newts expressed Nav channels with modified TTX binding sites, conferring extreme physiological resistance to TTX. We then screened bacterial culture media for TTX using LC-MS/MS and identified TTX-producing bacterial strains from four genera, including Aeromonas, Pseudomonas, Shewanella, and Sphingopyxis. We characterized the skin-associated microbiota from a toxic and non-toxic population of newts and established pure cultures of isolated bacterial symbionts from toxic newts. Here, we investigated whether symbiotic bacteria isolated from toxic newts could produce TTX. Interestingly, newts exhibit extreme population-level variation in toxicity attributed to a coevolutionary arms race with TTX-resistant predatory snakes, but the source of TTX in newts is unknown. Rough-skinned newts (Taricha granulosa) use tetrodotoxin (TTX) to block voltage-gated sodium (Nav) channels as a chemical defense against predation.
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