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. 2020 Apr 7;9:e53898. doi: 10.7554/eLife.53898

Figure 4. Protein alignment of Nav channels across representative vertebrates.

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 Nav channel shown here. 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. Data are missing for DI and DII of Nav1.1 in newts, which we did not recover in our sequencing efforts. The approximate locations of newt mutations are shown as orange circles, and the amino acid site of each mutation is numbered based on Nav1.6 from Mus musculus.

Figure 4—source data 1. GenBank accession numbers of vertebrate Nav channel protein sequences used in multiple sequence alignments and analysis.

Figure 4.

Figure 4—figure supplement 1. Parallel evolution of DIII and DIV P-loop substitutions in Nav1.6 of toxic newts and TTX resistant garter snakes.

Figure 4—figure supplement 1.

Structure of the Nav channel and multiple sequence alignment of Nav1.6 across representative vertebrate taxa. The consensus sequence is shown above the alignment, and orange labels indicate taxa that either possess TTX or consume TTX-laden prey. Identical amino acid substitutions were observed between rough-skinned newts (Taricha granulosa) and garter snakes (Thamnophis sirtalis) from Benton Co. OR, where T. granulosa populations are highly toxic. Furthermore, some of these mutations were also identified in the less toxic eastern newt (Notophthalmus viridescens) and the non-toxic axolotl (Ambystoma mexicanum). These observations suggest that the last common ancestor of Ambystomids and Salamandrids possessed replacements in DIII and DIV. Our physiological data indicate that these replacements provide low levels of resistance, and may have thus allowed low level exposure to TTX, possibility facilitating the evolution of TTX toxicity in some Salamandrid species. Phylogenetic relationships are based on full-length Nav1.6 assessed by RAxML with 1000 bootstrap replicates. Support values are shown on each node, and the scale bar reflects the mean number of nucleotide substitutions per site.